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+{"text": "Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of \u201cacquired\u201d resistance.EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec).We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.In patients with tumors bearing gefitinib- or erlotinib-sensitive  A specific secondary mutation in the kinase domain of the epidermal growth factor receptor can render cells insensitive to the two kinase inhibitors. This mutation was found in resistant tumors from three of six patients studied KRAS mutations have been associated with some cases of primary resistance to gefitinib or erlotinib [Somatic gain-of-function mutations in exons encoding the epidermal growth factor receptor (EGFR) tyrosine kinase domain are found in about 10% of non-small cell lung cancers (NSCLCs) from the United States ,3, with rlotinib , mechaniAcquired resistance to kinase-targeted anticancer therapy has been most extensively studied with imatinib, an inhibitor of the aberrant BCR-ABL kinase, in chronic myelogenous leukemia (CML). Mutations in the ABL kinase domain are found in 50%\u201390% of patients with secondary resistance to the drug (reviewed in ). Such mAlthough imatinib inhibits different kinases in various diseases  (reviewed in ), some tEGFR exons 18 to 24 in tumors from five patients who initially responded but subsequently progressed while on these drugs. These exons were also assessed in tumor cells from a sixth patient whose disease rapidly recurred while on gefitinib therapy after complete gross tumor resection. Because of the association of KRAS mutations with primary resistance to gefitinib and erlotinib [KRAS in tumor cells from these six patients. In an effort to explain the selective advantage of cells with a newly identified \u201cresistance\u201d mutation in EGFR\u2014a T790M amino acid substitution\u2014we further characterized the drug sensitivity of putatively resistant EGFR mutants versus wild-type or drug-sensitive EGFR mutants, using both a NSCLC cell line fortuitously found to contain the T790M mutation and lysates from cells transiently transfected with wild-type and mutant EGFR cDNAs.To determine whether lung cancers that acquire clinical resistance to either gefitinib or erlotinib display additional mutations in the EGFR kinase domain, we have examined the status of rlotinib , we alsoTumor specimens, including paraffin blocks, fine needle biopsies, and pleural effusions, were obtained through protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (protocol 92\u2013055  and protEGFR (exons 18\u201324) and KRAS2 (exon 2) analyses were as published [Genomic DNA was extracted from tumor specimens, and primers for ublished ,7. All sEGFR wild-type) for calibration, this assay detects the presence of the T790M mutation when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (data not shown).A specific exon 20 mutation (T790M) was also detected by length analysis of fluorescently labeled (FAM) PCR products on a capillary electrophoresis device , based on a new NlaIII restriction site created by the T790M mutation (2369 C\u2192T), using the following primers: EGFR Ex20F, 5\u2032-FAM-CTCCCTCCAGGAAGCCTACGTGAT-3\u2032 and EGFR Ex20R 5\u2032-TTTGCGATCTGCACACACCA-3\u2032. Using serially mixed dilutions of DNA from NSCLC cell lines , as per manufacturer's instructions. Minipreps of DNA from individual clones were sequenced using the T7 priming site of the cloning vector.The following primers were used to generate EGFR cDNAs using a QuikChange Site-Directed Mutagenesis Kit  and cloned into expression vectors as described [Two numbering systems are used for EGFR. The first denotes the initiating methionine in the signal sequence as amino acid \u221224. The second, used here, denotes the methionine as amino acid +1. Commercial suppliers of antibodies, such as the Y1068-specific anti-phospho-EGFR, use the first nomenclature. To be consistent, we consider Y1068 as Y1092. Likewise, the T790M mutation reported here has also been called T766M. Mutations were introduced into full-length wild-type and mutant escribed . The folescribed . All mutescribed . Cells wSee 2. For viability studies, cells were seeded in complete growth medium in black 96-well clear bottom ViewPlates  at a density of 5,000 (H1975 and H2030) or 7,500 cells per well (H3255). Following overnight incubation, cells were grown for 24 h in the supplemented RPMI-1640 medium with 0.1% serum. Cells (in supplemented RPMI-1640 medium containing 0.1% serum) were then incubated for 48 h in the continued presence of gefitinib or erlotinib.The NSCLC cell lines H1650, H1975, H2030, H2347, H2444, H358, and H1734 were purchased from American Type Culture Collection . H3255 was a gift of B. Johnson and P. Janne. Cells were grown in complete growth medium  supplemented with 10% fetal calf serum, 10 units/ml penicillin, and 10 \u03bcg/ml streptomycin) at 37 \u00b0C and 5% COCell viability was assayed using Calcein AM . Following incubation with gefitinib or erlotinib, monolayers were washed twice with PBS  and incubated with 7.5 \u03bcmol Calcein AM in supplemented RPMI-1640 (no serum) for 30 min. Labeling medium was removed, and cells were washed three times with PBS. Calcein fluorescence  was detected immediately using a Victor V multi-label plate reader (PerkinElmer). Three independent experiments were performed for each cell line; each experiment included four to eight replicates per condition.EGFR mutations in three of six individuals whose disease progressed on either gefitinib or erlotinib  initially presented with bilateral diffuse chest opacities and a right-sided pleural effusion. Transbronchial biopsy revealed adenocarcinoma. Disease progressed on two cycles of systemic chemotherapy, after which gefitinib, 250 mg daily, was started. Comparison of chest radiographs obtained prior to starting gefitinib .This 55-y-old woman with a nine pack-year history of smoking underwent two surgical resections within 2 y (right lower and left upper lobectomies) for bronchioloalveolar carcinoma with focal invasion. Two years later, her disease recurred with bilateral pulmonary nodules and further progressed on systemic chemotherapy. Thereafter, the patient began erlotinib, 150 mg daily. A baseline CT scan of the chest demonstrated innumerable bilateral nodules .This 55-y-old female \u201cnever smoker\u201d was treated for nearly 4.5 y with weekly paclitaxel and trastuzumab  for adenEGFR mutations, by direct DNA sequencing of exons 19 and 21 [EGFR mutation by direct sequencing .All three specimens from patient 2, including the original lung tumor and the two metastatic samples from bone and lung, showed an exon 19 deletion involving elimination of 11 nucleotides (2238\u20132248) and insertion of two nucleotides, G and C . These nBoth of the available tumor samples from patient 3 contained a deletion of 15 nucleotides (2236\u20132250) in exon 19  encoding the EGFR catalytic region in the available tumor specimens.To determine whether additional mutations in the EGFR sequence . However, we did not find any additional mutations in exons 18 to 24 of EGFR, including the C\u2192T change at position 2369 (data not shown). These results imply that alternative mechanisms of acquired drug resistance exist.In three additional patients (case histories not described here) with lung adenocarcinomas who improved but subsequently progressed on therapy with either gefitinib or erlotinib, we examined DNA from tumor specimens obtained during disease progression. In all three patients, we found KRAS2 occur in about one-fourth of NSCLCs. Such mutations rarely, if ever, accompany EGFR mutations and are associated with primary resistance to gefitinib or erlotinib [KRAS mutations confer acquired resistance to these drugs, we performed mutational profiling of KRAS2 exon 2 from tumor specimens from patients 1 to 3, as well as the three additional patients lacking evidence of the T790M mutation. None of the specimens contained any changes in KRAS  and KRAS exon 2 in eight established NSCLC lines .In our own analysis of H1975 (exons 18 to 24), the mutant 2369 T peak resulting in the T790M amino acid substitution was dominant, suggesting an increase in copy number of the mutant allele in comparison to the wild-type allele. The ratio of mutant to wild-type peaks was similar to that of the mutant 2573 G (corresponding to the L858R amino acid substitution) to wild-type T peaks  generated by the specific missense mutation. After PCR amplification with exon-20-specific primers spanning nucleotide 2369, wild-type sequence contains specific NlaIII sites, which upon digestion yield a 106-bp product A. PresenEGFR;We first used DNA from the H1975 cell line (which contains both T790M and L858R mutations) to confirm the specificity of the PCR-RFLP assay. As expected, analysis of these cells produced both the 97- and 106-bp fragments. By contrast, analysis of DNA from H2030 (which contains wild-type EGFR exons 18 to 24 by direct sequencing (data not shown).We next used this PCR-RFLP assay to assess various patient samples for the presence of the specific 2369 C\u2192T mutation corresponding to the T790M amino acid substitution. DNA from the progressing bone and lung lesions in patient 1 produced both the 97- and 106-bp fragments, but DNA from the original lung tumor did not B. The raEGFR cDNAs that encoded the exon 21 and 19 mutations found in patients 1 and 2, respectively. Corresponding proteins  were then produced by transient transfection with expression vectors in 293T cells, which have very low levels of endogenous EGFR [To determine how the T790M mutation would affect EGFR proteins already containing mutations associated with sensitivity to EGFR tyrosine kinase inhibitors, we introduced the specific mutation into Gefitinib inhibited the activity of wild-type and L858R EGFRs progressively with increasing concentrations of drug, as demonstrated by a reduction of tyrosine-phosphorylated proteins A and a dEGFR TK domain mutations (50 of about 0.01 \u03bcmol (50 of about 1 \u03bcmol (EGFR (exons 18 to 24) and mutant KRAS  appears to be extremely rare. We have not identified this mutation in 155 tumors (see above), and among nearly 1,300 lung cancers in which analysis of erformed ,5,6, onl. However, based upon analogous studies in CML, it is also possible that NSCLC subclones bearing this secondary mutation pre-exist within the primary tumor clone in individual patients, albeit at low frequency [How tumor cells bearing the T790M mutation emerge within gefitinib- or erlotinib-treated patients is a matter of investigation. Subclones bearing this mutation could arise de novo during treatmentrequency . In eithFrom analysis of the crystal structure of the EGFR kinase domain bound to erlotinib, it is has been shown that the wild-type threonine residue at position 790 is located in the hydrophobic ATP-binding pocket of the catalytic region, where it forms a critical hydrogen bond with the drug . The relThe T790M mutation could also affect the kinase activity or alter the substrate specificity of mutant EGFRs, such that a proliferative advantage would be conferred upon cells bearing the mutation. Consistent with this, the H1975 NSCLC cell line reported here to contain both T790M and L858R did not to our knowledge undergo any prior treatment with gefitinib or erlotinib; the doubly mutated cells must have become dominant over time through multiple passages in vitro. This scenario could explain the seemingly contradictory report by others who found the H1975 cell line to be highly sensitive to gefitinib ; our H19Recently, new small-molecule inhibitors have been identified that retain activity against the majority of imatinib-resistant BCR-ABL mutants. The new drugs bind to ABL in an \u201copen\u201d conformation, as opposed to imatinib, which binds ABL in a \u201cclosed\u201d conformation ,13. AnalIn some of the patient specimens analyzed, the actual sequencing peaks demonstrating the T790M mutation were smaller than originally anticipated. These results differ from those of acquired resistance mutation in CML , GIST 1,27, and EGFR, either within or outside the tyrosine kinase domain, are likely to exist. It is also possible that EGFR amplification itself plays a role in acquired resistance, since imatinib-resistant clones have been shown to lack resistance mutations but contain amplified copies of BCR-ABL [Since tumor specimens from three additional patients with acquired resistance to EGFR tyrosine kinase inhibitors did not demonstrate the T790M mutation, this specific lesion does not account for all mechanisms of acquired resistance to gefitinib or erlotinib. Given the paradigm established with imatinib, other drug-resistance mutations in  BCR-ABL ,28. NoneFigure S1(A) Patient 1. Serial chest radiographs from before (day 0) and during gefitinib treatment (14 d and 9 mo), demonstrating initial response and subsequent progression.(B) Patient 2. Serial CT studies of the chest before (day 0) and during erlotinib treatment (4 mo and 25 mo), demonstrating initial response and subsequent progression.(C) Patient 3. Serial chest radiographs before (day 0) and during adjuvant gefitinib treatment (3 mo), following complete resection of grossly visible disease. The left-sided pleural effusion seen at 3 mo recurred 4 mo later, at which time fluid was collected for molecular analysis.(951 KB PPT).Click here for additional data file.Figure S2EGFR exon 21 in tumor specimens from patient 1. DNA from the growing lung lesion and the pleural effusion demonstrated a heterozygous T\u2192G mutation at position 2573, leading to the common L858R amino acid substitution.(A) Status of (B) All three specimens from patient 2 showed the same heterozygous exon 19 deletion, removing residues 747\u2013749 and changing the alanine at position 750 to proline.(104 KB PPT).Click here for additional data file.Figure S3See legend for (153 KB PPT).Click here for additional data file.Protocol S1(566 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) accession number for the KRAS2 sequence discussed in this paper is 3845; the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the KRAS2 sequence discussed in this paper is NT_009714.16. Reference EGFR sequence was obtained from LocusLink accession number 1956 and GenBank accession number NT_033968.The LocusLink  and erlotinib , have been developed to inhibit activated EGFR, and studies have shown that they can shrink tumors in some patients. Most patients who respond to these drugs have tumors that carry an alteration (or mutation) in the EGFR gene, which somehow makes their tumors responsive to the drugs.In those patients in whom the drugs work, the tumors shrink initially, but after a while they stop responding and the cancer comes back. The cancer has, as researchers describe it, become resistant to the drugs. Understanding how tumors become resistant is important to develop new and better drugs.They asked patients who initially responded to erlotinib or gefitinib but then became resistant to consent to studies allowing further analysis of tumor tissue during and after drug treatment. They then re-examined the EGFR gene in these tumor samples.They found that tumors from all patients carried mutations in the EGFR gene that are known to make them responsive to the drugs. In addition, three of the post-treatment tumors had an identical second mutation in their EGFR gene. Biochemical studies showed that these secondary alterations made the original drug-sensitive EGFR less sensitive to drug treatment. The numbers are small but suggest that this secondary resistance mutation could be quite common. Tumor cells from the three other patients didn't have this mutation, which suggests that there are other ways for lung cancers to become resistant to gefitinib and erlotinib.Larger studies are needed to confirm that this particular mutation is a major cause of resistance against the two drugs. It is also important to find out what causes resistance in the other cases. And knowing about this resistance mutation will help researchers to develop drugs that will work even against tumors with the mutation.The following pages contain some information on the EGFR kinase inhibitors.http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htmU. S. Food and Drug Administration information page on Iressa (gefitinib): http://www.cancerhelp.org.uk/help/default.asp?page=10296Cancer Research UK information page about erlotinib (Tarceva):"}
+{"text": "The research article by Pao et al.  providesA 56-year-old female who had never smoked presented with nonproductive cough for one month. Her chest radiography revealed a mass in the right lower lung (RLL) . Chest tGenomic DNA was extracted from the tumor specimen of an original lung biopsy and a progressive tumor biopsy specimen. The tyrosine kinase domain (exons 18\u201321) was amplified and sequenced. Mutations were also checked against the corresponding DNA from blood lymphocytes at the diagnosis of lung cancer. The original diagnostic biopsy specimen contained a thymidine to guanine mutation at nucleotide 2573 of exon 21, resulting in L858R. In the second biopsy, an additional single-base change from cytosine to thymidine was identified at nucleotide 2369 in exon 20, resulting in T790M.This report strengthens the evidence of T790M as an acquired gefitinib-resistant mutation. Gefitinib responsiveness results in large part from the drug's effective inhibition of essential antiapoptotic signals transduced by the mutant receptor, and L858R is the most commonly detected mutation . The T79Pao et al. and Kobayashi et al. identified four cases with lung adenocarcinoma harboring pre-existing mutations of EGFR as delL747\u2013E749 plus A750P, delE746\u2013A750, or delL747\u2013S752, prior to the use of gefitinib or erlotinib ,6. All oThis correspondence was peer reviewed."}
+{"text": "EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.The T790MEGFR alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. L858R+T790MEGFR-driven tumors are transiently targeted by hsp90 inhibition. Notably, T790MEGFR-expressing animals develop tumors with longer latency than L858R+T790MEGFR-bearing mice and in the absence of additional kinase domain mutations.To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of T790MEGFR alone or in conjunction with drug-sensitive EGFR kinase domain mutations.These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring  Point mutations in the kinase domain of mutant epidermal growth factor receptors (EGFRs) are associated with acquired resistance to the EGFR inhibitors, gefitinib (Iressa) and erlotinib (Tarceva) in human lung adenocarcinoma T790M mutations in patients who never received gefitinib or erlotinib are rare T790M mutation T790M mutation in an L858REGFR-harboring lung adenocarcinoma cell line (H1975) that was developed and propagated in vitro prior to the development of EGFR kinase inhibitors T790MEGFR confers a growth advantage over cells expressing wildtype EGFRAlthough identified in the context of drug resistance, emerging data suggest that the T790M change may potentiate oncogenic activity, either by itself or in association with alterations in the EGFR kinase domain already known to confer gain-of-function properties T790M mutant in lung tumorigenesis in vivo, we have generated tetracycline (tet)-inducible transgenic mice that express in mouse lung epithelia the EGFRT790M mutant alone or in conjunction with a TKI-sensitive EGFRL858R mutant. We determined the effect of induction and de-induction of the transgenes in these animals by the exogenous administration and withdrawal, respectively, of the tet analog, doxycycline (dox). We further tested whether T790M-expressing lung tumors would respond to the kinase inhibitor, erlotinib, or an hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG). The latter drug has been previously shown to selectively degrade mutant EGFR in cell culture and xenografts experiments To study further the role of the EGFREGFR alleles, L858REGFR and del L747-S752EGFR. Both mutants are associated with sensitivity to erlotinib T790M mutant.A tet-inducible system has been used to regulate the expression in mouse lung epithelial cells of cDNAs encoding the commonly encountered mutant EGFR allele encoding the T790M mutation associated with EGFR kinase inhibitor resistance together with the L858R mutation associated with drug sensitivity . Transgene expression was induced in weaned double transgenic progeny  by administering dox via the animal diet We first generated a double mutant We observed that a bitransgenic mouse derived from line 51 became tachypneic and had an apparent large tumor burden on MRI after being administered a dox-containing diet for 17.5 weeks (data not shown). A colony from this line was subsequently expanded, and transgene-positive animals on dox for varying amounts of time were sacrificed for further analyses.EGFR expression was specific to lung tissues from line 51 animals, we performed RT-PCR with transgene specific primers on mRNA extracted from various tissues derived from multiple progeny. Transgene expression was detectable only in lung tissue . Moreover, we could not detect the transgene in control mice, i.e. in animals that harbored only the CCSP-rtTA or L858R+T790MEGFR transgenes alone .To determine whether mutant g tissue . MoreoveL858R-specific antibody . Mutant EGFR protein was also not detected in lung lysates from mice on dox for 17 weeks  and then subsequently fed a normal diet for 2 and 4 weeks, respectively. Expression of the mutant protein correlated with the detection of EGFR autophosphorylation at an important tyrosine residue (Y1092) . The timepoint at which induction of EGFRL858R+T790M protein can first be detected after initiation of dox appears to be somewhat variable, because we were able to detect the mutant protein after 2 weeks of induction in lungs from some animals (data not shown) but not in others . RT-PCR performed on lung-derived mRNA showed similar results regarding transcription of the transgene (data not shown). Nevertheless, these results collectively indicate that we were able to achieve inducible, lung-specific expression of mutant EGFR in C/L858R+T790M mice.Immunoblotting studies with an anti-EGFRantibody . Mutant antibody . Mutant . Lungs from bitransgenic mice maintained without dox exhibited normal lung morphology . By contrast, lungs from mice fed dox displayed heterogeneous adenocarcinomas  involving three main histological subtypes: solid , bronchioloalveolar , and papillary . Often, multiple histological subtypes were observed in the same mouse in adjacent lesions . Immunohistochemical analyses revealed that tumors were negative for CC26, a Clara cell protein  and positive for surfactant protein-C, a type II pneumocyte marker , indicating that the tumor cells had a type II cell-like phenotype. Tumors were not examined for the presence of bronchioalveolar stem cells, which display features of both type II pneumocytes and Clara cells We analyzed multiple untreated bitransgenic mice from line 51 fed dox for varying amounts of time (0\u201332 weeks), to determine the effect of mutant EGFR protein expression on mouse lungs . Lungs f. Lungs f. Lungs f. Lungs f. Lungs f. Lungs f. Lungs f. Lungs fL858R+T790M animals, lung lesions appeared mostly as dense solid consolidations ( and ). The patterns observed by MRI correlated well with tumor histopathology (data not shown).We previously used MRI to detect and monitor lung lesions in other lung tumor models L858R+T790M-induced lung tumors on continued expression of the mutant EGFR transgene, we monitored lung lesions by MRI in six bitransgenic animals (on dox for 13\u201332 weeks) after dox was removed. Serial MRIs showed that lung tumors progressively regressed in all animals ( and data not shown). Regression was seen as early as one week after removal of the inducer. By six weeks, MRI lesions appeared to have mostly resolved . The serial change over time in lung opacities seen by MRI\u2013presumably indicative of tumor volume (cm3) in an animal -- was readily quantifiable in individual animals using imaging software (see ).To examine the dependence of EGFR. Consistent with this observation, immunoblotting of lung lysates from the corresponding animals showed no detectable expression of mutant EGFR . However, microscopic islands of tumor cells could still be detected on tissue sections, even after mice were off dox for four weeks .In animals sacrificed after dox withdrawal, lungs displayed mostly degenerating tumors at the histological level . Consist. Consist. The EGFR inhibitor was given for 1 to 30 days at doses (25\u201350 mg/kg/d) known to be effective at inducing regressions in C/L858R mice that bear drug-sensitive tumors .We next examined the effect of erlotinib on lung tumors in C/L858R+T790M animals. Tumor-bearing animals with large lung opacities on MRI were administered placebo (n\u200a=\u200a3) or erlotinib (n\u200a=\u200a8) . The EGF, EGFRL858R+T790M-bearing tumors contained virtually all viable cells with no treatment effect  derived from a different founder (#12) with erlotinib at 50 mg/kg/d for 12 days. Consistent with the results discussed above, lung tumors in this animal did not show a response at either the radiological or histological levels (data not shown).To extend our findings to a separate line of C/L858R+T790M animals, we treated a lung adenocarcinoma-bearing mouse  and bitransgenic system as described above, we also generated mice expressing tetracycline-regulatable alleles carrying a mutant T790MEGFR cDNA alone. Thus far, we have identified 5 promising founder lines ; lines 8 and 37 have been characterized in most detail.Using the same transgenic plasmid construct . These lesions corresponded well with gross lung histology . In line 8, while one animal was found to have visible lung lesions after 18 weeks on dox, another 5 animals developed lung lesions only after 28\u201332 weeks. In lines 22, 45 and 71, lesions were detected after 52, 40 and 32 weeks, respectively (data not shown).Bitransgenic progeny on dox were screened for lung tumors as above. In most instances, animals from these lines developed detectable lesions only after long-term administration of dox, and we never observed the development of tachypnea or cachexia. In mice derived from line 37, lung lesions were not visible by MRI until about 28\u201332 weeks on dox . These lEGFR transgene . H&E staining of lung sections revealed invasive lung adenocarcinomas with a histological spectrum similar to those in C/L858R+T790M mice . Moreover, C/T790M tumors were dependent upon mutant EGFR for survival, as tumors regressed after dox withdrawal . Finally, as expected, tumors did not respond to erlotinib; none of five animals treated for 7 to 30 days with 50 mg/kg/d showed any tumor response .Similar to C/L858R+T790M animals, C/T790M animals (lines 37 and 8) displayed lung-specific expression of the mutant ransgene . H&E staransgene . H&E staransgene . H&E staT790M mutation, four of six tumors analyzed by dideoxynucleotide sequencing showed a secondary somatic activating EGFR mutation, arising in cis with the germline T790M mutation T790M mutation, we used oligonucleotide primers that span the human EGFR kinase domain to perform transgene-specific RT-PCR on mRNA derived from nine lung tumor nodules derived from nine different animals . Sequence analysis of the PCR products revealed the presence of the T790M mutation, but no additional kinase domain mutations were observed. Although we cannot currently exclude the possibility of other cooperating oncogenes that contribute to the lung tumors in these animals, these data indicate that expression of T790MEGFR alleles in mouse lung can lead to lung tumor formation in vivo in the absence of more common gain-of-function EGFR kinase domain mutations (i.e. deletion mutations in exon 19 or the common L858R point mutation in exon 21).Others have reported that in a family with multiple cases of lung adenocarcinoma associated with germline transmission of the . These data are consistent with the notion that the T790M mutation, when combined with activating EGFR kinase domain mutations, confers enhanced catalytic phosphorylating activity, as shown by others using insect cells infected with baculovirus expressing various EGFR intracellular domain constructs Interestingly, C/L858R+T790M (line 51) and C/T790M (line 8) animals on dox developed lung tumors with different latencies (\u223c17 vs \u223c32 weeks). Moreover, despite being on dox for less time, the number of nodules per lung that developed in C/L858R+T790M animals was greater than those observed in C/T790M mice T790M-driven lung adenocarcinomas is to use them to identify agents that can potentially overcome T790M-mediated resistance. Thus, as proof-of-principle, we treated tumor-bearing C/L858R+T790M animals with the geldanamycin analogue, 17-AAG. This ansamycin antiobiotic acts by inhibiting hsp90, a molecular chaperone which helps the folding of nascent polypeptides and stabilizes oncogenic kinases L858R+T790MOne goal of developing mouse tumor models that express EGFR, consistent with the notion that 17-AAG induces degradation of EGFR. Degradation was accompanied by variable effects on the phosphorylation of downstream components of the EGFR signaling pathway (i.e. Erk and Akt) . At the histological level, lungs from 6h-treated animals displayed evidence of treatment effect such as tumor necrosis . In contrast, lungs from placebo-treated animals displayed only viable tumor .We first treated tumor-bearing C/L858R+T790M animals with either placebo or 17-AAG, sacrificed mice 6 hours later, and examined the levels of total EGFR in lung lysates. As compared to extracts from lungs of mice treated with placebo, levels of EGFR were lower in extracts from lungs of drug-treated mice , consist, consist, consist that were similarly treated with 17-AAG (data not shown). Collectively, these data indicate that the hsp90 inhibitor, 17-AAG, has some antitumor effect against T790M-driven lung tumors. However, the dosing schedule used for these experiments induced only unsustained and modest disease control.To assess the durability of 17-AAG-induced treatment, additional animals were treated with 75 mg/kg/day of drug for 3 consecutive days per week for 1 week (n\u200a=\u200a6), 2 weeks (n\u200a=\u200a3), or 4 weeks (n\u200a=\u200a2). Control animals were treated with placebo for 3 consecutive days per week for 1 week (n\u200a=\u200a3) or 4 weeks (n\u200a=\u200a1). This dosing schedule was previously shown to deliver the maximum tolerated dose to mice T790MEGFR alleles also have been detected in a minority of tumors with primary resistance to these drugs T790M mutation has also been described Mutations that lead to substitution of methionine for threonine at position 790 in the EGFR kinase domain have been found in about 50% of tumors from patients with acquired resistance to the EGFR inhibitors, gefitinib and erlotinib T790MEGFR mutation in lung tumorigenesis, we have developed two new inducible mouse models of lung cancer that express transgenes encoding either T790MEGFR or L858R+T790MEGFR. The latter construct encodes the T790M mutant in combination with the drug-sensitive L858R mutant. Expression of either transgene induces formation of heterogenous lung adenocarcinomas that display histologies commonly found in human NSCLC, i.e. solid, papillary, and bronchioloalveolar subtypes. Tumors from mice expressing either allele are dependent upon mutant EGFR for survival. Moreover, compared to mice that express the drug-sensitive alleles, L858REGFR and del L747-S752EGFRT790M-EGFR and L858R+T790MEGFR-driven tumors are resistant to erlotinib.In order to understand further the role of the T790MEGFR transgene alone usually develop tumors with longer latency than animals expressing L858R+T790MEGFR(shown here) or even L858REGFR alone T790M can induce lung tumor formation in the absence of additional gain-of-function EGFR kinase domain mutations. However, because C/T790M mice develop tumors with a relatively long latency, it is likely that other genes may collaborate with T790MEGFR to induce lung tumorigenesis.Mice expressing the T790M facilitates transformation remains to be fully determined. Studies using global phosphoproteome analysis of kinase inhibitor-resistant BCR-ABL mutants suggest that the analogous T315I mutation in BCR-ABL substantially alters kinase function and is associated with a unique phosphosubstrate signature, such as a shift in phosphorylation of two tyrosines in the P-loop of BCR-ABL T790M similarly is associated with a unique phosphosubstrate signature in EGFR. Such studies could provide insight into disease progression as well as uncover new targets for therapy.How EGFRT790M-driven lung adenocarcinomas is to use them to identify agents that can potentially overcome T790M-mediated resistance. As proof-of-principle, we treated mice with the hsp90 inhibitor, 17-AAG, which can target mutant EGFRs, at least in cell culture or xenograft studies L858R+T790M-dependent tumors, as manifested by EGFR degradation, in vivo radiographic responses, and histological evidence for treatment effect. However, most tumor nodules in mice remained viable, and any responses seen by MRI appeared to be transient. Such results could be due to the short half-life of 17-AAG (6 hours) and an inability to dose the drug more frequently due to hepatotoxicity p value >0.05; Sawai, Solit, and Rosen, unpublished). These cells harbor L858R+T790MEGFR and are resistant to gefitinib/erlotinib in vitro Finally, one aim of developing mouse tumor models that express EGFRT790MEGFR alone or in conjunction with drug-sensitive EGFR kinase domain mutations.T790M-mediated resistance to EGFR inhibitors remains a significant clinical problem. We have generated two new mouse models that provide insights into clinical observations. The models could help accelerate the development of improved therapies for patients with lung cancers harboring EGFR cDNAs encoding T790MEGFR or L858R+T790MEGFRPmeI and ligated into the tet-op-mp1 vector EGFR, was further excised from each construct as previously described BssHII and injected into fertilized FVB F2 eggs by the MSKCC Transgenic Core Facility. For the L858R+T790MEGFR transgene, 60 pups were obtained. For the T790MEGFR transgene, two separate egg injections produced 212 pups. Tail PCR genotyping and/or Southern blotting identified six and twenty-four founders, respectively, for each transgene. Founders were subsequently crossed to mice that express the reverse tetracycline transactivator (rtTA) in type II pneumocytes (i.e. CCSP-rtTA) CCSP-rtTA mice were previously described All animals were housed in specific pathogen-free housing with abundant food and water, and they were treated with various drugs under guidelines approved by the MSKCC Institutional Animal Care and Use Committee and Research Animal Resource Center. Erlotinib (kindly provided by Genentech) was suspended in 0.5% (w/v) methylcellulose and injected intraperitoneally at the doses and times indicated. 17-AAG  was\u00a0dissolved in DMSO to yield 50mg/mL stock solutions and stored at \u221220\u00b0C. The EPL diluent was obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute. Prior to injection, 17-AAG was diluted with EPL solution at a minimum of 1:3 and\u00a0administered by i.p. injection.2 per institutional guidelines. In most instances after excision, the left lung was flash-frozen in liquid nitrogen. The right lung was inflated with 4% paraformaldehyde in PBS, fixed overnight at room temperature, placed in 70% ethanol, and sent for paraffin embedding and sectioning . In some animals, gross tumor nodules were macrodissected and flash-frozen, and the remaining lung tissues were processed for histological analysis. All lungs were sectioned in the same manner: 5 steps were taken, 100 microns apart. All steps were evaluated to determine whether tumors were present. Slides were reviewed by a board-certified pathologist with expertise in lung cancer (MFZ).Animals were sacrificed with a lethal dose of COTaq system (Invitrogen), with the following primers: RT-PCR F: 5\u2032-ACCAAGCCACAGCAGGTCCT-3\u2032 and RT-PCR R 5\u2032-TATGGTGTATGAGCGGCGGC-3\u2032. Control reactions were performed with Platinum Taq polymerase in the absence of reverse transcriptase.RNA was extracted from pulverized tissue samples using Trizol (Invitrogen) reagent. RNA was treated with DNase I (Sigma) to eliminate contaminating DNA. RT-PCR reactions were performed using the SuperScript III One-Step RT-PCR with Platinum EGFR kinase domain, RT-PCR analysis was performed as described above on mRNA from discrete lung nodules macrodissected from various mice. The following primers that span exons 18\u201321 were used: 2101F: 5\u2032-CCCAACCAAGCTCTCTTGAG-3\u2032 and 2948R: 5\u2032-AATGACAAGGTAGCGCTGGGGG-3\u2032. PCR products were then analyzed by direct dideoxynucleotide sequencing. All sequence tracings were manually reviewed in the forward and reverse directions.To seek potential mutations in the coding sequence of the For experiments involving 17-AAG, tumor lysates were homogenized in SDS lysis buffer (50mM Tris-HCl (pH7.4) and 2% SDS). For all other immunoblotting experiments, established protocols were performed 3) per animal was quantified by calculating the area of visible lung opacities present in each axial image sequence , using ParaVision 3.0.2 imaging software, and then multiplying the total sum of the areas by 0.09 cm (the distance between each MRI sequence).Mice were imaged according to established protocols by the MSKCC Small Animal Imaging Core facility There are no standard response criteria for evaluating the effect of drug treatment on lung tumors in mice. In humans, such criteria are based on uni- or bidimensional measurements, obtained from imaging studies where patients are conscious, placed in a certain position, and asked to hold their breaths at specific times. In mice, which are anesthesized, we have found that uni- or bi-dimensional measurements are not as accurate to assess tumor responses (data not shown). Therefore, we used tumor volume measurements and the following criteria to classify tumor responses to treatment: 1) complete response (CR): the disappearance of all target lesions; 2) partial response (PR): at least a 30% decrease in the volume of target lesions, taking as reference the baseline tumor volume; 3) progressive disease (PD): at least a 20% increase in the volume of target lesions, taking as reference the baseline tumor volume, and 4) stable disease (SD): neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the baseline tumor volume.Table S1Summary of C/L858R+T790M bitransgenic mice (line 51) treated with erlotinib.(0.06 MB DOC)Click here for additional data file.Table S2Summary of C/T790M bitransgenic mice treated with erlotinib.(0.05 MB DOC)Click here for additional data file.Table S3Summary of C/L858R+T790M bitransgenic mice (line 51) treated with 17-AAG.(0.09 MB DOC)Click here for additional data file.Figure S1Status of cell proliferation in lung tumors from C/L858R and C/L858R+T790M animals treated with erlotinib. Histological sections derived from the lungs of bitransgenic animals were stained with antibodies to the proliferation marker, Ki-67. Ki-67-positive cells in lungs were then quantitated by determining the average (with standard deviations) number of positive cells counted in three separate high power fields (hpf) at a magnification of 200x.(3.34 MB TIF)Click here for additional data file.Figure S2Representative MR images from bitransgenic mice treated with 17-AAG. Serial pre- and post-treatment images are shown. D-days; W-weeks.(7.55 MB TIF)Click here for additional data file."}
+{"text": "Transplantable organs are scarce everywhere. Therefore, countries have developed policies to support the efficient use of potential donors. Nevertheless, the shortage of organs remains. Were these policies in vain? The aim of this study is to assess the impact of donor policies on donor procurement in 10 Western European countries from 1995 to 2005.To assess the impact of the donor policies we studied the conversion of potential donors into effectuated donors. 80% of the donors died from CVAs or a (traffic) accident. We considered these mortality rates to be a good proxy for potential donors. Here we call the conversion of potential donors into actual donors 'the donor efficiency rate by proxy'.The mortality rates for CVA and (traffic) accidents have decreased in the countries under study. At the same time, in most countries the donor efficiency rates have steadily increased. The variance in donor efficiency rates between countries has also increased from 1995 to 2005. Four countries introduced a new consent system or changed their existing system, without (visible) long-term effects.The overall increase in donor efficiency means that the efforts to improve donor policies have paid off. However, substantial differences between countries were found. The success of donor policies in terms of the number of absolute donors is blurred by the success of policies on traffic safety and CVA treatment. It remains unclear which specific policy measures are responsible for the increase in donor efficiency rates. This increase is not related to having a presumed consent system. Furthermore, an analysis of countries that introduced a new consent system or changed their system showed no effect on donor efficiency. Transplantable organs are scarce throughout the world. The discrepancy between the number of people listed on a national organ transplant waiting list and the number of post mortem organ donations per year results in long waiting times for patients to receive an organ ,2. In otTo deal with the scarcity of organs, countries have developed national organ donation policies. In most countries these donor policies consist of a legislative system which regulates consent for donation  and addiOther policy measures are directed at optimizing the process of donor procurement. Examples of such measures are hospital programs like Donor Action ,9. and EIn many countries these efforts have intensified over time. However, the shortage of organ donors remains. According to several studies ,6,16,17.To assess the impact of donor policies and to compare the performance of the different countries, a valid and reliable measure is needed. In many studies the national donation rates PMI are used. Several studies have demonstrated that using the number of donors PMI does not produce a valid comparison ,18-30. TThe aim of this study is to assess the impact of the donor policies in 10 Western European countries on donor procurement from 1995 until 2005. This study is part of the national evaluation of the Dutch Organ Donation Act .To assess the impact of the donor policies we studied the conversion of potential donors into effectuated donors in 10 Western European countries. The number of confounding factors between countries was restricted by analysing only countries which share a more or less similar historical background and have more or less the same status of health systems.An exact measure for establishing a country's number of potential donors could be found by analysing all hospital medical records of deceased persons and identifying all potential donors -34. SuchAnother issue in establishing a country's number of potential donors is the selection of age groups. The number of people dying from a CVA increases with age, especially after the age of 65. Several countries implement senior donor programs ,37. Howeth Revision (ICD-10).The data for CVA and (traffic) accident mortality rates were derived from the WHO's Health for All Database (HFA-DB) . The ageFor some countries the mortality rates for certain years were missing in the WHO's HFA-DB. Because the mortality rates in all countries show a steady decrease we decided it was safe to estimate the missing mortality rates. The trend lines, which are based on estimated mortality rates, are shown in the graphs by a dotted line.Different countries use different definitions for their national organ donation rates. To counteract these variations in national definitions we collected new data on the national number of donors based on one uniform definition. We asked the national transplant centres to send us their 'numbers of post mortal organ donors of whom at least one solid organ had been successfully transplanted per year'. This definition was preferred because it accounts for differences between countries in the quality of procured organs and it has a better coverage of the data in the period 1995 to 2005. Because France and Sweden could not provide their data according to this definition for the entire period (1995\u20132005), they were asked for their 'numbers of post mortal organ donors of whom at least one solid organ had been recovered for the purpose of organ transplantation '.From the countries that could provide the rates according to both ways of measuring organ donation rates we learned that the difference between both rates is no more than 5% overall. As we use the same definition within countries, the use of different definitions between countries does not affect the national trends for organ donation. Therefore, we found it acceptable to use the rates for France and Sweden, although they were obtained by using a deviating definition.The rates per million inhabitants were calculated using the population size of the mid-year population given by the WHO HFA-DB .In this study we assess the conversion of potential donors into actual donors by the donor efficiency rate by proxy. The donor efficiency rate by proxy is calculated by using the following definition:  * 100. To determine significantly increasing or decreasing trends, the slopes of the donor efficiency trends of three time frames  were calculated using a standard regression analysis.As several authors report a positive impact of consent systems on donor procurement -7., we aFigure Figure There are large differences between the national organ donation rates PMI. As often mentioned in other studies, Spain has by far the highest rate per million inhabitants, followed by Austria and Belgium. These countries have twice as many organ donors PMI as for example Germany, the Netherlands, Sweden, Switzerland and the United Kingdom.The impact of donor policies, taking into account differences in relevant mortality between countries, is demonstrated by the donor efficiency rates by proxy in figure Table During the 1995\u20132005 period, most countries  showed a significantly rising trend. In the same period the Netherlands, Sweden and the United Kingdom had no significant increase in their donor efficiency rates. We even see that during 1995\u20131999 the Netherlands as well as the United Kingdom had a significant decrease in their donor efficiency rates. The decreasing trend in the Netherlands recovered after 2000, showing a significantly rising trend instead. Likewise, the Swedish donor efficiency significantly increased in this period. In addition to figure On average, we did not find obvious differences between countries with a presumed consent system and countries with an explicit consent system figure , nor didWe found a decrease in relevant mortality rates figure , which iAfter adjusting the organ donation rates for the changes in relevant mortality rates, most of the ten countries under review now demonstrate an increased efficiency in their donor procurement, implying a positive impact of donor policies in these countries. Not all countries show a steady increase in donor efficiency. The variance in donor efficiency rates by proxy increased from 1995 to 2005  consent system. In both countries the donor efficiency trend was already increasing before the implementation of the new consent system and continued to increase in the same way after the introduction , to increase the donor pool (by using older donors or non heart beating donors) or to inform the public about the relevant aspects of organ donation.Whereas the shortage of donor organs persists, it is important to find pointers for the improvement of donor procurement. Because little is known about which specific policy measures are successful for each country, an organization like the European Commission, for instance, should support initiatives that achieve international comparable data on national policy measures among EU-members. In 2007 the European Commission organized two meetings to discuss organ donation and transplantation at EU level . In addiNonetheless, policymakers should be aware of the fact that post mortal organ donation alone will not solve the long waiting times for patients in need of an organ. Therefore, other methods of donor procurement, such as encouraging living donation and the development of an artificial kidney, should be considered.The authors declare that they have no competing interests.RC is responsible for the study conception and design, carried out the data collection and drafted the manuscript. RDF is responsible for the study conception and design. All authors made substantional contributions to the analysis and interpretation of the data.RDF, SKMG, GAB and JZ made critical revisions to the manuscript for important intellectual content and provided supervision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Using the 5XFAD mouse model of AD that shows accelerated A\u03b2 deposition, we investigated the effect of chronic treatments (treatment onset at different ages and different durations) with the 5-HT4 receptor agonist RS 67333 during the asymptomatic phase of the disease. Chronic administration of RS 67333 decreased concomitantly the number of amyloid plaques and the level of A\u03b2 species. Reduction of A\u03b2 levels was accompanied by a striking decrease in hippocampal astrogliosis and microgliosis. RS 67333 also transiently increased sAPP\u03b1 concentration in the cerebrospinal fluid and brain. Moreover, a specific 5-HT4 receptor antagonist (RS 39604) prevented the RS 67333-mediated reduction of the amyloid pathology. Finally, the novel object recognition test deficits of 5XFAD mice were reversed by chronic treatment with RS 67333. Collectively, these results strongly highlight this 5-HT4 receptor agonist as a promising disease modifying-agent for AD.Amyloid \u03b2 (A\u03b2) accumulation is considered the main culprit in the pathogenesis of Alzheimer\u2019s disease (AD). Recent studies suggest that decreasing A\u03b2 production at very early stages of AD could be a promising strategy to slow down disease progression. Serotonin 5-HT Alzheimer\u2019s disease (AD) is currently recognized as one of the most socially devastating neurodegenerative disorders . AD pathAlzheimer\u2019s disease neuropathological hallmarks include extracellular deposits of amyloid \u03b2 (A\u03b2) peptides (amyloid plaques) and intracellular aggregates of hyper-phosphorylated Tau protein , and 2C (5-HT2C) receptors -3-[1-[2-[(methylsulfonyl)amino]ethyl]]-4-piperidinyl]]-1-propanone hydrochloride). All drugs were purchased from Tocris Bioscience .4 receptor activation in non-pathological conditions, four different groups of WT C57BL/6 mice (n = 6/group) received one intraperitoneal (i.p.) injection of vehicle , RS 67333 , RS 39604 (a 5-HT4 receptor antagonist), or both drugs . Animals were sacrificed 30 min after the injection, heads were quickly frozen in liquid nitrogen, skulls opened on ice, and frontal cortex and hippocampus dissected and stored at -80\u00b0C for further analysis.To induce acute 5-HTn = 5) were treated with RS 67333 from 1 to 4 months of age. In \u201cProtocol 2,\u201d mice (n = 6) were treated for 2 months, from 2 to 4 months of age. In \u201cProtocol 3,\u201d RS 67333 was administered for 1 month, from 2 to 3 months of age (n = 8). In addition, to antagonize RS 67333 effects, four different groups of mice (n = 4 each) received vehicle, RS 67333, RS 39604, or both drugs (the antagonist was administered 15 min before the agonist) according to protocol 2. At the end of each protocol, mice were anesthetized with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine in saline solution and perfused transcardially with PBS. Brains were quickly isolated on ice, the olfactory bulbs and cerebellum removed and the two hemispheres divided. One hemisphere was frozen on dry ice and stored at -80\u00b0C for biochemical analysis, while the other was post-fixed in 4% PFA for immunohistochemistry (IHC). WT mice, which do not develop plaques, were used to investigate the possible toxic effects of the drugs.5XFAD mice and WT littermates received chronic treatments with drugs or vehicle according to three different protocols. In each experiment, drugs or vehicle solution were administered i.p. twice a week (1 mg/kg). In \u201cProtocol 1,\u201d mice -tagged mouse APP695, as previously described  according to the manufacturer\u2019s instructions. The reaction readout was performed at 405 nm using an Infinite 2000 luminescence counter .This technique measures all secreted soluble forms of APP and does not discriminate between sAPP\u03b1 and sAPP\u03b2.COS-7 cells were grown in Dulbecco\u2019s modified Eagle medium (DMEM) supplemented with 10% dialyzed fetal calf serum (dFCS) and antibiotics. Cells were transfected with plasmids encoding HA-tagged 5-HTescribed , and thedura mater of the cisterna magna. Cerebrospinal fluid (CSF) was collected by capillary action and transferred to 0.5 mL microtubes, immediately frozen on dry ice and stored at -80\u00b0C until use. Once thawed, samples were heated at 60\u00b0C for 5 min as described in Mice were anesthetized and mounted onto a stereotaxic instrument. The neck skin was cut and subcutaneous tissue and muscles separated with the help of micro-retractors . Mice were then laid down so that the head formed an angle of about 135\u00b0 with the body . A capilg for 20 min and supernatants (the \u201csoluble fraction\u201d) collected and aliquoted for storage at -80\u00b0C. Pellets were resuspended by brief sonication in 10 volumes of 6 M guanidine HCl in 50 mM Tris-HCl, pH = 7.6 and centrifuged again at 265,000 \u00d7 g for 20 min. Supernatants (the \u201cinsoluble fraction\u201d) were aliquoted and stored at -80\u00b0C  with a protease inhibitor cocktail . The resulting homogenates were centrifuged at 540,000 \u00d7 at -80\u00b0C .40 , A\u03b242  or sAPP\u03b1  were used according to the manufacturer\u2019s instructions. Reactions were read at 620 and 450 nm using an Infinite 2000 luminescence counter. The obtained values were normalized to the protein concentration of each sample, measured using a BCA protein assay (Sigma-Aldrich). The sAPP\u03b1 ELISA kits enable the precise and selective quantification of sAPP\u03b1 versus sAPP\u03b2.ELISA kits from IBL International  for the dosage of A\u03b2Thirty-micrometer-thick sections were cut using a vibratome  and stored in cryoprotectant medium at -20\u00b0C. For the labeling of amyloid plaques, free-floating tissue sections of frontal cortex, hippocampus, and entorhinal cortex  were extensively washed in PBS and then incubated in blocking solution  for 1 h. Sections were stained with Hoechst dye  for 15 min to detect cell nuclei and then with freshly prepared thioflavin T solution  for 15 min. After washing in 70% ethanol for 5 min, samples were mounted on poly-lysine slides with coverslips. For GFAP  or IBA1  staining, free-floating brain sections were blocked as before and incubated with polyclonal rabbit anti-GFAP  or anti-IBA1 antibodies  at 4\u00b0C overnight. After thioflavin T staining and washing with 70% ethanol, the secondary Alexafluor 594 goat anti-rabbit antibody  was added for 2 h. PBS washes and mounting were performed as described before.2 in two tissue sections from the same brain area/animal (Image J software). GFAP and IBA1 expression were quantified in hippocampus (dentate gyrus) using the same method and results were expressed as area fractions. For representative images of thioflavin T and GFAP staining, mosaic sequential scans with a 10\u00d7 objective were taken. Detailed images of plaques were captured with a 40\u00d7 objective and selected Z-stacks were pooled together.Images were acquired with an AxioImager Z1 microscope . Analysis of thioflavin T staining was performed blindly and data are presented as the mean number of particles per mmThe cognitive performance of mice was tested using the novel object recognition (NOR) test . Animalspost hoc tests in the case of multiple comparison groups. In all other cases, the unpaired Student\u2019s t test was used. For all statistical tests, p < 0.05 was considered significant. Analysis was performed with GraphPad Prism 6.0a .All values are expressed as the mean \u00b1 SEM. Significant effects of treatments were determined by ANOVA analysis followed by Bonferroni\u2019s or Tukey\u2019s 4 receptor activation in non-pathological conditions, COS-7 cells that transiently express 5-HT4 receptors and SEAP-tagged APP were stimulated with increasing concentrations of the highly selective and widely characterized 5-HT4 receptor agonist RS 67333 . Then, the release of sAPP\u03b1 was investigated in vivo in WT C57BL/6 mice after a single administration of RS 67333 , as previously done for the two 5-HT4 receptor agonists prucalopride and ML 103022  and 1.73-fold  relative to control values . RS 67333-mediated sAPP\u03b1 release in both brain regions was prevented when the specific 5-HT4 receptor antagonist RS 39604 . Finally, acute 5-HT4 receptor activation by RS 67333  increased significantly sAPP\u03b1 release also in the 5XFAD transgenic mouse model of AD). Specifically, sAPP\u03b1 concentration in CSF from treated 5XFAD mice reached a peak 90 min post-injection  and returned to basal levels 4 h after drug delivery . sAPP\u03b1 level was also significantly higher  in the hippocampus of RS 67333-treated 5XFAD mice in comparison to controls . Conversely, A\u03b242 concentration in CSF and hippocampus of RS 67333-treated mice was not significantly different compared to controls , indicating that acute 5-HT4 receptor stimulation increases sAPP\u03b1 release in hippocampus and CSF, without affecting A\u03b2 levels in CSF during the 4 h following RS 67333 administration.To study sAPP\u03b1 release upon acute 5-HTRS 67333  or with RS 67333 , showed L 103022 . Thirty RS 39604  was injeFigure 2A). To test whether chronic treatment with the 5-HT4 receptor agonist RS 67333 could slow down amyloid plaque deposition, we designed three treatment protocols (protocols 1\u20133) that differed in terms of age at the onset of treatment and duration of treatment, but not in the amount and frequency of administration . Controls were 5XFAD mice treated with vehicle. Frontal cortex, hippocampus, and entorhinal cortex were analyzed as representative areas of 5XFAD mouse brain  and of human brain regions that are affected early by AD and are highly enriched in amyloid deposits , with a drastic reduction of amyloid plaque density (A\u03b2 load) in all the analyzed brain areas . In protocol 2 , a significant reduction of the plaque number was still observed in the frontal and entorhinal cortices , but the small decrease in the hippocampus was not significant . Further shortening of the treatment , only resulted in a non-significant trend toward a decrease of the plaque number . This suggests that both treatment onset at early age and treatment duration are crucial to obtain robust therapeutic effects.RS 67333 treatment (protocols 1 and 2) strongly decreased the number of amyloid plaques compared to vehicle. The most significant effects were observed with protocol 1 . Conversely, A\u03b240 and A\u03b242 decrease in the soluble fraction was not significant . In the group treated following protocol 2, only A\u03b242 levels were significantly reduced in the insoluble and soluble factions . One-month treatment with RS 67333 (protocol 3) was not sufficient to affect A\u03b240 and A\u03b242 accumulation in brains of 5XFAD mice . Collectively, these findings suggest that RS 67333 treatment might impact total brain A\u03b2 content.Consistent with the reduced amyloid plaque load, quantification by ELISA showed a clear reduction of both A\u03b24 receptors in the in vivo effect of RS 67333, we examined whether RS 39604, a specific 5-HT4 receptor antagonist inhibits the protective effect of the agonist in one of the protocols used in our study (protocol 2). Pre-treatment with RS 39604 (administered 15 min before each RS 67333 injection) of a group of 5XFAD mice treated for 2 months prevented the RS 67333-induced reduction in A\u03b242 levels  and the decrease in plaque formation in the entorhinal  and frontal cortices . The 5-HT4 receptor antagonist had no effect on the amyloid burden when administered alone for 2 months . Collectively, these data demonstrate that 5-HT4 receptor activation during the prodromal phase of disease reduces A\u03b2 accumulation and amyloid plaque load in 5XFAD mice.To demonstrate the involvement of 5-HTFigures 5A\u2013C) and microgliosis . In protocol 2, a slight but non-significant reduction of astroglial staining was seen in RS 67333-treated 5XFAD mice (data not shown).Increasing evidence indicates that plaque deposition induces astrogliosis and microgliosis in the brain of patients with AD. Over the years, these chronic inflammation processes are likely to contribute to AD progression . We thus4 receptors exerts pro-cognitive effects on learning and memory  could also improve cognitive performances via preventive reduction of A\u03b2 formation. These behavioral studies were performed using protocol 2, which is the most relevant protocol for translation into a treatment of the human pathology (drug delivery at the adult stage). 5XFAD mice and WT littermates were thus treated for 2 months with RS 67333 or vehicle and then their episodic-like memory was tested using the NOR test that is classically employed in mouse models of AD , in agreement with the fact that 5XFAD mice start to show altered cognitive functions at 4 months of age . RS 67333 did not have a pro-cognitive effect in WT littermates in these experimental conditions. Finally, the total exploration time during the training session did not differ significantly in the four groups , indicating that the recognition memory improvement in 5XFAD mice treated with RS 67333 is not secondary to an increase of the exploratory activity.As acute stimulation of 5-HTd memory , we thenls of AD . Trainins of age . Convers4 receptor agonist RS 67333 reduces the amyloid burden and inflammation markers (astrogliosis and microgliosis) and prevents memory impairment in 5XFAD mice. We also demonstrate that starting the treatment early  and its duration are both crucial to significantly reduce the levels of A\u03b2 species and plaque formation.In this study, we show that early, chronic administration of the 5-HT4 receptor activation is now well established in cell lines and primary neuronal cultures  can reduce amyloidogenesis in 10- to 12-month-old Tg2576 mice (another mouse model of AD) possibly through matrix metalloproteinase-9 (MMP-9) up-regulation (4 agonist (SSP-002392) in APP/PS1 mice that express human APP with the Swedish mutations and mutant human PS1  the model used (Tg2576) was far less aggressive and easier to cure than the mouse model that was used here (5XFAD). Therefore, our work brings new insights into the 5-HT4 receptor-induced non-amyloidogenic cleavage of APP and the potential of 5-HT4 agonists to slow down disease progression.Amyloid precursor protein non-amyloidogenic processing and sAPP\u03b1 release upon agonist-mediated 5-HTcultures . Moreovegulation . Anotheruman PS1 . Despite4 receptor agonist RS 67333 at low doses (1 mg/kg). This effect was reversed by a 5-HT4 receptor antagonist. Moreover, we show for the first time an increase in sAPP\u03b1 accumulation in the CSF of 5XFAD mice following RS 67333 administration. Kinetic studies indicated that sAPP\u03b1 was only transiently increased in CSF, a finding that might explain why sAPP\u03b1 production is difficult to detect in brain samples . The extent of 5-HT4 receptor-mediated sAPP\u03b1 release in CSF could also constitute a biomarker to follow the capacity to induce non-amyloidogenic APP cleavage in AD brains in which the 5-HT4 receptor density is known to be reduced  in all brain regions after early, chronic treatment with RS 67333 (up to 3 months). These data are in agreement with previous in vitro results showing that RS 67333 inhibits in a dose-dependent manner the generation of A\u03b240-42 species in primary cortical cultures of Tg2576 mice over a 2-day accumulation period  reaches a critical concentration, aggregation might begin to occur, leading to plaque formation. Therefore, the decrease in amyloid plaque formation, following chronic 5-HT4 receptor activation, might result from a shift of APP cleavage toward the non-amyloidogenic pathway. The apparent discrepancy with the absence of change in A\u03b242 level following acute treatment with the 5-HT4 receptor agonist might reflect the slower kinetic of A\u03b2 production, which requires two consecutive enzymatic cleavages. Further studies are needed to precisely characterize the effects of 5-HT4 receptor agonists on the release in CSF of other products of APP metabolism, an issue that we could not address in the present study due to insufficient amount of material available. Collectively, our results are consistent with a decrease in A\u03b2 accumulation that consequently diminish the number of amyloid plaques in diseased brains and with a preventive action of chronic treatment with RS 67333.Amyloid plaques seem to be the final response of the organism to the progressive increase in A\u03b2 concentration . Interes4 receptor activation, in contrast with recent results using the 5-HT4 agonist SSP-002392 in APP/PS1 mice . We observed the strongest effects with protocol 1, which combined very early treatment onset (1-month-old mice) and longest duration (3 months). In contrast, the 1-month treatment at a later stage of disease was insufficient to trigger significant improvement, consistent with the observation by 4 receptors undergo rapid and sustained desensitization in neurons (4 receptor agonist at moderate doses (1 mg/kg) and only twice a week, not daily. Finally, our findings demonstrate that a 2-month-long chronic treatment is already sufficient to prevent the appearance of cognitive deficits in 5XFAD mice. Collectively, our results clearly show that 5-HT4 agonists given at an early stage of the disease (before the appearance of cognitive decline) act preventively on A\u03b2 formation and can preserve memory performance in 5XFAD mice. The necessity of early intervention regarding AD mouse models has also been established for other therapeutic strategies such as \u03b3-secretase inhibition  is associated with lower A\u03b2 levels and reduced number of plaques in mice and humans . AmyloidThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Surgery remains the mainstay of soft tissue sarcoma (STS) treatment and has been the primary treatment for the majority of patients in Scandinavia during the last 30 years although the use of adjuvant radiotherapy has increased. Patient and treatment characteristics have been recorded in the Scandinavian Sarcoma Group (SSG) Register since 1987. When the effect of new radiotherapy guidelines from 1998 was evaluated, the reliability of surgical margin assessments among different Scandinavian institutions was investigated. Margins were reevaluated by a panel of sarcoma surgeons, studying pathology and surgical reports from 117 patients, randomly selected among 470 recorded patients treated between 1998\u20132003. In 80% of cases, the panel agreed with the original classification. Disagreement was most frequent when addressing the distinction between marginal and wide margins. Considered the element of judgment inherent in all margin assessment, we find this reliability acceptable for using the Register for studies of local control of STS. Soft Tissue Sarcomas (STSs) are optimally removed with a safety margin of healthy tissue encompassing the tumor. After surgery, the completeness of removal is evaluated by assessing the quality and thickness of this margin. During the last decades, the margin has most often been classified as intralesional, marginal, wide, or radical/compartmental referring to Enneking et al. .During the early years (1970s) of the Scandinavian Sarcoma Group's (SSG) existence, compartmental excisions according to Enneking were sometimes attempted. However, better referral practices, with more patients referred to tumor centers before surgery, has often made it possible to avoid the sacrifice of function such operations entail. Routine use of MRI in planning has enabled safe resection margins inside compartments \u20134. The sIt is widely accepted that the quality of the surgical margin is of prime importance for local control \u20138. To coDifferent routines for margin assessment are described , 8\u201312. MNot all studies detail these procedures. The surgeon may measure the thickness of the smallest margin of surrounding tissue on the fresh specimen, omitting areas of smaller distances where there is fascial coverage. The pathologist may measure the thickness of the cuff macroscopically on fresh or formalin-fixed specimen using a variable amount of slices. Finally, the smallest distance without fascial cover can be measured microscopically as the distance from tumor tissue to an inked surface. The number of slides necessary for the pathologists' conclusion may or may not be stated, even when detailed margin analyses are done .In Scandinavia, a wide surgical margin without radiotherapy was formerly considered adequate for treatment of localized STS. Radiotherapy was applied after surgery with intralesional or marginal margins. From 1998, adjuvant radiotherapy was recommended by the SSG for all deep extramuscular, high-grade sarcomas regardless of surgical margin or size. In the context of evaluating the efficacy of this change of policy regarding local recurrence rate , the preNine university hospitals with a sarcoma unit report to the Central Register. Compiled between 1986 and 2011, this Register contains data on 8322 bone and soft tissue sarcoma patients. They all had their final treatment for primary tumor at a sarcoma centre in Scandinavia. Detailed guidelines for reporting the variables exist, and these are discussed among local unit coordinators on a yearly basis. During the first five-year period after 1998, 470 patients were treated for a primary, previously not operated, soft tissue sarcoma of the extremity or trunk wall (liposarcoma grade I excluded) at four large Scandinavian institutions. These institutions have contributed 68% of the total content of the Register.Of the 470 patients, 117 (25%) were randomly selected for blinded reassessment of margins by four sarcoma surgeons representing the above-mentioned institutions. The evaluation was based on reports from the operation and the pathology evaluation. The original margin classification as reported to the SSG Central Register was in most cases based on collaboration between the local surgeon and the pathologist.Guidelines for margin assessment in this study were those sited in \u201cCentralized Registration of Sarcoma Patients in Scandinavia SSG VII: 2 Modified 31 March 1995.\u201d\u2009Intralesional: intracapsular, subtotal, piecemeal removal.\u2009Marginal: the dissection is close to the tumor in one or more places, perhaps all around . If during an operation, that is intended to be a wide excision, the tumor is unexpectedly exposed in one single place, or if histological examination reveals that the margin is marginal in one single place, the excision should be described as marginal, irrespective of how much healthy tissue is included elsewhere.\u2009Wide : the tumor is removed en bloc completely surrounded by a safety margin of healthy tissue. The tumor is not completely surrounded by an unbroken fibrous boundary. A wide margin for a subcutaneous sarcoma requires inclusion of the deep fascia beneath the tumor. For a tumor located between muscles, the muscles around the tumor are included in the surgical specimen.\u2009Myectomy : the muscle in which the tumor is located is removed unopened along with its fibrous boundary (sometimes bone).\u2009Compartmental: the tumor-involved compartment (defined according to SSS) is removed en bloc.The definitions of surgical margins are basically those of the Surgical Staging System  and those given by SSG 1981 in \u201cAdjuvant chemotherapy in soft tissue sarcoma\u201d (Bertil Stener).In the SSG, the margin should be assessed on the pathological specimen after fixation in formalin and ink-dying of the surface. The specimen is sliced at maximum 1\u2009cm increments and gross-sections or partial gross-sections are made from areas of closest margin at the surgeon's guidance. The pathology report must also include an evaluation of the quality of the margin and the tumors growth pattern at the periphery.The SSG originally defined \u201cthe cuff of healthy tissue\u201d at around 5\u2009cm. As in other parts of the world there has been a gradual change to acceptance of a smaller cuff. Today, the SSG defines the wide margin as a cuff of at least 10\u2009mm nonfascial tissue surrounding the tumour. The panel was blinded for the original assessment when independently scrutinizing the surgical and pathology reports. The surgeons did not evaluate margins from their own institution.During a second round of assessments, all four surgeons convened, and the 25 cases where they disagreed with each other during the independent round were discussed before a final agreement was reached. The originally recorded margin was then disclosed.Among the 117 patients, originally recorded margins were 8 intralesional, 43 marginal, and 66 wide. In 71% of cases, all three reviewers agreed and in 8% all three reviewers disagreed with the original classification. In 19/117 (16%), one reviewer disagreed, and in 6/117 (5%), two reviewers disagreed. When all reviewers disagreed, disagreement was most frequent when addressing the distinction between marginal and wide margins. Reclassification from marginal to wide and from wide to marginal was equally favored.In 17 of 25 cases of disagreement among the surgeons, one reviewer changed opinion after listening to the arguments of the other three. In 5/25 cases, two reviewers changed opinion, and in 3/25, there was no change of a differing opinion.The opinion changes were not necessarily in favor of the original classification.The final result was agreement with the original assessment in 80% of cases . After dAfter a median followup of 7, 8 years, 11 patients experienced a local recurrence of which the distribution among assessment groups is given in We present two different ways of margin reviewing. An independent approach and a discussion among reviewers. It is not obvious which is the most reliable, and we think both methods convey valuable information.Margin assessment years after the original recording is not the same as real-time evaluation, even if guidelines are the same. The judgment by the operating surgeon concerning the completeness of resection has elements of quality beyond his written report. This may be illustrated by studies of breast cancer surgeons' ability to determine the margin based on examination of the gross specimen \u201318.When many hospitals are classifying margins, heterogeneity might be more pronounced than for single institution materials. Among hospitals participating in this study, one institution systematically emphasized the pathologist's measurements of distances on formalin-fixed specimens to differentiate between marginal and wide margins. Consequently, this institution reported 15% less cases of wide margin than the others. Most cases where the panel suggested a change from marginal to wide margin represented patients from this institution. If the reassessment had been carried out without this institution, the panel would have disagreed with the original margin classification in only 7% of cases. This illustrates the need for a large sample size when multicenter margins are evaluated.The need for carefully defining both study population and how margins are classified is illustrated by local recurrence rates following various margins. Enneking et al.  originalNone of the intralesional margins in the present study represented cases with gross tumor left. They were all primarily treated at a sarcoma center, and an area of positive margin was typically present only in a small part of the specimen. The distinction between intralesional and marginal margins may, therefore, be of limited prognostic value in this material. From this point of view and without the institution emphasizing the pathologists measurements, the fraction of disagreeing margin assessments of prognostic significance could be regarded to be less than 4%.Among the cases that eventually had a local recurrence, none had all reviewers agreeing on a different margin than the original one.Studies of interobserver variability in assessment of surgical margin, attempting a differentiation between marginal and wide margins, are not published. When assessing only the likelihood of residual tumor at the resection margin, two reviewers disagreed on only 1/62 patients . When adIn a resent SSG series of treatment results in STS, the surgical margins were wide or better in 76% of subcutaneous lesions and in 58% of deep-seated lesions . AmputatSince the first SSG protocol for STS adjuvant treatment in 1981, the Surgical Staging System (SSS) has been in use when recording margins in Scandinavia [In 2006, positive/negative margins were introduced for the SSG Central Register. The intralesional margin is categorized into two types: macroscopic tumor tissue left behind or not. A marginal margin is recorded when the plane of excision passes outside the tumor, but in any part too close to the tumor to merit a wide margin. In other terminologies, the intralesional margin (both types) corresponds to a positive margin whereas the marginal margin corresponds to a negative margin. A wide margin is recorded when the excised tumor is surrounded all around by a cuff of healthy tissue or uninvolved fascia. The margin obtained by myectomy is regarded as a subtype of the wide margin and has been applied for strictly intramuscular lesions (not subjected to open biopsy) when the involved muscle, from origin to insertion, is completely removed. The necessary thickness of the cuff to merit a wide margin has been discussed during the years. In the latest soft tissue sarcoma protocol (SSG XX) active from 2007, a cuff thicker than 10\u2009mm in a formalin-fixed specimen is considered adequate for a wide margin to be recorded. Future studies may utilize improved methodology when evaluating the adequacy of planning treatment and reporting procedures, for the resection of soft tissue sarcoma . For multicenter studies of prognostic significance of surgical margins, written guidelines for margin assessment are important. The procedure of margin assessment in Scandinavia can be considered valid for measuring the real distance between the tumor surface and the resection margin. It is also reasonably reliable as there is a high level of agreement among sarcoma surgeons evaluating the procedure retrospectively. Considered the element of judgment inherent in all margin assessment, we find this validity and reliability acceptable for using the Scandinavian Sarcoma Group Register for studies of local control."}
+{"text": "Tumor surgical resection margin status is important for any malignant lesion. When this occurs in conjunction with efforts to preserve or conserve the afflicted organ, these margins become extremely important. With the demonstration of no difference in overall survival between mastectomy versus lumpectomy and radiation for breast carcinoma, there is a definite trend toward smaller resections combined with radiation, constituting \u201cbreast-conserving therapy.\u201d Tumor-free margins are therefore key to the success of this treatment protocol. We discuss the various aspects of margin status in this setting, from a pathology perspective, incorporating the past and current practices with a brief glimpse of emerging future techniques. P < 0.001) [The B04 study of the National Surgical Adjuvant Bowel and Breast Project (NSABP) has continued to demonstrate no significant differences in long-term survival between patients undergoing mastectomy versus lumpectomy with radiation therapy , 2. The < 0.001) , 4.Although pathologic assessment of margins for tumor is standard practice in evaluation of lumpectomies and mastectomy specimens, the obstacles for obtaining consistently accurate results are the very nature of the tissue (adiposity), the extent of in situ component , 6, and In this review of margin assessment, we will describe the various methods and settings in which margin assessment is performed and the advantages and disadvantages of each. We will also discuss some of methodologies employed to better predict which patients have higher risk of residual disease and shortened disease-free intervals.Classically, a margin was considered to be \u201cpositive\u201d if invasive tumor had been cut across by the surgical blade, but margins in which tumor was close but not transected were considered \u201cnegative for tumor\u201d  B-06 study. Currently a positive margin is generally interpreted to mean the presence of tumor, either invasive and/or ductal carcinoma in situ (DCIS), at the surgical resection line . HoweverWhat constitutes adequate clearance of tumor at the surgical margin? . Measure Skripenova and Layfield found residual invasive carcinoma in greater than 25% of patients with margins less than 2\u2009mm while only 16% had residual invasive carcinoma when the margin was greater than 2\u2009mm . The incUnderstandably, size, location, grade, and cosmesis all factor into the surgeon's decision of what constitutes an adequate clearance in any given patient. A survey of radiation oncologists in the U.S. and Europe shows a significant variation in the definition of a negative margin with European radiation oncologists seeming to prefer a larger tumor-free margin (>5\u2009mm) than their American counterparts . FinallyThe type of tumor transected or near the resection margin is significant in terms of residual disease (RD) found on reexcision . InvasivP < 0.0001). They also found a higher incidence of positive margins on lumpectomy in patients with eDCIS  [Studies have consistently shown that patients with extensive DCIS in the primary excision are at significantly higher risk for residual tumor than those without such extensive DCIS , 12\u201314.  = 0.05) . Rodriguez et al. defined extensive DCIS as DCIS having 1 or more dimensions measuring greater than 10\u2009mm . The preA few groups have also shown an association between high nuclear grade or histologic subtype of DCIS and the presence of residual disease \u201319. SahoThe effectiveness of pathologic margin assessment is impacted by utilization of imaging and other techniques in determining the extent of breast surgery. In the case of a palpable mass, one line of resection may be followed if done using palpation guidance and another if radiologic or ultrasound imaging is employed to assess the mass and surrounding tissue. Pre- or intraoperative detection of abnormal calcifications and tumor extension will alter the final excision margin to encompass more disease and reduce the risk of positive final margins and inadequate clearance of disease . Once a lesion of interest is excised, intraoperative palpation assessment with additional tissue taken from suspicious areas of the wall of the resection cavity, either using palpation or ultrasound guidance, can yield additional disease. Guidroz reported that surgeon assessment of the lumpectomy cavity with selective excision of additional tissue resulted in decreased need for second surgery following primary lumpectomy . Simply  In theory, a mass is clearly identifiable and the distance to various resection margins measurable. In reality, often the mass is irregular with ill-defined tentacles cast out in different directions . An adva Many institutions confirm resection of lesions using specimen imaging. Conventionally this is a single dimension X-ray with compression of the excision specimen . A small An imprint (touch) or a scraping of the specimen surface, placed on glass slides and stained using either hematoxylin and eosin or diffquick, can be used to evaluate for tumor cells in a specimen margin . This me Mammary tissue is notoriously technically difficult to cryosection because of its adiposity. Freezing also introduces tissue artifact in the form of architectural distortion and resistance of adipose tissue to sectioning. In addition, if the tissue submitted for evaluation is more than one centimeter in largest dimension, there is the added risk of sampling error. This method therefore is not popular amongst most pathologists. Surgeons, however, like the method because it enables rapid microscopic examination of tissue during surgery and it can be used to determine the extent of surgery to be performed in a single operative setting. However, the use of frozen section for multiple margin assessment is time consumptive and adds significantly to operating time. In order to provide good turnaround for multiple margin assessments, a pathology frozen section suite would have to be equipped with multiple cryosectioning units and have reserves in both equipment and personnel so as not to impact other surgeries. More importantly frozen section alters the appearance of tumors, particularly ductal carcinoma in situ and infiltrating lobular carcinoma and benign lesions such as intraductal papillomas and sclerosing adenosis . The abiCendan and his group performed a retrospective analysis of FS margin accuracy compared to permanent sections and showed an 84% concordance per case, with 24% of the patients requiring immediate reexcision intraoperatively of the lesion and approximately 20% of patients needing second surgery due to false negative margins. Expectedly, invasive lobular carcinoma and DCIS cases had higher rates of false negative FS margins. In addition, 51.2% of all patients with positive margins had at least one false-negative margin on either the primary or secondary excision . Osborn et al. compared the cost-effectiveness of routine FS analysis of breast margins against reoperation for positive margins assessed by routine examination of the resected specimen. Their experience has shown that the use of FS for margin assessment with the attendant increased operative time provide cost savings only when the reexcision rates are greater than 36% . The use Surgically, a shave margin is a thin piece of tissue obtained by shaving the surface of a lumpectomy cavity or other excision surface. This tissue will have two surfaces of interest: the original margin and the new margin surface. These two surfaces are differentially inked to maintain identification of the two margins. Most shave margins are large enough to require serial sectioning with submission of multiple tissue sections for microscopy to completely assess for presence or absence of disease. The pathologist can trace disease, if present, from the original margin to the new margin. Any disease present can be measured for distance from the \u201cfinal\u201d margin. A shave margin taken by a pathologist is a very thin slice of tissue from a margin surface in question and is usually a size that can be frozen for microscopic intraoperative examination or placed directly in a tissue-processing cassette. Any tumor present in the section examined would indicate a positive margin . In the  A perpendicular margin is a tissue section taken perpendicular to the margin surface. This type of margin section allows a pathologist to not only determine if a margin is positive or negative, but more importantly measure clearance of tumor from the margin. An excision specimen will be inked, either a single color or in multiple colors if the specimen is oriented and serially sectioned perpendicular to the longest axis of the tissue . This reIn the practice setting, pathologists will employ combinations of the above techniques to provide greater accuracy in determining margin status and the risk of residual disease being left in a patient. Good communication with the surgeon concerning how s/he is excising a lesion, whether there is additional tissue submitted separately for \u201cmargins\u201d and the size and type of carcinoma are key. Meticulous gross examination and/or image assessment of the tissue will discover areas suspicious for tumor involvement. Such areas will be the focus of microscopic examination, both in the frozen section suite and at microscopic \u201csign out\u201d of the specimen. The pathologist's goal in margin assessment is to provide an accurate assessment of margin status and accurate estimate of the risk of residual disease in each and every patient. Alternative methodologies for margin assessment have emerged recently. Intraoperative Optical Coherence Tomography (OCT) is a high resolution imaging technique involving real-time exvivo microscopic images up to 2\u2009mm beneath the tissue surface. In an initial analysis the method demonstrated a sensitivity of 100% and a specificity of 82% in evaluating disease at margins . MarginProbe. Quantitative diffuse reflectance spectroscopy is used to non-destructively image entire lumpectomy margins. The multichannel probe has a sensing depth of 0.5\u20132.2 (45\u2013600\u2009nm) and demonstrates a sensitivity and specificity of 79.45% and 66.7%, respectively, in an initial study. Dune Medical Devices, Inc., the sponsor of the MarginProbe is seeking premarket approval from the FDA [ the FDA . Margenthaler et al. retrospectively analyzed the margin status of 475 patients who underwent BCT and proposed a margin index as a more appropriate assessment of the optimum margin. The margin index was calculated using the formula: margin index = closest margin (mm)/tumor size (mm) \u00d7 100. A receiver operator curve (ROC) was created using the derived margin index and the presence or absence of residual disease in the reexcision specimen. A margin index >5, producing a sensitivity of 85% and specificity of 73%, was found to equate with a 3.2% risk of finding residual disease . While there is consensus on what constitutes a positive margin, there is still no consensus on what constitutes an adequate clearance. As neither the NSABP-B04 nor B06 trials ever defined clearance or close margin, we recommend that objective data be incorporated in routine reporting. We utilize the format of \u201csurgical resection margins are free of tumor/negative for carcinoma\u201d, and specifying the closest margin \u201cwith a clearance of \u201cX\u201d mm\u201d in the main report. Documentation of margin clearance is also a component of the College of American Pathologists' (CAP) Breast Cancer Case Summary protocol. We concur with Morrow et al. that systemic chemotherapy that reduces the risk of distant metastases also likely reduces the risk of local recurrence . HoweverWe therefore recommend compliance with CAP Breast Cancer summary protocols and that all mammary tumor excisions  routinely incorporate not only the margin status (positive/negative), but also document the width of clearance at the closest margins, particularly those less than 2\u2009mm which have been shown to carry a >25% risk of residual disease. Over the past fifty years, treatment of breast cancer has evolved from a single, radical procedure to techniques that limit the extent of surgery while improving disease free survival and overall survival of patients. With the introduction of limited surgical excision has come the need for accurate assessment of excision margins both intraoperatively and postoperatively. We have defined what constitutes a positive and a negative margin and why tumor clearance rather than just a \u201cnegative\u201d margin is important in eliminating residual disease. We have outlined the various methods of pathologic assessment of margins and the settings in which they are employed and two new techniques that have potential to provide assessment in real time."}
+{"text": "Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in cellular energy metabolism and provides approximately 40% of the proton-motive force that is utilized during mitochondrial ATP production. The dysregulation of complex I function \u2013 either genetically, pharmacologically, or metabolically induced \u2013 has severe pathophysiological consequences that often involve an imbalance in the production of reactive oxygen species (ROS). Slow transition of the active (A) enzyme to the deactive, dormant (D) form takes place during ischemia in metabolically active organs such as the heart and brain. The reactivation of complex I occurs upon reoxygenation of ischemic tissue, a process that is usually accompanied by an increase in cellular ROS production. Complex I in the D-form serves as a protective mechanism preventing the oxidative burst upon reperfusion. Conversely, however, the D-form is more vulnerable to oxidative/nitrosative damage. Understanding the so-called active/deactive (A/D) transition may contribute to the development of new therapeutic interventions for conditions like stroke, cardiac infarction, and other ischemia-associated pathologies. In this review, we summarize current knowledge on the mechanism of A/D transition of mitochondrial complex I considering recently available structural data and site-specific labeling experiments. In addition, this review discusses in detail the impact of the A/D transition on ROS production by complex I and the S-nitrosation of a critical cysteine residue of subunit ND3 as a strategy to prevent oxidative damage and tissue damage during ischemia\u2013reperfusion injury. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.  \u2022The current knowledge on active/deactive (A/D) transition of complex I is reviewed.\u2022The mechanism and driving force of the A/D conformational change are discussed.\u2022The A/D transition can affect ROS production and ischemia/reperfusion injury. Complex I  of the mitochondrial respiratory chain catalyzes NADH oxidation by regenerating NAD+. This giant enzyme is located in the inner mitochondrial membrane and remarkable recent progress in understanding its molecular structure The aerobic catabolism of carbohydrates, lipids, and proteins by eukaryotes, which provides energy for cellular needs, includes the transfer of electrons originating from metabolites to NADDuring NADH oxidation by complex I (forward reaction), electrons are transferred from the primary electron acceptor FMN via a chain of FeS-clusters to ubiquinone, the hydrophobic electron carrier in the inner mitochondrial membrane. The free energy change of this redox reaction drives the translocation of four protons across the membrane Since electron transfer from NADH to ubiquinone and proton translocation are spatially separated, conformational change-driven models of coupling are the consensus in the field + reduction ). Under physiological conditions, complex I can catalyze the production of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide and can also be a target of ROS in vitroThe catalytic properties of eukaryotic complex I are profoundly multi-facetted  \u2212\u00a01. If the enzyme is incubated in vitro at physiological temperatures or in situ when respiration is blocked, e.g., by lack of oxygen (ischemia), the A-form spontaneously converts into the deactive, dormant form (D-form). This form of the enzyme has a different conformation and can potentially be reactivated during slow (~\u00a01\u00a0min\u2212\u00a01) catalytic turnover(s) of NADH oxidation by ubiquinone in vitro, the D-form demonstrates a considerable lag-phase during continuous assay of the NADH:ubiquinone oxidoreductase reaction. This lag-phase represents the conformational transition of the D-form into the A-form during slow initial catalytic turnover(s), after which complex I becomes fully active in vitro can be rapidly shifted toward the D-form at physiological temperatures, but the addition of both substrates (NADH and Q) can reactivate the enzyme back into the A-form The A/D transition, which was first characterized for the mammalian enzyme 2.1N-ethylmaleimide (NEM), washed, and labelled with N-fluorescein maleimide after deactivation. After crude purification of complex I, and separation of the subunits, specific incorporation of the fluorescence label into an unknown subunit of approximately 15\u00a0kDa was observed et al. using doubled SDS-PAGE ND3) connecting the first and second transmembrane helices E.coli) The first characterization of a structural change during the A/D transition of mitochondrial complex I was undertaken by Vinogradov's group Despite the recent availability of partial structures of yeast and mammalian complex I, the exact location of the loop is not well defined, probably indicating it is highly flexible in eukaryotic enzymes 2 heterobifunctional cross-linker (N-succinimidyl 3-(2-pyridyldithio) propionate), a conformation-specific cross-linked product between the ND3 subunit and the accessory subunit 39\u00a0kDa (NDUFA9) was observed exclusively in the D-form of the enzyme Knowing the nature and approximate location of the critical SH-group, we exploited a range of thiol-specific crosslinking reagents to identify the neighboring subunits. Using a 6.8\u00a0\u00c5 SH/NHYarrowia lipolytica enzyme ND3) is in fact located above the transmembrane helixes of ND1 (ND1), could explain the rather basic pKa value of 10.2 determined for this thiol group in the D-form of the bovine enzyme Using a similar fluorescent labelling, we identified that the mitochondrially encoded subunit ND1 is also more exposed in the D-form of the enzyme s of ND1 . TherefoND3) ((i)Y. lipolytica structure, PSST middle alpha helix (aa 127\u2013135) and loop \u03b21\u2013\u03b22 of N-terminal \u03b2-sheet of 49\u00a0kDa subunit closely approach the hydrophilic loop THM 1-2ND3. However, currently, no data indicate that the position/exposure of these subunits is affected by the A/D transition. Either this is because respective parts of PSST and 49\u00a0kDa are buried inside the tertiary structure and are not accessible for modification, or the conformation of these subunits does not change during the A/D transition.Are additional subunits in that region also involved in the A/D transition? Based on the (ii)What could be the driving force of the activation? Assuming that in the D-form the entrance to the active center is somehow restricted Evidently, energy released from NADH oxidation by the D-form is used to drive the initial activation step resulting in the conformation change. However, the exact molecular mechanism that drives the transition is not clear. The final activation step is completed when the Q-binding site is enabled to catalyze terminal electron transfer from N2 during steady-state reaction and a ubiquinone molecule is present. Since reduced A-form of the idle enzyme is converted into the D-form with the same rate (iii)Is there any similarity between the D-form and proposed catalytic states of the enzyme? The resemblance of the A/D transition with the states of the enzyme occurring during catalytic turnover merits special consideration. Despite progress in resolving the complex I structure, the actual mechanism of energy transduction from the Q-binding site toward the proton translocating subunits at the membrane part is not completely understood and several models for the coupling have been suggested As shown in ND3) . It is nND3) . Based o2.2in vitro and in situ. (i) Reversible effects on kinetics of the deactivation/activation : several low and high molecular weight ligands may influence the kinetics of the A/D transition. (ii) Substrates availability: in steady state in the presence of limited amounts of NADH and ubiquinone, slow flux of electrons via complex I could maintain the enzyme partially in the A-state, shifting A\u00a0\u2194\u00a0D equilibrium to the left. (iii) Effect of irreversible covalent modifications of the D-form that prevent reactivation: the persistent inhibition of complex I by nitric oxide (NO) observed in In vitro treatment of such modified enzyme by reducing agents (glutathione or dithiothreitol) could reduce the critical thiol group and fully restore physiological activity. The lifetime of Cys-39\u00a0S-nitrosation in situ and the nature of enzymatic systems able to reverse covalent thiol modification are still obscure In the last 25\u00a0years, significant progress has been made in studying of the regulation of the A/D transition, and a number of physiology-relevant effectors have been identified for the mammalian enzyme. Due to some uncertainty in the current literature, we should emphasize the necessity to distinguish between three fundamentally different ways of affecting the equilibrium between the A and the D form in vitro). This is correct only for the idle enzyme in the absence of reduced nucleotides and ubiquinone. In conditions when complex I turnover is allowed in vitro , enzyme in SMP could be maintained in the A-form for a very long time After prolonged incubation of the idle enzyme preparation at different temperatures, only 10% of the enzyme stays in the A-form in situ. In conditions of tissue ischemia, mitochondrial redox centers are over-reduced due to the slowing of cytochrome c oxidase. Consequently, complex I turnover becomes restricted by the lack of electron acceptor ubiquinone. Accumulation of reduced ubiquinone not only decreases the availability of substrate for the enzyme but also inhibits the physiological oxidoreductase activity of complex I c oxidase in situ  would strongly synergize with hypoxia to induce the deactivation of complex I.Provision of the enzyme turnover is opposite to the situation when insufficient oxygen supply is provided in vitro. Recently, we analysed the effects of monovalent cations on the rate of activation. At neutral pH (7.0\u20137.5), only sodium was able to increase the rate of activation (D\u00a0\u2192\u00a0A conversion) while all other alkali cations were not. This stimulating effect of sodium was caused not by an increase in ionic strength but probably by specific effects on membrane subunits. At alkaline pH, all tested metal ions showed a pronounced inhibitory effect on activation, which could be explained by an increase in ionic strength.Several physiological low molecular weight effectors influence the kinetics of the A/D transition. Divalent cations and alkalinisation strongly inhibit the activation of enzymes from bovine in situ could be significantly modulated by the content of fatty acid metabolites that accumulate when the oxygen supply is restricted, and that the rate of deactivation in tissues is probably higher than in vitro. The time course of complex I A\u00a0\u2192\u00a0D conversion after cardiac arrest showed a faster rate in brain when compared to heart tissue It has been shown over the time that lipophilic compounds such as free fatty acids inhibit electron transport from NADH to oxygen via complex I Y. lipolytica structure, subunits NB4M and ACPM1 (B14 and SDAP orthologs in bovine) form a domain at the base of the hydrophilic arm above the critical ND3 loop. It has been suggested that NB4M could come in contact with the loop and may bind so-far-unidentified factors that can regulate the A/D transition The existence of specific high-molecular weight effectors of the A/D transition has not been studied yet. In principle, proteins from matrix or intermembrane side may interact with complex I and affect the kinetics of the A/D transition. Also, some of the numerous accessory subunits of complex I could regulate the A/D transition. A suppressing effect of methylation-controlled J protein on complex I activity observed in Another important question is whether any chemical/pharmacological compound could affect activation/deactivation of the enzyme in a turnover-independent way. Rotenone is a classical hydrophobic inhibitor of complex I that binds to the Q-pocket. It was shown that this inhibitor binds to the A-form of the enzyme with an almost two fold higher affinity than to the D-form ND3) rate of activation. Similarly, at alkaline pH (>\u00a08.5) and in the presence of divalent cations (1\u20135\u00a0mM Ca2\u00a0+ or Mg2\u00a0+), the rate of the D\u00a0\u2192\u00a0A transition is decreased by several orders of magnitude Pharmaceutical compounds that modulate the A/D transition rate can be of specific therapeutic interest. For a long time, metformin was the first-line drug for the treatment of type II diabetes. It has been reported that metformin, as well as some other biguanides, directly inhibits complex I 32.\u2212) and its dismutation product hydrogen peroxide (H2O2) 2O2 production and focuses specifically on the impact of the A/D transition and thiol redox modifications in I/R.The mitochondrial complex I has been recognized for decades as one of the main sources of reactive oxygen species inside mitochondria \u2013 largely superoxide  are the major sources of superoxide/H2O2 formation + ratio, which largely affects the ROS production by complex I (see below). The situation is complicated by the fact that either superoxide or hydrogen peroxide (H2O2) could be the primary reactive species produced by complex I. Of the total ROS production, lipid-activated purified bovine heart complex I releases 95% as superoxide Inhibitory analysis is still the most common tool for the studying production of ROS by mitochondria. Hydrophobic inhibitors like rotenone, piercidin A, or DQA bind at the Q-pocket and do not cause a noteworthy increase in the rate of superoxide production by purified complex I or bovine SMP + and most likely proton movement from intermembrane space to matrix drives this process. RET requires the presence of proton-motive force across the membrane and availability of ubiquinol (\u2212\u00a01 (Y. lipolytica and bovine complex I) Despite experimental data on purified enzyme, SMP, and intact mitochondria, there is still no absolute consensus in the field on the site and mechanism for ROS production by the enzyme . Complex I can operate in forward and reverse mode as a fully reversible proton pump Y. lipolytica and E. coli affecting redox potential of cluster N1a had no effect on superoxide production Y. lipolytica lacking a detectable cluster N2 exhibited the same rate of ROS production as a wild type enzyme What could be sources for ROS production in complex I? Thermodynamically, any of the eight FeS clusters of the enzyme may react with molecular oxygen, and the most obvious candidates are low potential cluster N1a + determines the rate of ROS in the forward mode, i.e., it is highest when [NADH]\u00a0>\u00a0[NAD+] 2O2 production has been observed in intact mitochondria + and QH2/Q ratio Tightly bound ubisemiquinones were proposed as a source of ROS in complex I by several groups 2O2 in complex I operating in the forward mode ++ partially overlays the isoalloxazine ring of the FMN molecule that protrudes into the cavity therefore hindering oxygen access.Most of the experimental data support the view that the reduced FMN is the source for superoxide/H2O2 generation during RET. Rotenone-like inhibitors acting at the Q-binding site also decrease the ROS production in RET + and acetyl-NAD+ decreased the rate of succinate-supported superoxide production in coupled SMP Complex I reverse reaction is associated with higher rates of superoxide generation +. In such conditions, the over-reduction of complex I redox centers (including FMN) could result in the generation of ROS. The situation is the opposite in intact mitochondria, where internal NADH/NAD+ is always present and therefore affects complex I ROS generation + pool 2O2 production could be attributed to the flavin site of complex I A critical issue of these investigations is the fact that the maximal rate of superoxide production by complex I in intact mitochondria 3.2Most experimental data on complex I-related production of ROS by the A and the D-forms could be explained in the view of the recently published structure of mitochondrial enzyme The effect of the A/D transition on the superoxide production by complex I was first investigated by Vinogradov's group The inhibition of NADH oxidation by rotenone or locking complex I in the D-form increased the amounts of produced superoxide by around 50% in bovine SMP When electrons were supplied from succinate by RET, locking complex I in the D-form almost completely stopped superoxide production 2]\u00a0=\u00a00  upon tissue reoxygenation may function as an intrinsic protective mechanism and decrease ROS production at the level of complex I. Moreover, the deactivation of the enzyme can prevent the burst of respiration via retention of electron transfer from the reduced NADH pool downstream of complex I  at the early reperfusion stage, when the oxygen level is high and metabolic intermediates are not balanced. Therefore, transition of complex I into the D-form in ischemia plays a significant role in attenuating the \u201coxidative burst\u201d that occurs during I/R Increased ROS production by mitochondria is associated with detrimental consequences in several pathophysiological situations including I/R injury in vivo is not exactly known at present, but it is likely to occur within minutes This mechanistic model explains very well the experimental data showing that the reversible inhibition of complex I during cardiac or cerebral postischemic reperfusion would protect mitochondria and decrease I/R damage in various animal models in vitro is only 1\u20132% of the full NADH-oxidase reaction and it drops dramatically with an increase in the succinate/fumarate ratio . The protein is shown in grey and subunits involved into A/D transition shown in color using a cartoon representation . Iron\u2013sulfur clusters are represented as yellow spheres. Probable conformational change of partially resolved hydrophilic loop of ND3 THM 1-2ND3 in the A and in the D-form is schematically shown at the end of the clip.Subunits involved into A/D transition of mitochondrial complex I based on The following are the supplementary data related to this article.Transparency document."}
+{"text": "Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I\u00a0+\u00a0III2\u00a0+\u00a0IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39\u00a0kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes  \u2022Supercomplex composition is not affected by mitochondrial complex I conformation.\u2022The D-form of complex I is selectively inhibited by tyrosine-reactive reagents.\u2022ND3, ND1 & 39\u00a0kDa subunits become exposed upon deactivation of complex I. The exact mechanisms of energy transduction between the redox reaction at the terminal cluster N2 and proton translocation in the membrane part are still not known. Recent studies Production of energy in most aerobic cells is provided by the joint activity of the mitochondrial respiratory chain and ATP-synthase. Most of the NADH produced in the mammalian cell, through aerobic catabolism, is oxidised by complex I or NADH:ubiquinone oxidoreductase \u2212\u00a01 at 25\u00a0\u00b0C) catalytic turnover(s) 4\u00a0min\u2212\u00a01in vitro, in cells, as well as in heart ex vivo in ischaemia An intriguing feature of mitochondrial complex I from several vertebrate species and yeast is the existence of two functionally and structurally different states of the enzyme: active, A-form and de-active or dormant, D-form Until now, the only known conformational difference between the A- and D-forms of mitochondrial complex I was the exposure of cysteine 39 (Cys-39) of the mitochondrially encoded ND3 subunit in the D-form Thermus thermophilus) is located in a hydrophilic interhelical loop that connects transmembrane segments (TMS) 1 and 2 and that forms part of a solvent-inaccessible ubiquinone reaction chamber. The formation of different semiquinone species as intermediates of electron transfer in complex I This cysteine of the ND3 subunit , digitonin was purchased from Serva and molecular weight ladder was from Fermentas.2.2Bovine heart SMP were prepared according to standard procedure 2.3340nm\u00a0=\u00a06220\u00a0M\u2212\u00a01\u00b7cm\u2212\u00a01) with 165\u00a0\u03bcM NADH in SET medium  or PBS pH\u00a07.0 buffer containing SMP (10\u201325\u00a0\u03bcg of protein/ml). Alternatively, complex I NADH:Q1 oxidoreductase activity was assessed: 25\u00a0\u03bcg SMP were diluted in 1\u00a0ml PBS pH\u00a07.5 supplemented with 10\u00a0\u03bcM NADH to convert the enzyme to the A-form. 1\u00a0mM KCN, 30\u00a0\u03bcM Q1 and 165\u00a0\u03bcM NADH were then added to the suspension and the NADH oxidation rate was measured in the linear part of the curve.Oxidation of NADH was determined spectrophotometrically (Varian Cary\u00ae 3000) as a decrease in absorption at 340\u00a0nm (\u03b52) or incubated with 15\u00a0\u03bcM NADH at pH\u00a07.2 prior to further dilution in measuring buffer. The measured rates correspond respectively to the activity of only the A-form and the total activity of complex I, i.e. the activity after NADH-dependent conversion of all complex I to the A-form.The A/D ratio in each sample was estimated essentially as in 2.42 and incubated at 35\u00a0\u00b0C for 30\u201360\u00a0min under constant shaking. This treatment resulted in almost complete deactivation of complex I. The resulting sample was then divided in two equal parts, one was kept on ice (D-form) and the other was aerobically re-activated in the presence of 400\u00a0\u03bcM NADH and a NADH regenerating system  for 20\u00a0min at room temperature with constant stirring To prepare SMP in which complex I is converted to the D-form, SMP were resuspended to 5\u00a0mg/ml in SET or PBS buffers pH\u00a08.0 supplemented with 1\u00a0mM malonate and 5\u00a0mM MgCl2.51 oxidoreductase activity as described above. The effects of these reagents on the D-form were assessed by measuring activity of the treated D-form after NADH-dependent activation.The effects of amino-acid specific reagents were evaluated by measuring the NADH:Qg at 4\u00a0\u00b0C for 30\u00a0min. SMP were diluted to 5\u00a0mg/ml in SET buffer pH\u00a07.5 and complex I was activated in the presence of an NADH-regenerating system. SMP containing the A-form of complex I were then incubated with 30\u00a0mM NEM at 15\u00a0\u00b0C for 15\u00a0min. After thiol blockage the reaction was terminated by the addition of 3 volumes of SET buffer pH\u00a07.5 supplemented with 35\u00a0mM cysteine. SMP were washed twice in SET buffer pH\u00a07.5. The sample was split into two portions prior to the penultimate centrifugation step. One portion was kept on ice (NEM-treated A-form) and the other one was de-activated (NEM-treated D-form). Both samples were resuspended at 5\u00a0mg/ml in SET buffer pH\u00a07.5 and treated with 2\u00a0\u03bcM Cy5-maleimide at 15\u00a0\u00b0C for 15\u00a0min in the dark. Labelled SMP were then collected by centrifugation and samples were immediately prepared for a BN-PAGE analysis.Sulfhydryl groups were labelled as described previously N-acetylimidazole (NAI) in PBS pH\u00a07.2. For NAI, the reaction was conducted at pH\u00a07.2, which maintains a good balance between the rate of the reaction and the stability of the O-acetyltyrosine produced For tyrosine labelling, SMP containing A- or D-forms of complex I were diluted to 5\u00a0mg/ml and treated with 2 to 15\u00a0\u03bcM of tetranitromethane (TNM) in SET buffer pH\u00a08.0 prior to activity measurements or incubated with 3\u00a0mM For lysine labelling, SMP containing A- or D-forms of complex I were diluted in PBS pH\u00a07.4 1 oxidoreductase activity.For activity measurements, SMP containing A- or D-forms of complex I were diluted to 1\u00a0mg/ml in PBS pH\u00a07.4 and incubated with 8\u00a0\u03bcM of fluorescein-NHS (F-NHS) for 1\u00a0h at 10\u00a0\u00b0C in the dark. Aliquots were taken every 10\u00a0min to measure the NADH:Q2.6g for 20\u00a0min at 4\u00a0\u00b0C and pellets were prepared for BN-PAGE analysis. Proteins were extracted with 2.5\u00a0g of DDM per gram of protein and Cy3/Cy5-labelled samples mixed to a 1:1 ratio (wt/wt). Complex I was isolated on a 4\u201313% BN-PAGE and differentially labelled subunits from both A- and D-forms were separated with double SDS-PAGE (dSDS-PAGE).SMP containing A- or D-forms of complex I were diluted to 1\u00a0mg/ml in PBS pH\u00a07.4 and incubated for 1\u00a0h at 10\u00a0\u00b0C in the presence of 8\u00a0\u03bcM of either Cy3-NHS ester or Cy5-NHS ester. The reaction was stopped by a 10\u00a0minute incubation on ice with 50\u00a0mM Tris\u2013HCl pH\u00a07.0. The suspension was then centrifuged at 20,000\u00a02.7ex/\u03bbem 635/670\u00a0nm for Cy5-NHS and 532/580\u00a0nm for Cy3-NHS using a fluorescence image scanner  and silver-stained as described previously The SMP pellet was processed according to 2.8Fluorescent scans were analysed with AIDA Image Analyzer (Raytest). For each set of experiments, the intensity of each spot was automatically quantified by the software and the background was subtracted from the signal for each spot. The intensity of fluorescence was then expressed as a percentage of the total fluorescence of all protein spots in the gel. This last step is a prerequisite for comparison since the dyes used do not have the same quantum yield (0.28 for Cy5 and 0.15 for Cy3). A- and D-form samples were both treated with Cy3 and Cy5-NHS esters and measures of all four signals were taken into account to overcome dye-dependent effects for quantification.2.9Gel spots from CyDye NHS treated A- and D-forms were excised and cut into 1\u00a0mm cubes. These were then subjected to in-gel digestion, using a ProGest Investigator in-gel digestion robot  using standard protocols The peptides were then separated using a nanoLC Ultra 2D plus loading pump and nanoLC as-2 autosampler equipped with a nanoflex cHiPLC chip based chromatography system (Eskigent), using a ChromXP C18-CL trap and column. The peptides were eluted with a gradient of increasing acetonitrile, containing 0.1% formic . The eluent was sprayed into a TripleTOF 5600 electrospray tandem mass spectrometer  and analysed in Information Dependent Acquisition (IDA) mode, performing 250\u00a0ms of MS followed by 100\u00a0ms MSMS analyses on the 20 most intense peaks seen by MS. The MS/MS data file generated was analysed using the ProteinPilot 4.5 Paragon algorithm (ABSciex) against the Swiss-Prot database Jan 2013 with no species restriction, trypsin as the cleavage enzyme and carbamidomethyl modification of cysteines.Bos taurus  with optional modifications on cysteine by carbamidomethylation, mono-, di- and tri-oxidations, modification by acrylamide, mono-oxidation on methionine and nitration of tyrosine.TNM treated active/de-active forms of complex I were isolated by BN-PAGE and subunits were separated by dSDS-PAGE. Spots containing the ND3 subunit were treated with DTT and cysteines were carbamidomethylated with iodoacetamide. This was followed by in-gel digestion with trypsin. Tryptic peptides were subjected to LC\u2013MS/MS analysis on an LTQ-Orbitrap XL (Thermo) mass spectrometer coupled to a nano-HPLC (Agilent). Briefly, peptides were separated in 30\u00a0min on an in-house C18 nano-flow column with an acetonitrile gradient (5\u201345%) containing 0.1% formic acid. This was followed by cleaning with 95% acetonitrile and reequilibration with 5% acetonitrile (15\u00a0min each step). Collision induced dissociation (CID) spectra were collected and evaluated by Mascot database searches against UniProt database of Label free quantification was conducted using Proteome Discoverer 1.3 Precursor Ions Area Detector node (Thermo Fisher scientific).33.12), two copies of complex III and several copies of complex IV (I\u00a0+\u00a0III2\u00a0+\u00a0IVx) Complex I is found to be a member of several supercomplexes as revealed by native electrophoresis after solubilisation of mitochondrial membranes with digitonin; a mild detergent known to preserve oligomeric association between membrane proteins To date, mitochondrial complex I is the only enzyme of the mitochondrial respiratory chain known to undergo a so called A/D conformational change. Whether the association of this enzyme with other complexes is influenced by its conformation remains unclear.2; I\u00a0+\u00a0III2\u00a0+\u00a0IV) independent of its conformational state.The supercomplex profiles of SMP containing A- or D-form complex I were analysed after separation by native electrophoresis. After labelling with a fluorescent lysine-reactive reagent, Cy5-NHS ester, A- and D-forms were solubilised and applied on a 4\u201313% BN-PAGE A. No difSince Coomassie in BN-PAGE quenches fluorescent signals in vitro as revealed by native electrophoresis. The A/D transition has no effect on the subunit composition of complex I, confirming our previous study Taken together these results suggest that the A/D conformational change(s) of complex I has no effect on the association of the enzyme with other respiratory chain complexes 3.2N-ethylmaleimide (NEM) are known to covalently modify Cys-39 of the ND3 subunit in the D-form of the enzyme i.e. complex III and complex IV), this cysteine could be enclosed and inaccessible to modification due to steric hindrance. We tested whether this cysteine is accessible to covalent modification if the D-form of complex I is a constituent of a supercomplex after solubilisation with digitonin.SH-reagents such as 1 supercomplex  are presented in A DIGE approach relies on the covalent labelling of two or more samples with a chemical compound bearing the same reactive part and a fluorophore with different spectral properties. After treatment, samples are combined and separated by denaturing electrophoresis. This mixing step circumvents the problem of variability between gels. The A- and D-forms of complex I in SMP were labelled either with cyanine fluorescent Cy3-NHS or Cy5-NHS esters respectively and pooled. Subunits were then separated by dSDS-PAGE . A recipFor the first time, we demonstrate that ND3 is not the only subunit to become exposed upon deactivation of mitochondrial complex I. A second membrane subunit, ND1 and the accessory subunit 39\u00a0kDa are shown to be associated with the structural reorganisation leading to the D-form of the enzyme.Protein labelling with NHS esters leads to the elimination of tryptic cleavage sites P34943). Among them, 28 are generated by a cleavage after lysine residues  form to a de-active (D) form. The role of this transition in enzyme regulation and functioning still remains to be characterised in detail. Despite recent progress detailing the existence of two forms of mitochondrial complex I T. thermophilus) is exposed to the matrix side in the D-form only, where it is susceptible to covalent modification by SH-reagents, nitric oxide metabolites and ROS The A to D transition of the enzyme in bovine SMP is characterised by high activation energy In the present study, we addressed two different questions to gain additional understanding of the A/D transition: (i) Does the de-activation of complex I affect the supercomplex assembly? (ii) Apart from ND3, are there any additional subunits associated with conformational changes upon de-activation?4.1Complex I has been proposed as an \u2018enhancer\u2019 of supercomplex formation In order to investigate this, the supercomplex profile of SMP containing the A- or D-forms of complex I were analysed after separation by two types of native electrophoresis. We did not find any differences in the migration profile and content of supercomplexes depending on the conformation of complex I. Our data suggests that the conformation of complex I does not affect the structural formation or stability of supercomplexes in bovine heart SMP. Indeed, the fact that no extra bands could be detected with in-gel activity staining or highly sensitive fluorescent labelling suggests that complex I can adopt two different conformations whilst being part of a higher molecular weight complex. It is important to note that, unlike NADH:ubiquinone reductase, the commonly used rotenone-insensitive NADH:tetrazolium reaction for in-gel activity measurements is catalysed by both forms of the enzyme with the same rate.Therefore, the A/D transition would correspond to changes in the enzyme structure rather than changes of the macromolecular organisation within supercomplexes.1 supercomplex, the complex III dimer has been shown to locate in the arc of the bent membrane part of complex I, allowing the ubiquinone binding sites of both complexes I and III to face each other 2\u00a0+\u00a0IV supercomplex. Our result indicates that the potential interaction between complex I and complex III in the mitochondrial membrane does not significantly affect the accessibility of Cys-39 in the D-form of the enzyme.The D-form of complex I is characterised by the exposure of Cys-39 of the ND3 subunit. Complex I is known to associate with complexes III and IV. In the S4.2To characterise the other potential differences in the accessibility of complex I subunits, we decided to test several amino acid specific covalent reagents. A- and D-forms of complex I in bovine heart SMP were treated with tyrosine-specific reagents, NAI and TNM. These compounds covalently modify tyrosine residues with a high level of specificity. We found that both reagents inhibited the re-activation of the D-form of the enzyme, suggesting other amino acids are exposed in the D-form. Modification of two tyrosines (Tyr-30 and Tyr-37) in close proximity to Cys-39 of the ND3 subunit may account for the sensitivity of the D-form to NAI and TNM. A mass spectrometric analysis of the TNM-modified ND3 subunit did not permit us to locate the modification site(s) on tyrosine residues in vivo upon oxidative stress This enabled us to identify sulfonic acid modification on Cys-39 after TNM treatment resulting in an irreversible de-activation of complex I. Since sulfonic acid modification could occur We used lysine specific fluorescent NHS-esters combined with a DIGE-like approach to gain further insight into the structural differences between these two conformations. We identified three subunits that were unequivocally more exposed in the D-form: ND3, ND1 and 39\u00a0kDa (NDUFA9).The differential exposure of the ND3 subunit confirmed our previous results 2/\u2014SH specific 6.8\u00a0\u212b crosslinker only when complex I is in the D-form. This observation could be accounted for by the individual movement of the hydrophilic loop of ND3 towards 39\u00a0kDa subunit or by a combined rearrangement of the two subunits. The work presented here strongly suggests that 39\u00a0kDa subunit undergoes concerted structural rearrangement along with ND3 during de-activation. The 39\u00a0kDa subunit is thought to be anchored to the membrane part of the enzyme via a single hydrophobic transmembrane helix T. thermophilus enzyme structure\u00a0The 39\u00a0kDa (NDUFA9) nuclear encoded accessory subunit was found to be associated with structural rearrangement during the A/D transition and therefore more accessible for chemical modification upon deactivation of complex I. A previous study performed in our laboratory The 39\u00a0kDa (NDUFA9) subunit belongs to the family of short-chain dehydrogenase/reductases. It contains a nucleotide binding Rossmann fold motif Yarrowia lipolytica protein have only a minor effect on the ubiquinone reductase activity of complex I, suggesting a stabilising function for this nucleotide rather than a role in biosynthesis of complex I Mutations affecting the NADPH binding site in T. thermophilus complex I) form the cavity at the quinone entrance side, accommodating the hydrophobic isoprenoid tail. The 49\u00a0kDa and PSST subunits (Nqo4 and Nqo6 in T. thermophilus complex I) accommodate the quinone head group from the N2 terminal cluster side T. thermophilus structure, three other ND1 lysine residues  are located within 10\u00a0\u212b of the hydrophilic loop of ND3 Another main finding of this study is that ND1, a core membrane subunit, is also affected by structural rearrangements upon deactivation of complex I. The recently obtained three-dimensional structure of the bacterial enzyme clearly revealed the quinone reaction chamber MT-ND1 are associated with the appearance of several severe syndromes, including LHON and MELAS. To date, 41 disease-associated mutations on MT-ND1 have been reported MT-ND1 leading to impairment in complex I activity (but not assembly) could also suggest a defect in the regulation of the A/D balance. The involvement of ND1 (and ND3) on the A/D transition demonstrated in this study raises questions as to the use of bacterial models in the study of human point mutations found on MT-ND1 and MT-ND3. Since the bacterial complex I does not display the A/D transition this may be misleading for the study of the overall functional consequences of such mutations, not at the level of the enzyme as such, but for global consequences on the respiratory chain.Mutations in human 4.3cat ~\u00a01\u201310\u00a0min\u2212\u00a01) necessary for activation of the D-form corresponds in fact to the formation of a sealed chamber able to accommodate ubiquinone.Deactivation results in rearrangements in ND3 as well as in ND1 subunits in the region of the quinone binding pocket and potentially accounts for the labelling of ND3 (by SH-reagents and NHS esters) and ND1 (by NHS esters) observed in the D-form only. It is possible that deactivation of mitochondrial complex I leads to the disruption of the enclosed quinone binding chamber due to a concerted structural rearrangement of these two crucial membrane subunits. The processes of reduction of the physiological ubiquinone molecule and transmission of the redox energy into putative proton translocating channel(s) could then be disrupted. Interestingly, 49\u00a0kDa and PSST subunits were equally labelled in both forms of complex I, suggesting rather selective conformational change(s) in the membrane part of the enzyme at the level of the entrance of the quinone binding cavity. It is tempting to speculate that the initial slow turnover  partially protects the A-form against de-activation and (ii) partially converts the D-form to an A-like conformation. This was observed by measurement of the reverse electron transfer activity after removal of rotenone with BSA. Rotenone could act (i) as a clamp for the A-form of the enzyme and (ii) induce conformational changes in the D-form similar to the transitory state so that the enzyme-inhibitor complex can undergo activation without a turnover i.e. formation of the pathway/binding site for the ubiquinone molecule and/or reduction of quinone at the terminal cluster N2.Therefore, the rate of activation would be limited by the time required for rearrangements of the enzyme structure, 5Y. lipolytica enzyme after treatment with lauryl dimethylamine oxide showed that the hydrophobic subunits of this connecting region (including ND1 and ND3) are the first ones to be dissociated Our results are in line with the known plasticity of the junction region (Q-module) between the hydrophilic module that operates the electron transfer from NADH to the terminal N2 FeS cluster (N-module) and the proton pumping module (P-module)"}
+{"text": "Complex I (NADH:ubiquinone oxidoreductase) is critical for respiration in mammalian mitochondria. It oxidizes NADH produced by the Krebs' tricarboxylic acid cycle and \u03b2-oxidation of fatty acids, reduces ubiquinone, and transports protons to contribute to the proton-motive force across the inner membrane. Complex I is also a significant contributor to cellular oxidative stress. In complex I, NADH oxidation by a flavin mononucleotide, followed by intramolecular electron transfer along a chain of iron\u2013sulfur clusters, delivers electrons and energy to bound ubiquinone. Either at cluster N2  or upon the binding/reduction/dissociation of ubiquinone/ubiquinol, energy from the redox process is captured to initiate long-range energy transfer through the complex and drive proton translocation. This review focuses on current knowledge of how the redox reaction and proton transfer are coupled, with particular emphasis on the formation and role of semiquinone intermediates in both energy transduction and reactive oxygen species production. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. \u2022Current knowledge of the redox reactions catalyzed by complex I is reviewed.\u2022Possible quinone reduction pathways are presented.\u2022The presence and number of semiquinone intermediates are deliberated.\u2022The involvement of cluster N2/semiquinones in coupled proton transfer is discussed.\u2022Evidence for reactive oxygen species production by semiquinones is examined. The two electrons from NADH oxidation are transferred through the enzyme and used to reduce ubiquinone to ubiquinol in the inner mitochondrial membrane, supplying the rest of the electron transport chain with electrons for the reduction of oxygen to water. The free energy produced by the redox reaction is captured and used to transport protons across the mitochondrial inner membrane, building the proton-motive force (\u0394p) that is consumed to support ATP synthesis and the import and export of metabolites and proteins to and from the mitochondrion. In addition, reactive oxygen species production by complex I is an important contributor to mitochondrial and cellular oxidative stress Complex I (NADH:ubiquinone oxidoreductase) Bos taurus heart mitochondria is the most studied mammalian complex I, and has been adopted as a closely-related model for the human enzyme. The forty-five (known) proteins in mammalian complex I B. taurus (regardless of the species concerned).Complex I from Thermus thermophilus and Escherichia coliB. taurusYarrowia lipolyticaThe core subunits form two distinct domains that are reflected in the L-shape of the complex see . Seven h2\u00a0+/1\u00a0+ and six [4Fe\u20134S]2\u00a0+/1\u00a0+ clusters) transfers electrons from the flavin to the quinone-binding site. An unusually-positioned additional [2Fe\u20132S]2\u00a0+/1\u00a0+ cluster is located on the opposite side of the flavin, separate from the main chain of clusters NADH is oxidized by a non-covalently bound flavin mononucleotide at the top of the hydrophilic domain, in the 51\u00a0kDa subunit. Then a chain of seven iron\u2013sulfur (FeS) clusters  then discuss the evidence for coupled chemistry at cluster N2 , and the mechanism of ubiquinone reduction. In particular, we examine evidence for the involvement of semiquinone intermediates in catalysis. We close by considering how current knowledge of the redox mechanism of complex I contributes to understanding its mechanisms of energy transduction and reactive oxygen species production.22.1\u2022 disfavors stepwise processes and because the structure of the hydrophilic domain of T. thermophilus complex I with a nucleotide bound in the flavin site In mitochondrial complex I, NADH oxidation by the flavin mononucleotide is both \u2018fast\u2019 B. taurus complex I have revealed that the apparent second order rate constant for NADH binding  rate of NADH oxidation observed at saturating NADH concentration and KM is the Michaelis constant, equivalent to the NADH concentration required for half the maximum rate) is ~\u00a07.5\u00a0\u00d7\u00a0107\u00a0M\u2212\u00a01\u00a0s\u2212\u00a01, approaching the diffusion-controlled limit, and that kcatNADH (which includes both reversible hydride transfer and NAD+ dissociation) is greater than 15,000\u00a0s\u2212\u00a01\u2212\u00a01NADH oxidation by the flavin occurs much faster than the full catalytic cycle (proton-coupled NADH:ubiquinone oxidoreduction) of complex I can turn. Thus, it is not rate limiting in catalysis, and to study the flavin site reactions NADH oxidation must be coupled to the rapid reduction of an \u2018artificial electron acceptor\u2019 directly by the flavin so it is not controlled by the slow downstream steps of the full cycle. Kinetic studies using + oxidoreduction, driven by \u0394p) establishes it only as an enzyme that can catalyze backwards, not as a thermodynamically-reversible catalyst that operates with a substantial rate in either direction under only minimal driving force + and NADH in solution \u2014 a feature that was instrumental in defining the reduced flavin as the site of reactive oxygen species production (see below) Here, we use a thermodynamic definition of the term \u2018reversible\u2019. The fact that complex I can catalyze \u2018reverse electron transfer\u2019  remain unclear. 2.2B. taurus enzyme B. taurus complex I, the electrons distribute predominantly onto alternating clusters . For an intramolecular electron transfer between two cofactors in a protein the rate is largely controlled by the distance and the reduction potential difference between the donor and the acceptor ters see  [31], . Toomplex I  [46],  cluster described above, in the [8Fe\u20137S] P-cluster in nitrogenases (upon formation of an amide nitrogen bond to one of the Fe subsites) Redox-coupled changes in the ligation of cluster N2 have been proposed on the basis of changes to the electron density of reduced N2 in the hydrophilic domain of complex I from Y.lipolytica by methionine  With one exception \u2212 (red). Pathway C also involves the dianion species, which (similarly to the double-protonated quinone) appears an unlikely intermediate at first sight but on whose formation a mechanism for the coupling reaction has been proposed [1].T. thermophilus complex I 4 for spin quantification may prove more reliable. The detection of more than one semiquinone species, and the fact that both anionic and neutral radicals have been reported (see below), may perhaps argue for pathway B. However, with so little agreement between studies and no unambiguous identification or description of the detected radicals it would be unwise to rule out pathways A and C at this stage.The atomic-resolution structure of [2].\u2212\u00a0and to show how they respond to pH. This information will not only provide insights into the pathway of ubiquinone reduction , relaxation properties and the protonation states of several radical species using CW-EPR data at a single microwave frequency. The rhombicity of the semiquinone signal is not evident at X-band frequencies (~\u00a09\u00a0GHz), and very high microwave frequencies are required to resolve differences in the g values of different types of semiquinone (evident only in the 5th significant figure). As an example of a case where the accurate determination of g values yielded mechanistic information, Stoll et al. g values of tryptophan radicals shift with hydrogen bonding. Indeed, deconvoluting X-band EPR spectra into the signals from different semiquinone species by using spectral subtraction procedures is unlikely to lead to a unique solution, especially when the linewidths are affected by saturation broadening and the total signal intensities are low. For example, Narayanan et al. P1/2), and the signal intensity as a proxy for the concentration). Importantly, power saturation curves also depend on the resonator used (owing to different power conversion factors for different resonators) and the degree of signal saturation depends on both the spin\u2013lattice (T1) and spin\u2013spin relaxation (T2) times, which are influenced by sample preparation conditions such as concentration, viscosity, level of oxygenation and temperature P1/2 values have not been included in It is undoubtedly a daunting task to extract and deconvolute [5].gz component of the N2 signal, attributed to interaction between N2 and a nearby fast-relaxing semiquinone species under conditions of high proton-motive force, has been reported in several papers  gz splitting was used to estimate the distance between the two paramagnets as ~\u00a012\u00a0\u00c5 3) and by treating both the N2 cluster and the semiquinone as point dipoles . For two point dipoles spaced 12\u00a0\u00c5 apart the dipolar coupling is ~\u00a030\u00a0MHz, and so with Dz  and gz collinear the maximum splitting of gz is ~\u00a060\u00a0MHz, smaller than the reported value of ~\u00a033\u00a0G (93\u00a0MHz). By including a relatively large exchange interaction of 55\u00a0MHz, and tilting the D tensor by 65o relative to gz, Yano et al. gz and the larger ~\u00a056\u00a0G apparent splitting of the SQ (semiquinone) signal. Although a large exchange coupling may be of mechanistic relevance as it indicates a facile pathway for electron transfer between N2 and bound semiquinone, the model proposed by Yano et al. requires further investigation because the complete EPR spectrum , while a semiquinone present in complex II would  be unaffected. However, many studies have not been comprehensive because they did not test the effects of inhibitors of different respiratory enzymes to exclude secondary effects and cross reactions, and Before discussing these points it is first instructive to consider the possible pathways by which ubiquinone reduction may occur and semiquinone species form. In summary, there are many unanswered questions about the presence, number, properties and roles of semiquinone intermediates formed during complex I catalysis. More detailed and quantitative analyses, in the absence of other respiratory enzymes complicating the picture, and by using methods such as pulse EPR and multiple microwave frequencies, as have been employed for the study of other respiratory enzymes, are clearly required.4.1Semiquinones have been investigated in detail in a number of other respiratory complexes but a comprehensive review of these studies is beyond the scope of this article. The two examples provided below serve to provide a flavor of the approaches employed to study semiquinones outside of the complex I field, with a focus on high-resolution EPR methods.4.1.1O site O spin-coupled state with a large exchange interaction. In the spin-coupled form, the semiquinone exhibits frequency-dependent g values and unusual properties such as a temperature dependent EPR signal that can be explained by the Leigh effect rather than the Curie law. Could some of the unusual temperature dependencies of the semiquinone signals in complex I , Grimaldi et al. inferred that the semiquinone is hydrogen-bonded to the observed histidine nitrogen, providing a further mechanistic clue. Subsequently, pulse X- and Q-band measurements characterized the exchangeable and non-exchangeable protons around the menasemiquinone, to produce a model for the  binding mode of the quinone substrate that may contribute to its exceptional redox properties Nitrate reductase A catalyzes oxidation of quinols and reduction of nitrate to nitrite. The relatively stable menasemiquinone radical has been characterized in detail using hyperfine EPR spectroscopic methods. Using native and 52 to form superoxide (O2\u2022\u2212) 2O22O2 production is relatively low, and addition of a complex I \u2018Q-site\u2019 inhibitor  causes it to increase. Conversely, if the mitochondria are respiring on succinate, under conditions that promote \u2018reverse-electron transfer\u2019  then H2O2 production is relatively high, and addition of rotenone or piericidin A abolishes it. The model used to explain these observations was for one or more O2-reactive semiquinones bound in complex I \u2018upstream\u2019 (on the NADH-side) of the inhibitor binding site. However, in addition to the question of whether any such semiquinones exist in complex I  were proposed to react with OB. taurus complex I to show that the superoxide produced upon addition of NADH is from the fully reduced flavin, and that it occurs by a slow, second-order reaction with O2, with the relative concentration of reduced flavin set by a (rapid) pre-equilibrium between the flavin, NADH and NAD+2 reduction 2 in the isolated enzyme and in the membrane-bound enzyme, particularly because no proton motive force is present, Pryde and Hirst then recapitulated data from the isolated enzyme using complex I in SMPs, confirming the flavin-site mechanism and finding no evidence for any further, proton motive force-dependent sites of superoxide production Kussmaul and Hirst used isolated + pool becomes more reduced; the complex I flavin also becomes more reduced and mitochondrial H2O2 production increases. Conversely, during reverse electron transfer by mitochondria, electrons are driven into complex I from the reduced ubiquinone/ubiquinol pool, supported by a high proton motive force \u2014 so adding an inhibitor \u2018cuts off\u2019 the flavin from further reduction and H2O2 production ceases. The mechanism of superoxide production established by work on isolated complex I thus provides a firm basis for understanding superoxide production by complex I in mitochondria, and for understanding the link between mutations that cause loss of complex I activity and increased reactive oxygen species production in mitochondrial-disease patients The flavin-site model is qualitatively consistent with data from intact mitochondria, because the flavin site is upstream of the rotenone and piericidin A binding sites. During the oxidation of NADH-linked substrates by mitochondria, inhibition of complex I prevents NADH oxidation and the mitochondrial NADH/NAD2 in solution, generating O2\u2022\u2212, H2O2 and semiquinone species Finally, a second route for complex I-mediated superoxide production, redox-cycling induced by the reduction of a redox-active molecule by the reduced flavin, is relevant to studies of the semiquinones formed by complex I during the reduction of hydrophilic ubiquinone substrates. Hydrophilic ubiquinones, such as ubiquinone-1 and -2 and decylubiquinone, are reduced by the complex I flavin (as well as at the ubiquinone-binding site) then reoxidized by O6A key question to answer about coupled electron\u2013proton transfer in complex I is whether the reaction proceeds by a single-stroke or double-stroke mechanism. In a single-stroke mechanism a single step in the redox reaction drives all the proton transfer steps; all the \u223c\u00a0800\u00a0meV of \u2018redox\u2019 free energy is transferred to the protein at once, then all four proton transfers follow spontaneously. In a double-stroke mechanism the energy is delivered in two stages, which may or may not be equivalent. Some insights may be gained from considering the two possible sites of coupling discussed here, cluster N2 and ubiquinone. N2 is rapidly reduced by the chain, and it is predominantly reduced during steady-state catalysis, so N2 oxidation (by ubi(semi)quinone) is more likely to be coupled to proton transfer than N2 reduction. For ubiquinone, possible coupling points are ubiquinone binding, reduction of ubiquinone , and ubiquinol dissociation.T. thermophilus complex I 1FO-ATP synthase provides an example of energy storage within a bioenergetic enzyme  For each redox cycle of one NADH oxidized and four protons pumped, cluster N2 must be reduced and reoxidized twice (assuming it is restricted to one electron transfers). Thus, proton translocation coupled to N2 is most consistent with a two-stroke mechanism. In the simplest model, both transitions are equivalent and they both drive two channels to each transfer one proton. The two steps in the reduction of ubiquinone may also be envisaged to drive two channels in a similar manner, as embodied in the \u2018two-state stabilization-charge\u2019 mechanism proposed by Brandt 2\u00a0\u2212 species, within a single site 2 transitions, and proton transfer into the site. Verkhovsky et al. further proposed that the quinone species moves between two binding sites, which confer tight- and weak-binding properties upon it, a feature in common with the mechanism proposed earlier by Ohnishi Nonetheless, identification of the fourth proton channel in complex I has favored proposals for single-stroke mechanisms. Verkhovsky et al. have proposed that the translocation of all four protons is driven after the reduction of the quinone  2\u00a0\u2212). Questions to answer include: Do SQ intermediates ever accumulate to significant levels? How many different species can be identified and are they anionic or neutral? Where are they located and which residues do they interact with? What are the reduction potentials associated with SQ formation ? When semiquinones are present, is N2 oxidized or reduced? Does N2 interact with the bound ubiquinone species, other than just as a simple electron donor? Understanding the intermediates of ubiquinone reduction by cluster N2 in complex I is a crucial part of defining the mechanism of the enzyme, and for understanding how energy provided by the redox reaction is harnessed to drive proton translocation at distant sites.To distinguish between current and future proposals for the coupling mechanism, a clear picture of the structures, thermodynamics and kinetics of the reaction intermediates formed during catalysis will be crucial. It will be important to establish, beyond any doubt, whether N2 plays a role in the coupling reaction \u2014 either directly by coupled chemistry linked to N2 redox cycling, or as part of a \u2018catalytic unit\u2019 comprising cluster N2 and bound ubiquinone. Depending on which steps are coupled to generation of the proton motive force, SQ intermediates will either be stabilized by it  or formed only transiently regardless of the proton motive force (for example, if proton translocation is linked to protonation of QTransparency document."}
+{"text": "Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen).The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39\u00a0kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site.+/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability.The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NADCysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.  \u2022The potential mechanism of complex I A/D transition is discussed.\u2022An \u2014SH group exposed in the D-form is susceptible to covalent modification.\u2022The role of A/D transition in tissue response to ischaemia is proposed. In mitochondria, this enzyme is responsible for the oxidation of NADH produced through glycolysis, the Krebs cycle and \u03b2-oxidation of fatty acids. Complex I reduces ubiquinone (Q) and transports protons across the inner membrane, contributing to the proton-motive force The presence of these two forms of complex I results in unusual kinetic parameters when mitochondrial preparations catalyse the inhibitor-sensitive NADH:Q oxidoreductase reaction. Depending on the preparation \u201chistory\u201d  the initial NADH oxidation proceeds with a lag-phase A [7]. HoSH). Addition of a small (5\u201310\u00a0\u03bcM) pulse of NADH before the assay results in activation of the enzyme during one or several slow turnovers when NADH is oxidised and ubiquinone is reduced. Pre-treatment with NADH completely eliminates the lag-phase, the enzyme then becomes fully active and catalyses the oxidation of NADH at a linear rate  or at alkaline pH, the turnover-dependent activation takes longer and the lag phase is more pronounced (SH). Divalent cations have no effect on the activity of the A-form.The occurrence of the A/D transition was initially demonstrated for bovine complex I in preparations of heart submitochondrial particles (SMP) ear rate A, A [11,onounced A, DSH. Dc oxidase. As a result, the turnover of complex I becomes restricted because of the lack of the electron acceptor ubiquinone. Therefore, the enzyme would be readily converted into the D-form 2\u00a0+ or Mg2\u00a0+) the rate of the D\u2192A transition is decreased by several orders of magnitude 2\u00a0+ or Mg2\u00a0+A convenient method for the determination of the A/D ratio in membrane preparations of complex I is based on the fact that at alkaline pH (>\u00a08.5) and in the presence of divalent cations (1\u20135\u00a0mM Ca(ii)Since the chemical modification of the D-form results in irreversible arrest of enzyme activation, the percentage of the A-form only can be estimated after treatment of the preparation with SH-reagents, such as NEM or DTNB. The residual NEM-insensitive NADH oxidase or NADH:Q oxidoreductase activity would correspond to only the A-form of complex I. The total enzymatic activity (A\u00a0+\u00a0D) of the preparation can be measured after activation of the enzyme with a pulse of NADH followed by the addition of NEM.Based on (i) the inhibitory effect of alkaline pH and divalent cations on the activation rate and (ii) on the sensitivity of the D-form to SH-reagents, there are currently only two diagnostic tests for the estimation of the A- and D-form fractions in preparations of complex I. The content of both forms can be estimated by measuring the NADH oxidase activity of the untreated preparation in conditions arresting the activation (the A-fraction only). This should be compared with the same sample in the same measuring conditions after complex I is fully activated . Preparations of intact mitochondria require permeabilisation or inner membrane disruption prior to measurement in order that the active site of complex I is available for NADH The inhibitory effect of SH-reagents on the D-form of complex I in bovine heart SMP can be assessed by NADH oxidase or NADH:Q oxidoreductase activity which are usually affected to a similar extent Given necessary optimisation, these two methods can be used for the estimation of the A/D content in cells, muscle fibres or tissue slices. After anoxic incubation or conditions of metabolic or chemical hypoxia 2\u00a0+ or Ca2\u00a0+ for measuring complex I activity The A/D transition should be taken into account when measuring complex I-dependent activities in crude mitochondrial membrane preparations isolated from tissues obtained from animal models. It is likely that any chemical treatment/pharmacological intervention affecting oxygen metabolism or animal models of oxygen deprivation  would result in a significant shift in the A/D equilibrium. Depending on the degree of de-activation of the enzyme in situ and the particular conditions of mitochondrial membrane isolation, a significant fraction of the enzyme is expected to be in the D-form in the final preparation. It should be stressed that when using a medium at alkaline pH or/and supplemented with Mg2.2SH) 2\u00a0+ or Mg2\u00a0+. Divalent cations possibly neutralise negative lipid charges at the membrane surface and therefore affect the process of conformational changes during re-activation. The inhibitory effect of divalent cations on the activation was found in the following order: Ni2\u00a0+\u00a0>\u00a0Co2\u00a0+\u00a0>\u00a0Mn2\u00a0+\u00a0>\u00a0Ca2\u00a0+\u00a0\u2248\u00a0Mg2\u00a0+\u00a0>\u00a0Ba2\u00a0+. It should be stressed, that nickel or cobalt ions are hypoxia-mimetics and widely used to induce HIF-1\u03b1 stabilisation in studies of the cellular oxygen sensing mechanism The conversion from the D- to the A-form of complex I was shown to be inhibited by either an increase in pH or by the presence of divalent cations A, DSH [1Free fatty acids not only affect the NADH oxidase and NADH:Q oxidoreductase activities of complex I 2\u00a0+ on the D\u2192A transition was re-evaluated using pig brain SMP 2\u00a0+ drastically potentiates the effect of free fatty acids possibly due to the formation of an ion-pair that would increase fatty acid concentration in the membrane, aggravating its effect on complex I Recently the effect of Ca2.3Paracoccus denitrificansThermus thermophilusThe proton-translocating NADH:ubiquinone oxidoreductase is found in many different organisms from bacteria to mammals. These enzymes share basic catalytic properties including similar turnover number and apparent affinity constant for substrates and inhibitors 2.4The electron transfer from NADH to ubiquinone catalysed by complex I is coupled to the translocation of four protons across the membrane as measured experimentally Escherichia coli and Thermus thermophilus), in the presence of NAD(P)H, the quantity of cross-linked products between the hydrophilic subunits was significantly reduced and susceptibility to tryptic degradation was increased E. coli in the presence of NADH or NAD+In bovine and bacterial complex I  was shown to be the most mobile, moving away from NuoI (TYKY) in the presence of NAD(P)H. The NuoB subunit moves closer to other subunits, potentially NuoG (75\u00a0kDa) and hydrophobic subunits such as NuoA (ND3) or NuoH (ND1) In the Taken together, these studies clearly showed a rearrangement of the subunits around the quinone binding pocket after the binding of substrate and inhibitors in the bacterial and mammalian enzyme. However, the changes in the enzyme structure are probably relatively small, explaining results found with zero-length cross-linkers. Indeed, the use of cross-linker reagents with a spacer arm longer than 10\u00a0\u00c5 is unlikely to unveil intramolecular differences N-ethylmaleimide and its fluorescent derivative, they were able to show that a subunit of around 15\u00a0kDa was modified by the SH-reagent only in the D-form of the enzyme. The residue differentially labelled in the D-form of complex I only was further identified by mass spectrometry as the Cys-39 of the ND3 subunit The first observation of a possible structural difference between the A- and D-forms of mitochondrial complex I was found by Vinogradov's group T. thermophilus) and PSST (Nqo6) The ND3 subunit is known to participate in the formation of the quinone reaction chamber, along with ND1, 49\u00a0kDa  was used To study the potential conformational changes in the A/D transition of complex I, a 6.8\u00a0\u00c5 \u2014SH/\u2014NHUsing fluorescent lysine specific reagents and a DIGE-like approach, we recently demonstrated the differential exposure of three different subunits depending on the conformation of complex I in SMP For the first time, ND1 was found associated to structural rearrangements upon de-activation of the enzyme. These three subunits are located at the junction between the hydrophilic and membrane arms of complex I and ND1 and ND3 are directly involved in the formation of the quinone binding pocket The junction region (Q-module) between the proton pumping (P-module) and hydrophilic module that operates the electron transfer from NADH to the terminal N2 FeS cluster (N-module) probably displays a high degree of flexibility Rotenone is a quinone-like specific inhibitor of complex I. SMP containing either the A- or the D-form of the enzyme were subjected to incubation with rotenone, the inhibitor was washed off and the content of both forms was determined. This resulted in a partial protection of the A-form against de-activation but also converted the D-form to an A-form-like conformation It would then be of strong interest to perform cross-linking studies in both hydrophilic and hydrophobic parts of the mammalian complex I depending on the conformation of complex I. It is tempting to speculate that the structural rearrangement leading to the A-state might be similar to the one observed on the bacterial enzyme in the presence of NADH 2.5As demonstrated by extensive studies in Schagger's laboratory 1 supercomplex (I1\u00a0+\u00a0III2\u00a0+\u00a0IV1) shows that the complex III dimer sits in the arc of the bent membrane part of complex I so that the ubiquinone binding sites of both complexes face each other The estimated apparent energy of the spontaneous A\u2192D transition for bovine heart mitochondrial complex I is 270\u00a0kJ/mol 3c oxidase The physiological role of the A/D transition is still under discussion. As shown previously, in tissues ex vivo 3.1c oxidase. Complex I turnover becomes restricted by the lack of electron acceptor ubiquinone. Accumulation of reduced quinone then not only decreases the availability of substrate for the enzyme, but also inhibits the physiological oxidoreductase activity of the enzyme In conditions of limited oxygen concentration  indicating that reintroduction of oxygen causes re-activation of the D-form of the enzyme As shown previously, chemical or pharmacological inhibition of complex I during ischaemic incubation would protect mitochondria from damage after reperfusion c oxidase. Therefore, the lag-phase in complex I activity, although probably very short in situ, would decrease the generation of ROS by the mitochondrial respiratory chain and lessen the oxidative damage. It is possible that the reversible de-activation of complex I in the absence of oxygen may function as a protective valve for complex I electron transfer pathway upon tissue reoxygenation.Following ischemia/anoxia, reoxygenation may initiate abnormal bursts of NAD(P)H oxidation, ROS generation and the formation of other reactive metabolites capable of damaging mitochondrial constituents including complex I. The de-activation of complex I could then be a physiological mechanism to maintain a low enzymatic activity when oxygen concentration rises. The presence of most of complex I in the D-state during reintroduction of oxygen to the tissue would have two important consequences. Firstly, the slow activation of complex I during the early phase of reoxygenation would reduce the burst of respiration downstream of the enzyme. Secondly, it would delay rapid formation of potentially ROS-promoting tightly bound ubisemiquinones in complex I + can be reduced to NADH 2O2 production which is prevented by complex I inhibitors 2O2+ is reduced it is unlikely that the reverse electron transfer occurs directly from ubiquinol to the nucleotide. However, during reoxygenation, de-activation can temporarily prevent reduction of FMN in a fraction of the enzyme. Since respiratory chain complexes are distributed heterogeneously in the inner mitochondrial membrane along the cristae and therefore can be exposed to different redox conditions (NAD(P)H/NAD(P)+ ratio) and/or potential Another important consideration is the effect of de-activation on reverse electron transfer. In the presence of membrane potential, ubiquinol is able to reduce redox-centres of complex I such as all iron sulphur clusters and flavin, so that NADA critical consequence of I/R process is oxidative stress and therefore special endogenous mechanisms should have evolved in order to ameliorate the damage. This damage is unlikely to be directly due to a decrease in the respiratory chain activity and ATP production per se, but due to oxidative stress following reintroduction of oxygen in tissues in the post-ischaemic overreduced state 3.3Covalent modification of thiol groups via reversible glutathionylation, oxidation, nitrosation Following the discovery that the glutathionylation of some complex I subunits increased superoxide production by the enzyme 2O2 and superoxide were found to inhibit only the D-form of complex I 2O2. Once Cys-39 of the ND3 subunit is modified, complex I is thought to be unable to undergo the D\u2192A conversion and catalyse the physiological NADH:ubiquinone reaction. Thus, Cys-39 may be an early mitochondrial target for oxidative/nitrosative stress during I/R.Recently, complex I conformation-specific thiol modifications were identified in mitochondrial membranes isolated from normoxic and ischaemic mouse heart Inhibition of complex I-mediated respiration was demonstrated in cells after they were incubated with activated macrophages c oxidase This was recapitulated in HEK cells producing endogenous NO SNO) S\u204e), however, is irreversible at the timescale of I/R. Prolonged exposure of the D-form of the enzyme to low steady-state levels of endogenous SH-reactive molecules such as S-nitrosoglutathione, peroxynitrite and ROS would lead to modification of a certain fraction of the enzyme. Depending on the nature of the covalent modification, the fraction of modified enzyme would gradually increase subject to the presence of the effector and activity of the thiol-regenerating system. Complex I has a high degree of flux control over oxidative phosphorylation +/NADH ratio and also the membrane potential and ATP production. Depending on the magnitude and time-frame of the process, the degree of oxidative damage and the course of recovery in ischaemic tissues after reperfusion could be significantly affected.The kinetics of the A/D transition can be significantly affected in different conditions but the degree of modification of the D-form by ROS or NO metabolites in situ is not clear at present. S-nitrosation of the ND3 subunit is probably reversible via reduction by glutathione and thioredoxin systems and may be protective B, DSNO [4The transition of mitochondrial complex I from the A- to the D-state is a unique feature of some eukaryotic enzymes, including mammals. The mechanism of the A/D transition of the enzyme is far from being understood, however one of the accessory subunit, 39\u00a0kDa (NDUFA9) and two mitochondrially encoded subunits, ND3 and ND1, all located in the area of the quinone binding site are involved in conformational rearrangements. It is unlikely that the A/D transition is induced by changes in the respiratory chain supercomplex assembly. The driving force of the A/D transition, the mechanism of the electron transfer blockage in the D-form and other potential accessory subunits involved remain to be clarified.Results from our and other laboratories suggest that accumulation of the D-form of complex I occurs in mitochondria-rich tissues during oxygen deprivation. The rate of accumulation of the D-form of the enzyme during ischaemia can be modulated by elevated temperature, the presence of free fatty acids and divalent cations. This would affect the redox state of cofactors and reactive centres upstream and downstream of the enzyme. The A/D transition may represent a natural mechanism providing a fast mitochondrial response to oxygen deprivation. Deceleration in the activity of the respiratory chain due to the slow activation of the D-form during the initial phase of reoxygenation would prevent mitochondrial generation of ROS, decreasing oxidative damage. However, the cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by ROS and NO metabolites. Accumulated D-form could react with natural effectors in mitochondria or with pharmacological agents during periods of hypoxia or reoxygenation. This would modulate the process of reactivation of the enzyme and therefore determine the outcome of the I/R event."}
+{"text": "Manipulation of cell\u2013cell interactions has potential applications in basic research and cell-based therapy. Herein, using a combination of metabolic glycan labelling and bio-orthogonal click reaction, we engineer cell membranes with \u03b2-cyclodextrin and subsequently manipulate cell behaviours via photo-responsive host-guest recognition. With this methodology, we demonstrate reversible manipulation of cell assembly and disassembly. The method enables light-controllable reversible assembly of cell\u2013cell adhesion, in contrast with previously reported irreversible effects, in which altered structure could not be reused. We also illustrate the utility of the method by designing a cell-based therapy. Peripheral blood mononuclear cells modified with aptamer are effectively redirected towards target cells, resulting in enhanced cell apoptosis. Our approach allows precise control of reversible cell\u2013cell interactions and we expect that it will promote further developments of cell-based therapy. Reversible manipulation of cell-cell interactions has potential applications in basic research and cell-based therapy. Here the authors control cell-cell adhesion in vitro with light, by modifying the surface sugars of cells to display \u03b2-cyclodextrin, which recognises one isoform of light-isomerizable azobenzene linkers. Dynamic cell\u2013cell interactions are imperative for correct cell behaviour. The failure of cell communications can cause uncontrollable cell growth and cancer134Apart from molecular biological techniques to genetically engineer cells6911131417trans and cis forms, can be reversibly interconverted on photoirradiationtrans isomer forms a stable inclusion complex with CD, while the bent cis isomer does not fit in CD2022Azobenzene represents a well-known class of photo-switchable compounds, the two isomers of which, the To realize this, tailoring cell surfaces with \u03b2-CD is a prerequisite. Non-covalent cell-surface modification approaches based on lipid insertion and liposome-to-cell fusion have received increasing attention451213141524262728314GalNAz) to enrich cell surface glycoconjugates with the azide tag34Herein, we take advantage of metabolic labelling approach and bio-orthogonal click reaction to tailor cell membranes with host molecules . The str4GalNAz was selected on the basis of its known incorporation into mucin-type O-linked glyco-proteins in mammalian cells via the N-acetylgalactosamine  salvage pathway , azobenzene binding could be blocked by adamantine. As expected, no distinct azo-DNA-FAM binding was observed for \u03b2-CD-modified cell exposed to adamantine. These results confirmed the successful \u03b2-CD modification and azobenzene binding. We thereafter quantified the number of cell-bound \u03b2-CD through quantification of azo-DNA-FAM. \u03b2-CD modified cells were treated with azo-DNA-FAM, and subsequently analysed by flow cytometry. The mean fluorescence intensities of cells were compared with known standards  to determine the number of azo-DNA-FAM per cell6 molecules were modified onto the cell surface   . Next, wm, 15\u2009W) . By mean) (3\u2009M\u22121 . The pho) (3\u2009M\u22121 . We demo) (3\u2009M\u22121 . The \u03b2-C) (3\u2009M\u22121 . Both grtrans-azobenzene (high) and \u03b2-CD and cis-azobenzene (low), photoinduced dispersion and re-aggregation of the cells could be achieved by ultraviolet irradiation, followed by Vis irradiation  this method allows modulating intercellular contacts in space and time; (2) cell\u2013cell adhesion can be reversible controlled by light; (3) due to the presence of \u03b2-CD, we envision that a series of stimuli-responsive host-guest recognition can be introduced to meet multi-level demand. Our design opens a new avenue to control contact-dependent cell\u2013cell reversible interactions and will promote further studies on cell communications.4GalNAz) was purchased from Invitrogen . FAM alkyne and Copper(II)-TBTA complex (10\u2009mM in 55% aq. dimethylsulfoxide) were purchased from Lumiprobe. MCF-7 cells/Hela cells were cultured using the general DMEM medium containing 50\u2009\u03bcM Ac4GalNAz for three days to enrich the azido groups in O-linked glycoproteins. The Ac4GalNAz-labelled cells were washed three times with 1X PBS, then incubated with PBS containing 50\u2009\u03bcM Copper(II)-TBTA complex, 2\u2009mM sodium ascorbate, 25\u2009\u03bcM FAM alkyne  in the dark at rt for 10\u2009min, followed by three washes before being used for the following experiments. The \u03b2-CD modified cells were incubated with azo-DNA-FAM (25\u2009\u03bcM) for 60\u2009min, followed by three washes before fluorescence imaging and flow cytometry analysis. Fluorescence imaging experiments were performed on a Zeiss LSM700 confocal laser scanning microscope. Flow cytometry analysis was performed on BD FACS Aria. For experiment of attaching \u03b2-CD-labelled cells to an azobenzene-patterned substrate, the \u03b2-CD-labelled cells were detached from 6-well plates by incubation with 1\u2009mM EDTA-PBS for 10\u2009min at 37\u2009\u00b0C. Then cells were incubated with the modified substrates for 4\u2009h. The unbound cells were gently removed by rinsing with buffer for 1\u2009min. Cells were stained by AM fluorescent dye for 10\u2009min and viewed using an Olympus BX-51 optical system microscope . For ultraviolet triggered release, cell-attached substrate were irradiated with ultraviolet light  for 10\u2009minN-azidoacetylgalactosamine , the \u03b2-CD-labelled cells were detached from 6-well plates by incubation with 1\u2009mM EDTA-PBS for 10\u2009min at 37\u2009\u00b0C. Green-stained and red-stained \u03b2-CD-modified MCF-7 were mixed together at a 1:1 ratio in the presence of different concentrations of azo-PEG-azo, and shaken at 300\u2009rpm for 60\u2009min at 25\u2009\u00b0C. Aliquots were analysed by optical microscope. For ultraviolet triggered disassembly, cell aggregates were irradiated with ultraviolet light  for 10\u2009min and shaken in the dark at 300\u2009rpm for 30\u2009min at 25\u2009\u00b0C. Aliquots were analysed by optical microscope. Flow cytometric analysis was performed on BD FACS Aria.The \u03b2-CD-modified Hela cells were incubated with 20\u2009\u03bcM azobenzene labelled MUC 1 aptamer for 20\u2009min. After washing, MCF-7 cells were added for heterotypic cell adhesions. For easily distinguishing, Hela cells and MCF-7 cells were stained with green (fluorescein) and red (lissamine rhodamine B) dyes respectively. For ultraviolet triggered release, the sample was irradiated with ultraviolet light  for 10\u2009min. After that, the substrate was gently washing with PBS for 15\u2009s to remove the released cells.4GalNAz for three days to enrich the azido groups. The azido-labelled cells were washed three times with PBS, then incubated with PBS containing 50\u2009\u03bcM Copper(II)-TBTA complex, 2\u2009mM sodium ascorbate, 50\u2009\u03bcM alkynyl-PEG-\u03b2-CD in the dark at rt for 10\u2009min, followed by three washes. Then the \u03b2-CD-modified PBMCs were incubated with 20\u2009\u03bcM azobenzene labelled MUC 1 aptamer for 20\u2009min. After washing, the resulted aptamer-modified PBMCs (or unmodified PBMCs) were added to each well of the 6-well plate containing MCF-7 cells. MCF-7 cells and PBMCs were then allowed to incubate together at 37\u2009\u00b0C for one hour (shaken at 300\u2009rpm) and further longer. The cell-cell contacts and resulting cell lysis were viewed using an Olympus BX-51 optical system microscope . Measurement of cytotoxicity of MCF-7 cells on incubation with aptamer-modified or unmodified PBMCs was done using a non-radioactive cytotoxicity assay (CytoTox 96\u00ae Non-Radioactive Cytotoxicity Assay)Human peripheral blood mononuclear cells (PBMCs) were obtained through density gradient centrifugation and cultured with RPMI-1640 media, followed by modified with aptamer MUC 1 through the above labelling strategy. Briefly, PBMCs were cultured with RPMI-1640 containing 50\u2009\u03bcM AcTo gain more insight on the details, SEM characterization was included in our study. The specimens were fixed with 4% glutaraldehyde for 3\u2009h. Next, the specimens were washed with sterile water for three times and then were dehydrated by addition of ethanol in a graded series  and then treated with tert-butanol. After drying under vacuum, the specimens were coated with platinum and examination on SEM.The authors declare that the data supporting the findings of this study are available from the corresponding author on request.How to cite this article: Shi, P. et al. Spatiotemporal control of cell\u2013cell reversible interactions using molecular engineering. Nat. Commun.7, 13088 doi: 10.1038/ncomms13088 (2016).Supplementary Figures 1-18, Supplementary Methods, Supplementary References"}
+{"text": "Vitamin B6 status was determined using fasting plasma concentrations of pyridoxal 5\u2019-phosphate (PLP). Mean  plasma PLP concentration was 61.0  nmol/L. The prevalence of B6 deficiency (plasma PLP < 20 nmol/L) was 1.5% and that of suboptimal B6 status (plasma PLP = 20\u201330 nmol/L) was 10.9%. Body mass index, South Asian ethnicity, relative dietary B6 intake, and the use of supplemental B6 were significant predictors of plasma PLP. The combined 12.4% prevalence of B6 deficiency and suboptimal status was lower than data reported in US populations and might be due to the high socioeconomic status of our sample. More research is warranted to determine B6 status in the general Canadian population.Low periconceptional vitamin B6 (B6) status has been associated with an increased risk of preterm birth and early pregnancy loss. Given many pregnancies are unplanned; it is important for women to maintain an adequate B6 status throughout reproductive years. There is limited data on B6 status in Canadian women. This study aimed to assess the prevalence of B6 deficiency and predictors of B6 status in young adult women in Metro Vancouver. We included a convenience sample of young adult non-pregnant women (19\u201335 years;  Vitamin B6 (B6) has an obligatory role in the endocrine system, immune competence, and heme biosynthesis. In the form of pyridoxal 5\u2019-phosphate (PLP), B6 serves as a coenzyme for >140 reactions in human metabolism, including in the interconversion of amino acids, synthesis of neurotransmitters, regulation of energy homeostasis, and formation of heme ,3. Othern = 247), maternal B6 supplementation (2.6 to \u226550 mg/day) was associated with a 217 g higher infant birth weight [Maternal B6 adequacy is crucial at conception and throughout pregnancy to ensure healthy pregnancy outcomes. In a meta-analysis of maternal B6 interventions , Cycle 2.2, Nutrition Focus, in 2004 reported that 18% of Canadian adult women did not meet the Estimated Average Requirement  of B6 from dietary sources; however, there was no biochemical measurement of B6 status . In the In several other countries , various socioeconomic and lifestyle factors have been associated with plasma PLP concentrations, including oral contraceptive use , use of This study aimed to determine the prevalence of suboptimal B6 status and B6 deficiency in a convenience sample of young adult women in Metro Vancouver, using plasma PLP, a biochemical marker of B6 status. Demographic, dietary, and lifestyle predictors associated with plasma PLP were also assessed.This study used data from a descriptive cross-sectional study conducted between 2012 and 2013. The original study was designed to determine the prevalence and predictors of low vitamin B12 status in a convenience sample of young adult women of South Asian and European descent. The recruitment and methods of the original study have been described in detail . In brieFasting venous blood samples were obtained from all participants during a single clinic visit. Samples were collected into lithium heparin vacutainers. Plasma was separated using centrifugation and stored at \u201380 \u00b0C until analyses. Blood samples of 202 subjects were available for plasma PLP analysis. Plasma PLP was measured by quantification of its semicarbazide derivative using high-performance liquid chromatography (HPLC) with fluorescence detection . The assA demographic questionnaire, the \u201cInternational Physical Activity Questionnaire (IPAQ)\u2014Short Last 7 Day Self-Administered Format\u201d ,23, and n = 8) indicating secondary school education or less than secondary school education, education was dichotomized. Low education was considered as below a bachelor\u2019s degree, and high education as equal or higher than bachelor\u2019s degree. Due to the low number of participants (n = 36) in the lowest household income bracket, household income was dichotomized into the following categories: Low income, total annual household income < $30,000 if 1\u20132 people, <$40,000 if 3\u20134 people, <$60,000 if \u22655 people, and high income, total annual household income \u2265 $30,000 if 1\u20132 people, \u2265$40,000 if 3\u20134 people, \u2265$60,000 if \u22655 people. Participants who reported use of oral contraceptives were classified as oral contraceptive (OC) users. Duration and frequency of OC use were recorded. Participants who reported current consumption of any nutritional vitamin or mineral supplements were classified as nutritional supplement users. Participants were asked to bring in nutritional supplement bottles. Brand names and frequencies of intake were recorded and B6 content (yes/no) was obtained retrospectively by researchers from web-based product information. Participants who were taking supplements containing B6 were classified as supplemental B6 users. Subjects were asked about their smoking habit and categorized as non-smoker, former smoker, current occasional smoker (1\u20139 cigarettes/day), current regular smoker (10\u201319 cigarettes/day) or current frequent smoker (\u226520 cigarettes/day). Due to the high number of non-smokers (n = 174), smoking data were dichotomized to non-smoker and smoker (including current smokers and former smokers).The demographic questionnaire collected information on age, ethnicity, immigration, education, household income, oral contraceptive use, and supplement use. South Asian and European ethnicities were defined as having at least three grandparents from a single ethnic group. South Asian ethnicity included Bangladeshi, Bengali, East Indian, Goan, Gujarati, Hindu, Ismaili, Kashmiri, Nepali, Pakistani, Punjabi, Sikh, Sinhalese, Sri Lankan, and Tamil ethnic groups. Because of the low number of participants  and duration (time per day). Participants were categorized into three physical activity levels; low, medium, and high, according to the IPAQ analysis protocol .A semi-quantitative FFQ was used to determine dietary B6 intake. The questionnaire contained 78 food items and has been validated to assess micronutrient intakes in the Canadian population ,25. Anthropometric measurements, including height, weight, and waist circumference, were taken by research staff during the clinic visit. Body mass index (BMI) was calculated based on weight and height. The primary outcome of the presented analyses is B6 status and described using the direct biomarker plasma PLP and established cut-offs for adequate B6 status, suboptimal B6 status , and B6 t tests were used for dichotomous variables, one-way ANOVA followed by Tukey\u2019s Honest Significance test for categorical variables with more than two levels, and simple linear regression for continuous variables. If the P value from bivariate analysis was \u22640.2, the variable was carried forward to the stepwise multiple linear regression model. The full model with plasma PLP concentration as the dependent variable was controlled for relative dietary B6 intake, South Asian ethnicity, first generation immigrant status, BMI, education status, household income status, smoking status, and supplemental B6 use. Backward elimination procedure was used to establish the best fit multiple linear regression model. The estimated percentage change in plasma PLP concentration was presented for each variable after adjustment for other variables. Statistical significance was set at a two-sided p value of <0.05. All statistical analyses were performed using R software .Bivariate analysis was conducted to identify variables associated with plasma PLP concentration  and relative dietary B6 intake. Two sample n = 202) were highly educated, with 71% having a bachelor\u2019s degree or higher  concentration of plasma PLP was 61.0  nmol/L . The prep < 0.001). Women of South Asian descent had significantly lower plasma PLP concentration ) compared with women of European descent , p = 0.002). There was no significant difference in plasma PLP concentration based on education level, household income, physical activity level, OC use, or smoking status in bivariate analyses. There was no significant difference in the prevalence of B6 deficiency, suboptimal B6 status or both combined (plasma PLP < 30 nmol/L) compared to adequate B6 status based on any demographic or lifestyle factors.Users of supplemental B6 had significantly higher plasma PLP concentration compared to non-users of supplemental B6; mean (95% CI) plasma PLP concentrations were 48.5  and 111.5 , respectively (p = 0.045). Plasma PLP concentration and relative dietary B6 intake were positively but weakly correlated .Quartiles of dietary B6 intake were 0\u20131.1 mg/day, 1.1\u20131.4 mg/day, 1.4\u20131.7 mg/day and >1.7 mg/day, respectively. Individuals with low household income had significantly lower dietary B6 intake compared to individuals with high household income .Dietary vitamin B6 intake derived mainly from meat and meat alternatives in this sample of young adult women of South Asian and European descent, as shown by multiple linear regression of dietary B6 intake . There wR\u00b2) of the variance in plasma PLP concentration.Relative dietary B6 intake, BMI, ethnicity and the use of supplemental B6 were significant predictors of plasma PLP concentration . Relativp = 0.026), after adjusting for relative dietary B6 intake, BMI and the use of supplemental B6.Women taking supplemental B6 are expected to have ~113% higher plasma PLP after adjusting for relative dietary B6 intake, BMI and ethnicity (compared to ~127% higher plasma PLP in the unadjusted model) . South ASince the vitamin B6 status of Canadian women was previously unknown, and the relationship between demographic, dietary, and lifestyle factors and plasma PLP had not been assessed, we conducted a study of B6 status in a convenience sample of 202 healthy young Canadian adult women. We identified a combined prevalence of B6 deficiency and suboptimal B6 status of 12.4%. We also found that body mass index, South Asian ethnicity, relative dietary B6 intake, and the use of supplemental B6 were significant predictors of plasma PLP.As defined by the Institute of Medicine, a plasma PLP concentration of <20 nmol/L corresponds with B6 deficiency . The pren = 1236) [Suboptimal B6 status has been associated with an increased risk of several chronic diseases, including cardiovascular disease , colorec = 1236) . The low = 1236)  and our The use of supplemental B6 was a strong predictor of plasma PLP concentration both before and after multivariate adjustment. This is consistent with large scale studies that reported higher plasma PLP concentrations and a lower prevalence of B6 deficiency in nutritional supplement users  and suppr = 0.32, p < 0.001) was observed [Relative dietary B6 intake was not a strong predictor of plasma PLP concentration both before and after multivariate adjustment. The weak association may partially be due to the use of a semi-quantitative FFQ. The validation study of the FFQ reported an 11% underestimation of relative dietary B6 intake compared to the use of dietary recalls . Althougobserved . We are Our study is the first to report that women of South Asian descent may have lower plasma PLP concentrations compared with women of European descent. The difference in B6 status between South Asian and European women was not related to the consumption of different food sources of B6; meat and meat alternatives were the main dietary B6 sources in these healthy adult women. The negative association between South Asian ethnicity and plasma PLP concentrations remained significant after adjustment for relative dietary B6 intake, BMI, and supplemental B6 use. Although the South Asian population was only 56, compared to the European population of 146 in this study, the sample size was sufficient to give over 80% of power with a small effect size of 0.2 and significance level of 0.05 in the multiple linear regression model. Ethnicity has been shown to correlate with nutritional biomarker levels of other micronutrients .Body mass index was inversely associated with plasma PLP concentration after adjustment for relative dietary B6 intake, supplemental B6 use, and South Asian ethnicity. An inverse relationship of BMI and plasma PLP was also reported in the NHANES; every 25% increase in BMI was associated with a 13% decrease in plasma PLP . The volCompared to other studies, we did not find OC use to be a significant predictor of B6 status. In recent literature, lower-dose OC use was associated with low B6 status . In the The predictors of B6 status we assessed explained only 32% of the variability in plasma PLP. Our results might be confounded by unexplained biological factors or genetic modifiers. One recent study reported some variants in tissue nonspecific alkaline phosphatase gene influenced plasma PLP concentration, but the clinical implications of these variants were unclear .Some weaknesses of this study include the recruitment of a convenience sample of relatively healthy women of high education and the lack of data on genetic variants. Over 71% of our study participants obtained a bachelor\u2019s degree or higher, where a comparatively lower proportion (35%) of women aged 25 to 64 in Metro Vancouver reported this high level of education, according to the National Household Survey . We usedn = 202). Periconceptional B6 adequacy is crucial for healthy pregnancy outcomes; and women with higher BMI and South Asian ethnicity might be more likely to have low B6 status. The lower prevalence of B6 deficiency and suboptimal B6 status in these women compared to reports from representative samples in other countries may be due to the high socioeconomic status of these women. Given the central roles of B6 in key biological functions and health, more research is warranted to determine B6 status in the general Canadian population. We report a 12.4% combined prevalence of B6 deficiency and suboptimal B6 status in healthy young adult women in Metro Vancouver ("}
+{"text": "Background: The aim of this study is to determine the frequency of intracranial artery stenosis in patients with acute ischemic stroke in Iran.Methods: A total of 169 patients with acute ischemic stroke were eligible to participate and were enrolled in this study from January 2012 to February 2013. All the patients were admitted to the Nemazee \u200eHospital, affiliated to Shiraz University of Medical Sciences, Iran. They underwent transcranial Doppler (TCD) ultrasonography. Mean flow velocity (MFV) of basilar artery, vertebral artery, middle cerebral artery (MCA), anterior cerebral artery (ACA), and posterior cerebral artery (PCA) were evaluated.Results: A mean of patients\u2019 age was 67.80 \u00b1 8.14 years. There were 83 men (49.1%) and 86 women (50.9%). Overall, 43 patients (25.4%), with a mean age of 66.7 \u00b1 6.2 years, had intracranial stenosis. The number of men and women with intracranial stenosis was comparable (52.4% men vs. 47.6% women). Hypertension (P < 0.001), hyperlipidemia (P < 0.001), and diabetes mellitus (DM) (P < 0.001) were major risk factors for intracranial stenosis.Conclusion: The prevalence of intracranial artery stenosis in patients with acute ischemic stroke is 25.4% which is comparable with previous reports from Iran and other Middle East countries. The incidence of stroke is increasing in developing countries in the Middle East.4 Approximately 10% (12.9%) of ischemic strokes occur secondary to atherosclerotic intracranial arterial stenosis.5 It has been demonstrated that the risk of developing ischemic stroke in those with middle cerebral arteries stenosis in about 24% in 6-year follow-up.6 As the investigation of intracranial artery stenosis is highly important, some studies have evaluated cerebral hemodynamics and the cerebral blood flow to predict ischemic CVAs.7 It has been suggested that the patients with more than 50% intracranial arteries stenosis should undergo more rigorous prevention strategies.8The previous studies have reported that intracranial etiologies are responsible for the majority of stroke cases, i.e., disturbances in intracranial circulation cause most strokes.9 Among these methods, TCD is an available, simple and non-invasive method for the assessment of the intracranial blood flow and hemodynamic changes.10,11 This non-invasive technique can be used for follow-up and continuous monitoring, can be performed in a bedside setting, and also can be applied for targeted thrombolysis.12 The European Federation of Neurological Sciences (EFNS) summarized that TCD is very useful for assessing stroke risk of children between 2 and 16 years of age with sickle cell disease, detection and monitoring of vasospasm after subarachnoid hemorrhage, evaluation of the intracranial artery stenosis and blood flow, diagnosis of the right-to-left shunts and for monitoring the cerebral reperfusion after thrombolysis therapy of middle cerebral artery (MCA) after acute ischemic stroke.13 The results of TCD can assist physicians to determine preventive and therapeutic strategies.14Several methods including transcranial Doppler (TCD) ultrasonography, cerebral angiography, computed tomography angiography (CTA), and magnetic resonance angiography (MRA) have been introduced to evaluate the intracranial blood flow in asymptomatic patients.15 Although many studies have documented that the intracranial artery stenosis can lead to ischemic stroke, some others have shown that the stenosis of extracranial arteries in thrombotic stroke is more prevalent.16 Caucasian patients with ischemic stroke have a higher prevalence of intracranial stenosis compared to other ethnic groups.15 There are few studies reported from the Middle East which have addressed intracranial arteries blood flow in ischemic stroke.17,18 The aim of this study is to determine the frequency of intracranial artery stenosis in patients with acute ischemic stroke in Iran.Several reports have indicated that the prevalence of intracranial artery stenosis varies between different geographical regions and is higher in blacks and Asians compared with Caucasians.19 The patients with condition confounding the clinical presentation including previous brain injuries, the patients with tumor or other conditions mimicking stroke, and those with a cardioembolic source of stroke were excluded.This prospective cross-sectional study was performed in the Neurology Section of Nemazee Hospital, affiliated to Shiraz University of Medical Sciences, Iran, from January 2012 to February 2013. The medical research ethics committee as well as Institutional Review Board of \u2026 approved the study protocol. All the patients provided their informed written consents before inclusion in the study. A total of 169 adult patients with definite diagnosis of ischemic stroke were included. The Recognition of Stroke in the Emergency Room scale (ROSIER) was used to confirm cerebral infarction, also known as ischemic stroke, as a focal neurological deficit of sudden onset that persisted beyond 24 hours in surviving patients and documented by a brain CT or an MR imaging.20 and associated medical diseases were assessed. The patients were also evaluated regarding the cardiovascular diseases and comorbidity, such as arrhythmias and impulse conduction disorders, mitral and/or aortic valve disease, left ventricular hypertrophy, and coronary heart disease (CHD).Cerebrovascular risk factors such as cigarette smoking, hypercholesterolemia [history of hypercholesterolemia and/or fasting total cholesterol level > 200 mg/dL or total triglyceride (TG) level > 200 mg/dL or low-density lipoprotein (LDL) > 130 mg/dL], hypertriglyceridemia (history of hypertriglyceridemia and/or fasting triglycerides level > 180 mg/dL), arterial hypertension , DM [diagnosis according to the criteria of the National Diabetes Data Group (NDDG)],We used odd/even day randomization technique to select the patients randomly. The demographic data of all the patients including age, gender, place of residence, educational status, and risk factors were recorded at the time of admission. CT scans of the brain were performed for the patients and the findings were reported based on the territory of the involved artery. 21 Therefore, we considered MFV of \u2265 80 cm/s for MCA as abnormal in favor of stenosis. In the same way, MFV of \u2265 80 cm/s for ACA was considered as stenosis.18 For vertebral artery, MFV of \u2265 70 cm/s was considered as abnormal while MFV \u2265 80 cm/s was considered as stenosis for basilar.22 For PCA, MFV of \u2265 60 cm/s was considered as stenosis.18 The patients, who were not evaluated due to technical problems, were regarded as poor window and the frequency was reported. Extracranial arteries were not evaluated in this study.All patients underwent TCD in the following days of admission. All TCD examinations were performed at maximum 5 days after ischemic stroke. TCD studies of intracranial arteries were performed via temporal and occipital windows using a DWL Multi-Dop T unit with a 2-MHz probe. The anterior cerebral artery (ACA), MCA, and posterior cerebral artery (PCA) were evaluated bilaterally through the temporal window. The vertebral and basilar arteries were assessed via the occipital window. The depth, peak systolic velocity (PSV), mean flow velocity (MFV), end diastolic velocity, and pulsatility index (PI) were measured for each artery separately. In this study, we considered the focal MFV  of arteries for the diagnosis of intracranial stenosis according to previously published data.-square test was used to compare the proportional data between those with and without intracranial artery stenosis. Independent t-test was used to compare parametric data between corresponding categories. Multivariate logistic regression analyses were carried out to control the potentially confounding effect of different risk factors. A two-sided P < 0.050 was considered statistically significant.SPSS software  was used for data analysis. Descriptive data are presented as mean \u00b1 standard deviation (SD). Chi-month period. A mean of patients\u2019 age was 67.80 \u00b1 8.14 (ranging from 29 to 92) years. There were 83 men (49.1%) and 86 women (50.9%). A total of 169 patients with acute ischemic stroke were eligible for participation and were included to undergo TCD during a 13The quality of the procedure was adequate in 139 patients (82.2%) but it was inadequate in 30 patients (17.8%) (poor window). Intracranial vessels could not be evaluated by TCD in 17.8% of the patients (poor window). No significant difference in the frequency of intracranial stenosis was observed between the two genders . There iThe frequency of poor window was significantly higher among the women . The patients with intracranial stenosis had significantly higher prevalence of hypertension , hyperlipidemia (P < 0.001), and DM (P < 0.001) compared with the patients who had no stenosis .This study suggests that the hypertension is the most common risk factor for cerebrovascular diseases as it was found in 55.0% of our patients following hyperlipidemia, DM and smoking. TCD findings revealed that MCA and ACA are two most common arteries which are stenosis in patients with acute ischemic stroke. 23 Some part of this variation may be due to different modalities and different criteria used in these studies. The pattern of its epidemiology and distribution is similar to ischemic strokes.24 It is more common among the Hispanics, people of African descent, and among Asians compared to Caucasians.25 Several studies have addressed the prevalence of intracranial artery stenosis in patients with acute ischemic stroke.24,25 Our findings replicate the findings of a study conducted by Zarei et al.17 that showed stenosis of intracranial arteries was detected in 29% of the patients with acute ischemic stroke in Iran. However, in our study, the prevalence of intracranial artery stenosis was significantly lower than the results of other previously published studies from other countries in the Middle East.17,18,26-30 Gujjar et al.18 reported a prevalence of 79% for intracranial artery stenosis in patients with ischemic stroke in Oman. Intracranial artery stenosis is considered among the most common causes of ischemic stroke worldwide. The incidence and prevalence of intracranial artery stenosis varies between geographical regions and ethnicities even in Asian countries.17,18,31 Wityk et al.15 conducted a study in 672 patients with ischemic stroke in Pakistan and reported that the prevalence of intracranial arteries stenosis was only 12%. However, Iranmanesh et al.16 reported that intracranial artery diseases in Caucasians are as high as 16-25%. Wasay et al.31 reported a low prevalence of intracranial vessels in ischemic stroke in Pakistan that is similar to the low frequency of carotid artery disease in patients with stroke in Southeast Asia.32,33 They also reported that there is a correlation between carotid atherosclerosis and risk factors such as hypertension, smoking status, and DM that is consistent with previous studies.32,33 It has been reported that the prevalence of intracranial stenosis in patients with ischemic stroke is 33-50% in China, 47% in Thailand, 48% in Singapore, and 10-25% in Korea.19 O\u2019Leary et al.34 conducted a study in the USA and showed that the prevalence of intracranial stenosis in patients with stroke and transient ischemic attack is similar to asymptomatic population.34 In their study, 1189 members of the Framingham cohort (asymptomatic), aged 66-93 years, were examined that showed there were no diseases in 30%, < 50% stenosis in 62%, 50-74% stenosis in 5%, 75-99% stenosis in 2%, and 100% stenosis in 1%.34 These results are comparable with our results in which only 25.4% of our patients with ischemic stroke had intracranial artery stenosis. In the present study, hypertension, hyperlipidemia, and DM were major risk factors for intracranial stenosis, but smoking was not. Controlling risk factors were protective against developing intracranial stenosis. The risk factors reported in this study is comparable with other previous studies.32-34The previous studies have reported that Asian populations have a higher prevalence of intracranial artery stenosis.17 reported that multiple intracranial stenosis were more common that single artery stenosis. Suh et al.35 also reported a prevalence of 21% for single stenosis, 79% for multiple stenosis, 52% for intracranial lesions, and 48% for the extracranial area in patients with ischemic stroke. Their results also showed that anterior circulation was involved in 59%, but posterior circulation was involved in 41% of the patients.35 In our study, the MCA was the most common site of stenosis followed by ACA. Right MCA was involved in 16% but left MCA was involved in 9.5% of our patients. In the same way, the right ACA was involved in 11.5% and the left in 7.7%. Zarei et al.17 showed that MCA was the most common involved artery with a prevalence of 11% bilaterally and 5% unilaterally. Gujjar et al.18 reported the involvement of anterior circulation in 22 patients (11.8%) and posterior circulation in 22 patients (11.8%). The anterior circulation (MCA + ACA) was involved in 64 patients (37.8%) in our study while the posterior circulation  was involved in 17 patients (8.2%) that are higher than any other previously reported incidence rates.18 The prevalence of ACA and MCA stenosis was higher in our study than what has been reported by Baumgartner et al.,36 however, the incidence of vertebral and basilar stenosis was comparable.Zarei et al.37 When applying TCD results especially for MCA, we should keep in mind that there are other causes which can increase MFV of MCA other than atherosclerosis such as recanalization of an occluded MCA.38 Therefore, it is recommended that the patients with intracranial stenosis diagnosed in TCD to be followed so that they could be differentiated form recanalization of the MCA.38 The diagnostic accuracy of TCD should be regarded when applying it. The sensitivity and specificity for TCD have been reported 90 and 88%, respectively.39It has been shown that the accuracy of TCD is the highest for the evaluation of MCA compared with other intracranial arteries which adds to the value of TCD as a non-invasive technique.18 reported a moderate accuracy for TCD in determining the pathology of the intracranial vessels. They reported some abnormality in about two-thirds of their study population when using TCD, while only 56% of those patients had changes in arteries corresponding to infarct location. The findings of these studies are in consistence with other studies.36,40 The incidence of poor window in our study was 18.3% that is comparable to previous studies from Western countries (11-20%).36,40,41 However, it is less than the East Asian populations (37%).42 Gujjar et al.18 reported a moderate sensitivity of TCD compared with MRA, with a relatively higher specificity. Alexandrov et al.21 compared the results of TCD with conventional angiography in a group of 84 patients with acute ischemic stroke. They reported high sensitivity (87.5%) and specificity (88.6%), together with high positive (87.5%) and negative (88.6%) predictive values.21Gujjar et al.31A study compared the MRA and transcranial color-coded Doppler sonography in 135 MCAs among 120 patients with acute stroke and reported that angle-corrected velocities correlated well with different grades of stenosis on MRA (P = 0.006). An angle corrected MCA PSV of > 120 cm/s correlated with MRA evidence of intracranial stenosis with high specificity (90.5%) and positive predictive value (93.9%), but relatively low sensitivity  and negative predictive value (55.1%).We note some limitations to our study. First, Doppler studies are operator-dependent, and the skill of the operator plays an important role in TCD results. To solve this problem, we had one single neurology resident interpret all the TCDs. Second, we did not record the outcome of the patient to correlate the TCD results with the severity and outcome of the ischemic stroke. In addition, we did not get the follow-up data of the patients and thus the predictive value of TCD could not be interpreted. Third, we performed conventional CT-scans of the brain at the time of admission and did not repeat the imaging in the following hours. This might lead to the high frequency of not formed ischemic region in brain CT-scans. Fourth, we did not perform any other vascular imaging of the brain other than TCD. Thus, the results of TCD could not be correlated with other neuroimaging techniques such as MRA or CTA. Because we used only MFV for the diagnosis of stenosis and MFV may not increase in severe stenosis so we may not detect some cases with very severe stenosis. The prevalence of intracranial artery stenosis in patients with acute ischemic stroke diagnosed by TCD is 25.4% which is comparable with previous reports from Iran and other Middle East countries."}
+{"text": "With aging, the proportion of intracranial artery stenosis alone decreased; at the same time, the proportion of extracranial artery stenosis and extracranial plus intracranial artery stenosis increased . Hypertension  and family history of stroke  were risk factors for intracranial artery stenosis. Male, aging, and smoking were factors more related to extracranial artery stenosis. Aging  and hypertension  were related to posterior circulation artery stenosis. Intracranial arteries and anterior circulation arteries were susceptible to stenosis in Chinese patients with ischemic stroke. However, the distribution pattern of atherosclerotic stenosis was dynamic and varied with aging. Aging and different risk factors contribute to this distribution pattern.The aim of this multicenter study was to demonstrate the distribution pattern of atherosclerotic stenosis and its trend with aging between extracranial and intracranial arteries and its distribution between the anterior and posterior circulations in Chinese patients hospitalized with ischemic stroke. In addition, the risk factors for the distribution pattern were illustrated. From June 2015 to May 2016, 9,346 patients with ischemic stroke from 20 hospitals were enrolled. Carotid artery ultrasonography and transcranial color-coded sonography/transcranial Doppler were used to evaluate the extracranial and intracranial arteries. The distribution pattern of atherosclerotic stenosis and its trend with aging were analyzed. Logistic regression was used to analyze the risk factors for the distribution pattern. Among the 9,346 patients, 2,882 patients (30.8%) had at least one artery with a degree of stenosis \u226550%. Among patients with arterial stenosis, the proportion of patients with intracranial artery stenosis was higher than those with extracranial artery stenosis (52.6%  The latest Global Burden of Disease 2015 Study (GBD 2015) reports that the prevalence of ischemic stroke increased by 15.8% between 2005 and 2015. In China, ischemic stroke is one of the leading causes of mortality . HoweverNoninvasive carotid artery color Doppler ultrasonography (CDU) and transcranial color-coded sonography (TCCS)/transcranial Doppler (TCD) have been widely used in China as screening methods for detecting extracranial and intracranial artery stenosis in patients with stroke . In thisClincalTrials.gov with the identification number NCT02397655. The study protocol was approved by the institutional ethics committee of Xuanwu Hospital, Capital Medical University .This study was registered on and released by From June 2015 to May 2016, 9,346 hospitalized patients with ischemic stroke from 20 hospitals were continuously enrolled. The 20 hospitals involved in this study represented North, South, East China, Central China, Southwest, Northeast, and Northwest China. The diagnoses of ischemic stroke were made by a neurologist according to the American Heart Association/American Stroke Association (AHA/ASA) guidelines [The risk factors collected in this study included hypertension, diabetes mellitus, dyslipidemia , and smoking (defined as a patient who had smoked continuously for 6 months \u2265 1 cigarette a day). In addition, family history of ischemic stroke (defined as the parents or siblings of the patient having a history of stroke) was recorded.All the CDU and TCCS/TCD examinations were performed by experienced ultrasound physicians (with more than 5 years of experience) and followd the vascular ultrasound protocol described by Zwiebel and Pellerito , and theIntracranial arteries, including the middle cerebral artery (MCA), the terminal of the ICA (TICA), anterior cerebral arteries (ACAs), posterior cerebral arteries (PCAs), the intracranial segment of the VA, and the basilar artery were examined by TCCS with a 1-5 MHz phased array probe or TCD  with a 1.6 MHz probe. The PSV and EDV of the arteries were recorded. According to the ultrasound criteria ,12, and To ensure the quality of the study, we have taken the following actions: (1) All the hospitals participated in this multicenter study were the base hospitals for stroke prevention and treatment awarded by the Stroke Screening Prevention Project Committee, National Health and Family Planning Commission of China, suggesting that these hospitals have a high standard of the techniques required for stroke prevention and treatment. (2) All the physicians performing vascular ultrasound have more than 5 years of experience in this field and received uniform training for CDU and TCCS/TCD before the study, with a diagnostic accuracy \u2265 90% (compared with CTA or MRA). (3) This study used the uniform ultrasound criteria for diagnosing artery stenosis -12. All t test was used to compare the difference between two groups. Numerical values with non-normal distribution are shown as the median (interquartile range), and a non-parametric test was used to compare the difference between two groups. The Pearson chi-square test was used to compare the enumeration values, and the chi-square trend test was used to investigate the linear trend of the enumeration values with aging. Logistic regression analysis was used to define the independent risk factors for the atherosclerosis distribution pattern. A P value <0.05 was considered statistically significant.The Statistical Package for Social Sciences (SPSS version 22.0) software was used for the statistical analysis. Numerical values with normal distribution are shown as the mean \u00b1 SD, and Among the 9,346 patients, 2,882 patients (30.8%) had at least one artery with a degree of stenosis \u226550%. The demographic and clinical characteristics of the groups with and without stenosis are listed in P<0.001). Moreover, we categorized the patients into five age groups, as follows: 40-49, 50-59, 60-69, 70-79, and \u226580 years. In all groups except for the 40-49-year group, the prevalence of artery stenosis in male patients was higher than in females , and the proportion of stenosis in the anterior circulation arteries was higher than that in the posterior circulation (52.2% vs. 26.2%). With respect to the gender difference, male patients had a higher proportion of extracranial artery stenosis, while females had a higher proportion of intracranial artery stenosis . There was no difference in the distribution pattern of anterior and posterior circulation artery stenosis between male and female subjects   .2=6.698, P=0.001). Similarly, the percentage of anterior artery stenosis alone declined and the percentage of posterior circulation artery stenosis and anterior plus posterior circulation artery stenosis increased with aging .As shown in P=0.008) and family history of stroke  were risk factors for intracranial artery stenosis. Male, aging, and smoking were more closely associated with stenosis of the extracranial arteries (P<0.001), and hypertension  were more related to stenosis in the posterior circulation arteries , cross-sectional, and multicenter registry study, we investigated the prevalence rate of large artery atherosclerotic stenosis and demonstrated its distribution pattern characteristics in Chinese patients hospitalized with ischemic stroke. The present study included almost all the arteries susceptible to atherosclerotic stenosis when demonstrating the distribution pattern. In addition, we demonstrated the trends of distribution pattern with aging and analyzed the risk factors contributing to this distribution pattern. Prevalence of stroke has been reported to be more common among men.  We also Significantly more patients had intracranial artery stenosis than extracranial artery stenosis, with an approximate ratio of 5:3. These findings corroborate with the previous notion that intracranial atherosclerosis is more prevalent in Asian patients . AlthougThe differences among the traditional risk factors are believed to affect the atherosclerotic stenosis distribution . The preFurthermore, this study showed that the proportion of stenosis in the anterior circulation arteries was higher than that in the posterior circulation arteries in the Chinese population. Aging and hypertension were more related to posterior circulation artery stenosis. In studies in Korea , Japan , and a svs.50.0%), which is consistent with the CICAS study  [vs. extracranial OR 0.672, P<0.001), which was consistent with the meta-analysis results  [With regard to the gender difference of the distribution pattern, the present study showed that female patients had a higher proportion of intracranial artery stenosis than males (60.8% P=0.028) . MoreoveP=0.028) . AlthougP<0.001) .The novelest and important discovery in this study is that we demonstrated the atherosclerotic artery stenosis distribution pattern trends with aging. Although the total frequencies of artery stenosis with \u2265 50% were not increasing with age, the distribution pattern of atherosclerotic stenosis between extracranial and intracranial arteries and its distribution between the anterior and posterior circulations showed an obvious trend along aging. With aging, the proportion of extracranial artery stenosis was gradually closer to that of intracranial artery stenosis. In this study, the ratio of frequencies of extracranial to intracranial arteries stenosis from 1:3 in patients 40-49 years old to near 1:1 in patients age \u2265 80 years old. Moreover, logistic analysis showed that aging was an independent risk factor for the distribution pattern. This new finding suggested that aging is a key factor that should be considered when demonstrating the distribution pattern of atherosclerotic artery stenosis. These distribution pattern trends might be attributed, at least partially, to the age-specific risk factor profiles and age associated accumulation of the number of risk factors. Genetic factors are believed to contribute to the development of intracranial artery stenosis , which iThe limitations of the study are that all the patients enrolled in the study had obvious symptoms and needed to be treated in hospital. Some patients with ischemic stroke but without symptoms or patients with mild symptoms that could be treated in an outpatient clinic were not enrolled. Thus, the results may not completely represent all patients with stroke in China. In addition, the correlation between this stenotic lesion with the location of cerebral infarction needs to be further studied.In conclusion, intracranial arteries and anterior circulation arteries were susceptible to stenosis in Chinese patients with ischemic stroke. The distribution pattern of atherosclerotic stenosis was dynamic and varied with aging. Aging and different risk factors contribute to this distribution pattern. Specifically, hypertension and family history of stroke were more related to intracranial artery stenosis. Male, aging, and smoking were factors more related to extracranial artery stenosis. Aging and hypertension were risk factors for posterior circulation artery stenosis."}
+{"text": "Neuroprotective activity could be quantified by the specific index (SI) value, that is, the ratio of the 50% cytotoxic concentration to the 50% effective concentration. Alkaline extract from the leaves of Sasa senanensis Rehder (SE), having had hormetic growth stimulation, showed the highest SI value, followed by epigallocatechin gallate. The SI value of curcumin and resveratrol was much lower. This simple overly method, that can prepare massive differentiated neuronal cells, may be applicable for the study of the differentiation-associated changes in intracellular metabolites, and the interaction between neuronal cells and physiological factors.Previous studies of the neuroprotective activity of polyphenols have used ununiform culture systems, making it difficult to compare their neuroprotective potency. We have established a new and simple method for preparing differentiated PC12 cells by removing the toxic coating step. Cells were induced to differentiate with the nerve growth factor (NGF) in a serum-free medium, without a medium change, but with a one-time overlay supplementation of NGF. The optimal inoculation density of the cells was 6\u201312 \u00d7 10 Improvement in the daily nutritional supply and the living environment resulted in the prolongation of our life span, but necessarily increased the number of elderly populations having cognitive diseases ,2,3. AlzPlatinum drugs such as cisplatin, carboplatin and oxaliplatin have been important parts of combination chemotherapy regimens to treat different types of solid tumors, but they can cause serious neurotoxicity in the dorsal root ganglion by the formation of adducts to DNA . Taxanesp-coumaryl, p-conifery, and sinapyl alcohols. Some polysaccharides in the cell walls of lignified plants are linked to lignin, and recover as lignin-carbohydrate complex\u2014after extraction with alkaline solution . Fixed cells were washed twice with PBS(\u2212), and then treated for 30 min with 400 \u03bcL of 0.2 mg/mL RNase A (preheated for 10 min at 100 \u00b0C to deactivate DNase) to degrade RNA. Cells were then washed twice with PBS(\u2212) and stained for 15 min with 0.005% propidium iodide (PI) in the presence of 0.01% NP-40 in PBS(\u2212), which prevents cell aggregation. After filtering through Falcon\u00ae cell strainers (40 \u03bcM)  to remove aggregated cells, PI-stained cells were subjected to a cell sorter . Cell cycle analysis was performed with the Cell Sorter Software version 2.1.2 .Cells  of triplicate or quadruplicate samples. Statistical analysis was performed using Student\u2019s The present study demonstrated that plant extracts, having hormetic growth stimulation, showed higher neuroprotective activity than lower molecular weight polyphenols. The overlay method, that can prepare massive differentiated neuronal cells, may be applicable for the study of differentiation-associated changes in intracellular metabolites by metabolomics, and the interaction between neuronal cells and physiological factors."}
+{"text": "Medication errors, adverse drug events, and nonadherence lead to increased health care utilization and increased risk of adverse clinical outcomes, including graft loss, in solid organ transplant recipients. Veterans living with organ transplants represent a population that is at substantial risk for medication safety events and fragmented care coordination issues. To improve medication safety and long-term clinical outcomes in veteran transplant patients, interventions should address interorganizational system failures and provider-level and patient-level factors.This study aims to measure the clinical and economic effectiveness of a pharmacist-led, technology-enabled intervention, compared with usual care, in veteran organ transplant recipients.This is a 24-month prospective, parallel-arm, cluster-randomized, controlled multicenter study. The pharmacist-led intervention uses an innovative dashboard system to improve medication safety and health outcomes, compared with usual posttransplant care. Pharmacists at 10 study sites will be consented into this study before undergoing randomization, and 5 sites will then be randomized to each study arm. Approximately, 1600 veteran transplant patients will be included in the assessment of the primary outcome across the 10 sites.This study is ongoing. Institutional review board approval was received in October 2018 and the study opened in March 2019. To date there are no findings from this study, as the delivery of the intervention is scheduled to occur over a 24-month period. The first results are expected to be submitted for publication in August 2021.With this report, we describe the study design, methods, and outcome measures that will be used in this ongoing clinical trial. Successful completion of the Improving Transplant Medication Safety through a Technology and Pharmacist Intervention study will provide empirical evidence of the effectiveness of a feasible and scalable technology-enabled intervention on improving medication safety and costs.ClinicalTrials.gov NCT03860818; https://clinicaltrials.gov/ct2/show/NCT03860818PRR1-10.2196/13821 Over the past 20 years, the use of contemporary immunosuppression has reduced the risk of rejection by more than 80%, but long-term allograft survival remains suboptimal ,2. CurrePrevious research has demonstrated that transplant recipients are burdened with numerous risk factors for the development of medication safety events. These include taking more than 10 medications concomitantly with more than 30 doses ingested per day, being prescribed narrow therapeutic index medications that are prone to drug interactions, taking chronic immunosuppressants with known debilitating side effects, and having frequent dosage adjustments. In addition, long-term ambulatory transplant recipients usually receive care across multiple health care organizations; thus, fragmented care, omissions, duplications, and discrepancies in medication regimens are common among these patients. We have also established this as a major issue facing veteran transplant recipients. Veteran transplant recipients who receive care from a transplant center outside their primary Veterans Health Administration (VHA) location are particularly at risk for these types of errors ,7,9-11.dual users ) will provide data to assess outcomes occurring within the VA health care system, including hospitalizations, ER visits and costs, and mortality. CDW data will also be used to assess interventions by querying pharmacists\u2019 progress notes. To ensure encounters are captured in a comprehensive manner, we will also link the VA CDW data to Centers for\u00a0Medicare\u00a0and Medicaid Services (CMS) claims data and capture non-VA ER and hospitalization encounters (after study completion). The CMS data will provide non-VA health care utilization, including hospitalizations and ER visits, as well as non-VA cost estimates. Scientific Registry of Transplant Recipients data will provide all baseline donor, recipient, and transplant characteristics and clinical outcomes, including acute allograft rejection, graft loss, and death. Queries answered by intervention pharmacists will include the number of alerts received, how many were considered clinically relevant/actionable, time to conduct the intervention, and general intervention types.The outcomes to be assessed for this study all relate to evaluating the impact of the intervention. The primary outcome measure for this study will include the overall rate of ER visits and hospitalizations, compared between the intervention and control groups, while adjusting for baseline patient, provider, and facility characteristics. As previously stated, we will link the VA CDW data to the CMS (Medicare) claims data to capture both VA and non-VA ER and hospitalization encounters and provide a more accurate assessment of health care utilization. ER visits and hospitalizations will be assessed and compared as described in the section Sample Size and Statistical Analysis Plan.Secondary outcomes to be assessed include a cost-benefit analysis of health care costs between the intervention and control groups as well as an assessment of the dashboard system\u2019s functionality and efficacy. Overall health care costs accrued during the 24-month study and those accrued in the 24 months before study initiation will be analyzed and compared between the control and intervention groups. Cost data will be standardized using the VA Health Economics Resource Center definitions, which normalize regional differences in costs because of variation in cost of living indices. As with the primary outcome, we will also acquire and link CMS claims data to gain a comprehensive assessment of costs, including those that accrue from non-VA care (after study completion). Another secondary outcome is to assess the success of the dashboard systems expansions and utilization. To do so, we will evaluate the dashboard\u2019s functionality by measuring and reporting descriptive statistics for the alert numbers, alert relevance, time, and the actions taken with regards to the alert and the intervention magnitude. Alert and intervention information will be entered by intervention pharmacists through the dashboard interface, which will then be brought into VINCI for formal analysis. These measures will allow us to ascertain if the expanded dashboard is meeting expectations, with regards to functionality and efficiency. We will also assess the number of potential immunosuppression safety issues that occur and compare these between the 2 study arms. To do so, we will use the dashboard to provide monthly measurements of the following: percentage of patients with missing laboratory assessments; percentage of patients with alarming laboratory values without follow-up scheduled; and mean adherence to immunosuppression, based on refill timeliness and estimated using the PDC; percentage of patients with a significant drug interaction without an immunosuppressant level; and percentage of patients with hospital discharge or ED visit without follow-up scheduled. These will be measured in all patients and compared between the intervention and control groups at monthly intervals. On the basis of the projected enrollment numbers, we expect to have ample power to detect a statistically significant difference between the intervention and control groups with regards to the primary aim of hospitalization and ER visit rates. We used data from a recent national study conducted between 2009 and 2012 . These rFor comparative statistical assessments of utilization outcomes , the 2 groups will be compared using a generalized linear mixed models (GLMM) approach . This apFor the cost analysis, we will also use multivariable modeling and propensity score calibration . We willFor the assessment of the functionality of the dashboard system and the time required to complete the intervention, we will use standard descriptive statistics for these measurements, including mean (SD), median (interquartile range), proportion (percentage), and 95% CI. Missing data will be handled using several techniques, including multiple imputation and maximum likelihood . MissingThis study is ongoing. Institutional review board approval was received in October 2018 and the study opened in March 2019. To date, there are no findings from this study, as the delivery of the intervention is scheduled to occur over a 24-month period. The first results are expected to be submitted for publication in August 2021.The use of the dashboard monitoring system to conduct automated near real-time surveillance for immunosuppressant safety issues and alert pharmacists when such issues arise is innovative in several ways. First, this technology leverages the currently underused enormity of data that are already embedded within the VA electronic health record system. Owing to the complexity involved in the clinical management of transplant recipients, there are substantial numbers of laboratory values and medication regimen alterations that occur within each patient. Automating the monitoring of these data to identify trends and potential patient issues allows for improved efficiency. Medication refill adherence and relevant drug interaction monitoring improve the comprehensive assessment of medication adherence and safety. Finally, monitoring for hospitalization and ER visits will allow for appropriate follow-up with the transplant teams when necessary. The use of a pharmacist-led intervention is also innovative. Although the use of clinical pharmacists to improve medication safety and outcomes is well documented, there are limited studies analyzing the effectiveness of such interventions within the transplant population, and none specifically within veteran organ transplant recipients. The limited studies that demonstrate improved medication outcomes using pharmacists\u2019 led interventions among transplant patients (a number of which are from our research group) predominantly focus on the acute perioperative hospitalization phase ,26,41-44There are several challenges with health services research that have the potential to undermine the intervention. First, as this intervention seeks to improve medication safety through modifying human behaviors, there is potential for implementation issues associated with the pharmacist-led intervention; there may be actions by the patient or provider that may limit or undermine the impact of the intervention. To maintain the fidelity and consistency of the intervention, we will use structured interventions based on identified barriers, develop a detailed standard operating procedure manual for the intervention, and conduct face-to-face training with the pharmacists. Systems barriers also have the potential to limit the intervention impact. As these patients are routinely managed across multiple health care systems, both inside and outside the VA, it is important that the pharmacist-led intervention facilitates medication safety and coordination across these systems. We will train the pharmacists on the best methods to ensure optimal coordination of care for these patients and provide tools that we currently use to improve the efficiency of outside care documentation.Supported by previous research, the use of a technology-enabled, pharmacist-led intervention provides a promising and innovative approach to improve medication safety and reduce drug-related problems in veteran organ transplant recipients. Successful completion of the ISTEP study will provide empirical evidence of the effectiveness of a feasible and scalable technology-enabled intervention on improving medication safety and costs. We envision this technology can be used in the monitoring of all US transplant patients receiving care within the VA. Our long-term goal is to leverage the use of this technology to develop a VA-specific pharmacist learning collaborative to substantially improve immunosuppressant safety and outcomes within veteran organ transplant recipients."}
+{"text": "Moreover, comparative receptor-independent and receptor-dependent structure\u2013activity studies were conducted to explain the observed variations in inhibiting the potential of the investigated carbamate series. The principal objective of the ligand-based study was to comparatively analyze the molecular surface to gain insight into the electronic and/or steric factors that govern the ability to inhibit enzyme activities. The spatial distribution of potentially important steric and electrostatic factors was determined using the probability-guided pharmacophore mapping procedure, which is based on the iterative variable elimination method. Additionally, planar and spatial maps of the host\u2013target interactions were created for all of the active compounds and compared with the drug molecules using the docking methodology. A series of new benzene-based derivatives was designed, synthesized and comprehensively characterized. All of the tested compounds were evaluated for their in vitro ability to potentially inhibit the acetyl- and butyrylcholinesterase enzymes. The selectivity index of individual molecules to cholinesterases was also determined. Generally, the inhibitory potency was stronger against butyryl- compared to acetylcholinesterase; however, some of the compounds showed a promising inhibition of both enzymes. In fact, two compounds carbamate and 28, benzyl (1-(3-chlorophenyl)-1-oxopropan-2-yl) (methyl)carbamate) had a very high selectivity index, while the second one (28) reached the lowest inhibitory concentration IC Several clinically implemented drug and pesticide molecules contain amide (\u2013CONH\u2013) and/or carbamate (\u2013OCONH\u2013) groups that can be variously substituted to form privileged structural fragments ,2,3,4. TPotency modeling of prospective drug molecules seems to be useful as an initial screening test (rank order) and a decisive factor in avoiding \u201cchasing\u201d false positives and/or eradicating \u201cbad actors\u201d at the early stages of drug design or development ,20. The The current paper can be regarded as a follow-up of the recently reported carbamates as prospective ChEIs ,30,31; tThe concept of the synthesis of the designed compounds was based on a modification of the structure of the selected \u03b1-aminoketones. Cathinone is biologically active natural product and its derivatives have broad pharmacological properties. All carbamates were formed in a four-step synthesis that begins with the basic reagents see . In the first step, the corresponding aromatic ketone was obtained in the Friedel\u2013Crafts reaction. Next, the bromination of the obtained aromatic ketone with elemental bromine led to a 2-bromo derivative, which was subjected to the ammonolysis of the obtained halide without secretion. In the final step, the amine in a free form reacted with the appropriate chloroformate to produce desired carbamate. The final products were purified using column chromatography with a mixture of ethyl acetate and hexane, respectively. The structures of the obtained compounds are presented in \u00ae) and galantamine . These standards were selected because of their different structures since rivastigmine is a classical acylating pseudo-reversible carbamate ChEI that inhibits both AChE and BChE, whereas galanthamine is a non-acylating competitive reversible ChEI as well as an allosteric ligand at the nicotinic ACh receptors. The choice of these reference drugs, which have different mechanisms of action, can provide relevant results. The findings are summarized in 50 [\u03bcM]) or the concentration of the inhibitor that was required for the 50% inhibition of the mentioned enzymes. The AChE- and BChE-inhibiting activity for all of the tested carbamates was evaluated and compared with the internal standards rivastigmine . Specifically, two compounds, 23 and 28, had a very high selectivity index (SI = 13.93 and 15.31). In fact, compound 28 had the lowest IC50 value and had an approximately seven-fold higher inhibitory activity against BChE than RIV (IC50 = 5.51 vs. 38.40 \u00b5M), which corresponds quite well with GLT (IC50 = 2.77 \u00b5M). Similarly, a five-fold higher inhibitor activity against BChE relative to RIV was also observed for compound 31 (IC50 = 7.02 \u00b5M) and was more than three-fold higher for compounds 23 and 32 as well. It should be emphasized that compound 32 exhibited a promising inhibitory activity against AChE with an IC50 value of 32.01 \u00b5M\u2014approximately two times better compared to RIV (IC50 = 56.1 \u00b5M). Among the naphthalene derivatives, phenyl carbamates 36\u201338 revealed a three-fold higher inhibitor activity towards BChE relative to RIV, whereas only one benzyl carbamate, 39, exhibited a two-fold higher anti-BChE activity to RIV. In fact, compounds 36 and 39 might serve as selective inhibition agents towards BChE with respect to AChE (SI = 7.83 vs. 5.55). Moreover, compounds 37 and 38 also exhibited a high activity against AChE. Not surprisingly, the substitution of the aryl ring had a direct impact on the variations in the potency of the investigated compounds. It was observed that the methyl-substituted carbamates generally exhibited a lower inhibitory ability compared to their phenyl or benzyl counterparts. Interestingly, compounds 17 and 29, which have the methoxy group in the same position were weaker inhibitors\u2014the presence of a hydrophilic electron-donating -OCH3 substituent of the phenyl ring at the para-position decreased the potency of the compounds, especially against the BChE enzyme  resulted in an improvement in the IC50 value for the BChE enzyme, thus suggesting the significance of the hydrophobic interactions with the enzyme. The placement of an electron-withdrawing chlorine substituent in the meta-position of the phenyl ring appeared to be strongly preferable, especially for the BChE inhibition activity (compounds 16 or 28).All of the considered derivatives exhibited a very good to moderate inhibitory activity. The ICzyme see . On the 1\u201341 was investigated as the training set using the CoMFA and CoMSA approach. In this case, both methods performed comparably for the AChE/BChE activity ; however, in general, the modeling of pBChE data produced superior outcomes of the statistical metrics (CoMFA 3+ or H+). It was clear that relying exclusively on data fitting with cross-validated leave-one-out (CV-LOO) procedure is not satisfactory, and therefore the external validation by splitting the molecules into training/test subsets was conducted to assess the predictive power of a model using the SDEP, MAE and 9), and, therefore, the overall number of samplings was restricted to a relatively small fraction of approximately 3 \u00d7 106 systematically generated populations (1 out of 1000). Not surprisingly, the generated 2, 5, 6, 13, 15, 28, 31, 38, and 41, respectively.The principal objective of the ligand-based study was to comparatively analyze the molecular surface (CoMSA) to gain insight into the electronic and/or steric factors that potentially determine the inhibitory AChE/BChE activities of the investigated compounds. The findings for the surface descriptors were compared with their force field counterparts (CoMFA), when modeling thinhibiting potency for the multiple training/test subsets. First, the tability . From a tability . In othetability . Hence, In general, the specified molecules covered the entire structural space as well as the BChE activity range (5.51\u2013440.65 \u00b5M) of the investigated compounds evenly, which partially explains the good ability and predictability of the model.41\u00d72777 with the rows representing molecules (objects) and the columns preserving the numerical values of the variables (parameters) -4-oxidanyl-phenyl]~{N}-ethyl-~{N}-methyl-carbamate) were retrieved from the Protein Data Bank repository (PDB entry: 6eul). The drug analog was subsequently (re)docked in the active site AC2 of the enzyme chain A, which is composed of six amino acid residues  and a 1,2-ethanediol molecule (EDO605), using the AutoDock Vina program . Moreove28) that was tested are illustrated in 1 substituent) seemed to be valid for the hydrophobic interactions with the Asp70 amino acid residue, which is in line with our previous receptor-independent findings  and spatial (3D) maps of the host\u2013target interactions were created for all of the active compounds using Schr\u00f6dinger Maestro software and the Protein-Ligand Interaction Profiler (PLIP) and were then compared with the drug (GLT and RIV) molecules . Overallfindings .meta/para-positioned carbamate derivatives exerted on the enzyme reaction site; however, the close proximity of a positively charged nitrogen atom of His438 methyl derivatives. Unfortunately, the obtained docking findings did not provide a clear explanation of the variations that the f His438 c might h2beware of q + calculated: 258.1106. m/z, found: 258.1102 m/z.Methyl methyl(1-oxo-1-phenylbutan-2-yl)carbamate (2). Yield 83%; 1H-NMR : \u03b4 7.90 ; 7.71\u20137.41 , 5.35 , 3.47 , 2.79\u20132.44 , 1.92-1.62 , 1.05-0.72 ; 13C-NMR : \u03b4 204.0; 161.8; 140.7; 138.4; 133.9; 133.9; 133.1; 133.1; 65.9; 57.8; 34.9; 25.6; 15.5; HR-MS (ESI): for C13H17NO3Na [M+Na]+ calculated: 258.1106 m/z, found: 258.1103 m/z.Methyl ethyl(1-oxo-1-phenylbutan-2-yl)carbamate (3). Yield 85%; 1H-NMR : \u03b4 8.01 ; 7.62\u20137.25 , 5.54 , 3.79 , 3.14 , 2.10\u20131.92 , 1.08\u20130.81 , 1.08-0.81 ; 13C-NMR : \u03b4 198.8; 157.3; 135.9; 133.3; 128.5; 128.5; 127.9; 127.9; 60.1; 52.9; 38.1; 21.8; 15.0; 10.4; HR-MS (ESI): for C14H19NO3 Na [M+Na]+ calculated: 272.1263 m/z, found: 272.1263 m/z.Methyl methyl(1-oxo-1-(m-tolyl)propan-2-yl)carbamate (4). Yield 86%; 1H-NMR : 7.75 ; 7.44\u20137.28 ; 5.79\u20135.25 , 3.95\u20133.45 , 2.77 , 2.30 ; 1.69\u20131.22 ; 13C-NMR : 199.5; 156.9; 138.5; 135.4; 134.1; 128.8; 128.5; 125.6; 55.0; 52.9; 29.3; 21.3; 13.5; HR-MS (ESI): for C13H17NO3 Na [M+Na]+ calculated: 258.1106 m/z, found: 258.1105 m/z.Methyl (1-(3-chlorophenyl)-1-oxopropan-2-yl)(methyl)carbamate (5). Yield 89%; 1H-NMR : 8.15\u20137.69 ; 7.64\u20136.99 ; 5.77\u20135.28 , 3.76 , 2.76 , 1.46\u20131.19 ; 13C-NMR : 198.1; 156.8; 136.9; 135.0; 133.2; 130.0; 128.4; 126.5; 55.3; 53.1; 29.4; 13.3; HR-MS (ESI): for C12H14ClNO3 [M+H]+ calculated: 256.0740 m/z, found: 256.0741 m/z.Methyl (1-(4-methoxyphenyl)-1-oxopropan-2-yl)(methyl)carbamate (6). Yield 79%; 1H-NMR : 7.97 ; 7.01\u20136.87 ; 5.84\u20135.14 , 3.88 ; 3.76 , 2.93-2.58 , 1.37 ; 13C-NMR : 197.3; 163.7; 156.8; 130.8; 130.5; 128.2; 113.9; 113.9; 55.4; 54.4; 53.0; 29.1; 13.4; HR-MS (ESI): for C13H17NO4 Na [M+Na]+ calculated: 274.1055 m/z, found: 274.1043 m/z.Methyl methyl(1-oxo-1-(p-tolyl)propan-2-yl)carbamate (7). Yield 87%; m.p.: 36-37.5 \u00b0C; 1H-NMR : 7.87 ; 7.28 ; 5.65 , 3.76 , 2.76 , 2.44 ; 1.81\u20130.70 ; 13C-NMR : 198.7; 156.8; 144.2; 132.8; 129.4; 129.4; 128.6; 128.6; 54.8; 53.0; 29.2; 21.7; 13.4; HR-MS (ESI): for C13H17NO3 Na [M+Na]+calculated: 258.1106, m/z, found: 258.1108 m/z.Methyl (1-(4-chlorophenyl)-1-oxopropan-2-yl)(methyl)carbamate (8). Yield 87%; 1H-NMR : 7.91 ; 7.50\u20137.16 ; 5.72\u20135.31 , 3.75 , 2.75 , 1.49\u20131.23 ; 13C-NMR : 197.9; 156.8; 139.8; 133.6; 129.9; 129.9; 129.0; 129.0; 54.0; 53.1; 29.2; 13.2; HR-MS (ESI): for C12H14ClNO3 [M+H]+ calculated: 256.0740 m/z, found: 256.0743 m/z.Methyl (1-(4-bromophenyl)-1-oxopropan-2-yl)(methyl)carbamate (9). Yield 87%; 1H-NMR : 7.77 ; 5,35 , 3.58\u20133.26 , 2.85\u20132.44 , 1.35\u20131.24 ; 13C-NMR : 203.3; 160.8; 139.8; 136.9; 136.9 135.1; 135.1; 132.3; 61.9; 57.9; 35.8; 18.2; HR-MS (ESI): for C12H14BrNO3 [M+H]+ calculated: 300.0235 m/z, found: 300.0222 m/z.Methyl -1-oxopropan-2-yl)(methyl)carbamate (10). Yield 76%; 1H-NMR : 7.54 ; 7.28 ; 6.87 ; 6.06 ; 5.77\u20135.26 , 3.77 ; 2.94\u20132.48 ; 1.61 ; 13C-NMR : 197.0; 156.8; 152.4 148.2; 129.9; 125.0; 108.2; 101.8; 100,4; 54.5; 53.0; 29.1; 13.5; HR-MS (ESI): for C13H15NO5Na [M+Na]+ calculated: 288.0848 m/z, found: 288.0842 m/z.Phenyl ethyl(1-oxo-1-phenylpropan-2-yl)carbamate (11). Yield 87%; 1H-NMR : 8.03 ; 7.68\u20136.71 ; 5.69 ; 3.56-3.15 , 1.56 ; 1.20 ; 13C-NMR : 199.1; 154.9; 151.4 135.5; 133.4; 129.4; 129.4; 128.8; 128.8; 128.5; 128.5; 125.5; 121.6; 121.6; 55.5; 39.1; 15.6; 14.6; HR-MS (ESI): for C18H19NO3 Na [M+Na]+ calculated: 320.1263m/z, found: 320.1258 m/z. Phenyl methyl(1-oxo-1-phenylbutan-2-yl)carbamate (12). Yield 89%; 1H-NMR : 8.07 ; 7.65\u20137.59 ; 7.51 ; 7.43-7.35 ; 7.29\u20137.20 ; 7.14\u20137.09 ; 5.62 ; 2.91 ; 2.09\u20131.81 ; 1.08 ; 13C-NMR : 197.8; 155.5; 151.3 135.7; 133.5; 129.3; 129.3; 128.8; 128.8; 128.6; 128.6; 125.5; 121.7; 121.7; 60.4; 29.8; 20.9; 10.4; HR-MS (ESI): for C18H19NO3Na [M+Na]+ calculated: 320.1263 m/z, found: 320.1260 m/z.Phenyl ethyl(1-oxo-1-phenylbutan-2-yl)carbamate (13). Yield 88%; 1H-NMR : 8.21\u20137.90 ; 7.62 ; 7.54\u20137.48 ; 7.46\u20137.37 ; 7.33\u20137.21 ; 7.18\u20137.09 ; 5.61 ; 3.63\u20133.12 ; 2.12-1.82 ; 1.13 ; 1.06 ; 13C-NMR : 198.6; 155.4; 151.4 135.8; 133.5; 129.4; 129.4; 128.8; 128.8; 128.6; 128.6; 125.4; 121.6; 121.6; 60.4; 38.8; 21.8; 15.3; 10.5; HR-MS (ESI): for C19H21NO3Na [M+Na]+ calculated: 334.1419 m/z, found: 334.1411 m/z.Phenyl methyl(1-oxo-1-phenylpentan-2-yl)carbamate (14). Yield 76%; 1H-NMR : 8.11\u20138.02 ; 7.62 ; 7.54\u20137.47 ; 7.47\u20137.35 ; 7.32\u20137.20 ; 7.13\u20137.08 ; 5.73 ; 2.92 ; 1.98\u20131.81 ; 1.49\u20131.40 ; 1.04 ; 13C-NMR : 199.0; 155.4; 151.4; 135.7; 133.5; 129.5; 129.5; 128.8; 128.8; 128.6; 128.6; 125.4; 121.7; 121.7; 58.6; 30.2; 29.8; 19.2; 13.9; HR-MS (ESI): C19H21NO3 [M+H]+ calculated for: 312.1600 m/z, found: 312.1605 m/z.Phenyl methyl(1-oxo-1-(m-tolyl)propan-2-yl)carbamate (15). Yield 84%; m.p.: 52.5-53.5 \u00b0C; 1H-NMR : 7.94\u20137.73 ; 7.40 ; 7.28 ; 7.23 ; 7.09 ; 5.74 ; 2.94 ; 2.44 ; 1.48 ; 13C-NMR : 199.3; 154.9; 151.4; 138.6; 135.4; 134.3; 129.3; 129.3 128.9; 128.7; 125.7; 125.4; 121.6; 121.6; 55.4; 30.0; 21.3; 13.6; HR-MS (ESI): for C18H19NO3Na [M+Na]+ calculated: 320.1263 m/z, found: 320.1277 m/z.Phenyl (1-(3-chlorophenyl)-1-oxopropan-2-yl)(methyl)carbamate (16). Yield 92%; 1H-NMR : 8.01 ; 7.91 ; 7.63\u20137.56 ; 7.49\u20137.42 ; 7.42\u20137.35 ; 7.27\u20137.19 ; 7.13\u20137.06 ; 5.68 ; 2.93 ; 1.49 ; 13C-NMR : 198.0; 155.0; 151.3; 137.0; 135.1; 133.5; 130.2; 129.4; 129.4; 128.5; 126.5; 125.6; 121.6; 121.6; 55.6; 30.0; 13.4; HR-MS (ESI): for C17H16ClNO3 Na [M+Na]+ calculated: 340.0717 m/z, found: 340.0714 m/z.Phenyl (1-(4-methoxyphenyl)-1-oxopropan-2-yl)(methyl)carbamate (17). Yield 78%; m.p.: 53\u201356 \u00b0C; 1H-NMR : 8.04 ; 7.39 ; 7.30\u20137.19 ; 7.11 ; 6.97 ; 5.74 ; 3.90 ; 2.93 ; 1.47 ; 13C-NMR : 199.3; 154.9; 151.4; 138.6; 135.4; 134.3; 129.3; 129.3 128.9; 128.7; 125.7; 125.4; 121.6; 121.6; 55.4; 30.0; 21.3; 13.6; HR-MS (ESI): for C18H19NO4Na [M+Na]+ calculated: 336.1212 m/z, found: 336.1222 m/z.Phenyl methyl(1-oxo-1-(p-tolyl)propan-2-yl)carbamate (18). Yield 82%; m.p.: 42.5\u201344 \u00b0C; 1H-NMR : 7.94 ; 7.39 ; 7.30 ; 7.23 ; 7.14\u20137.07 ; 5.74 ; 2.93 ; 2.44 ; 1.48 ; 13C-NMR : 198.5; 154.9; 151.4; 144.4; 132.8; 129.5; 129.5; 129.3; 129.3; 128.7; 128.7; 125.5; 121.7; 121.7; 55.0; 29.9; 21.7; 13.6; HR-MS (ESI): for C18H19NO3Na [M+Na]+ calculated: 320.1263 m/z, found: 320.1262 m/z.Phenyl (1-(4-chlorophenyl)-1-oxopropan-2-yl)(methyl)carbamate (19). Yield 91%; m.p.: 52\u201354 \u00b0C; 1H-NMR : 7.98 ; 7.48 ; 7.43\u20137.36 ; 7.24 ; 7.08 ; 5.68 ; 2.93 ; 1.48 ; 13C-NMR : 197.7; 154.9; 151.3; 140.0; 133.7; 130.0; 130.0; 129.4; 129.4; 129.1; 129.1; 125.6; 121.6; 121.6; 55.2; 29.9; 13.4; HR-MS (ESI): for C17H16ClNO3Na [M+Na]+ calculated: 340.0717 m/z, found: 340.0712 m/z.Phenyl (1-(4-bromophenyl)-1-oxopropan-2-yl)(methyl)carbamate (20). Yield 88%; m.p.: 54\u201355 \u00b0C; 1H-NMR : 7.90 ; 7.65 ; 7.40 ; 7.25 ; 7.08 ; 5.67 ; 2.93 ; 1.48 ; 13C-NMR : 198.0; 155.0; 151.3; 134.1; 132.1; 132.1; 130.0; 130.0; 129.4; 129.4; 128.8; 125.6; 121.6; 121.6; 55.2; 29.9; 13.4; HR-MS (ESI): for C17H16BrNO3Na [M+Na]+ calculated: 384.0211 m/z, found: 384.0226 m/z.Phenyl -1-oxopropan-2-yl)(methyl)carbamate (21). Yield 74%; m.p.: 94.5\u201395.5 \u00b0C; 1H-NMR : 7.84\u20137.74 ; 7.42\u20137.36 ; 7.29\u20137.19 ; 7.11 ; 5.75 ; 2.92 ; 2.41\u20132.25 ; 1.47 ; 13C-NMR : 198.8; 154.9; 151.4; 143.2; 137.2; 133.2; 130.0; 129.6; 129.3; 129.3; 126.3; 125.4; 121.7; 121.7; 55.1; 29.9; 20.1; 19.8; 13.7; HR-MS (ESI): for C19H21NO3Na [M+Na]+ calculated: 334.1419 m/z, found: 334.1418 m/z.Phenyl -1-oxopropan-2-yl)(methyl)carbamate (22). Yield 76%; m.p.: 94\u201395 \u00b0C; 1H-NMR : 7.75\u20137.63 ; 7.54\u20137.48 ; 7.39 ; 7.24 ; 7.12 ; 6.89 ; 6.08 ; 5.69 ; 2.93 ; 1.46 ; 13C-NMR : 196.8; 154.9; 152.2; 151.4; 148.4; 129.1; 129.4; 129.4; 125.5; 125.0; 121.7; 121.7; 108.2; 108,0; 102.0; 54.7; 29.8; 13.7; HR-MS (ESI): for C18H17NO5Na [M+Na]+ calculated: 350.1004 m/z, found: 350.0998 m/z.Benzyl ethyl(1-oxo-1-phenylpropan-2-yl)carbamate (23). Yield 82%; 1H-NMR : 8.01 ; 7.81 ; 7.60\u20137.26 ; 5.76 ; 5.31\u20135.05 ; 3.32\u20133.10 , 1.43 ; 1.05\u20130.98 ; 13C-NMR : 199.3; 156.2; 136.7 135.5; 133.3; 128.7; 128.7; 128.5; 128.5; 128.5; 128.5; 128.0; 127.6; 127.6; 67.4; 55.4; 38.7; 15.5; 14.6; HR-MS (ESI): for C19H21NO3 [M+H]+ calculated: 312.1600 m/z, found: 312.1645 m/z.Benzyl methyl(1-oxo-1-phenylbutan-2-yl)carbamate (24). Yield 87%; 1H-NMR : 8.03-7.87 ; 7.62 ; 7.52\u20137.29 ; 5.52 ; 5.29\u20135.10 ; 2.76 ; 1.99\u20131.73 ; 0.98-0.92 ; 13C-NMR : 198.4; 156.4; 137.3 136.1; 133.1; 128.6; 128.6; 128.4; 128.4; 128.2; 128.2; 127.8; 127.5; 127.5; 66.8; 60.7; 29.6; 20.5; 9.8; HR-MS (ESI): for C19H21NO3Na [M+Na]+ calculated: 334.1419 m/z, found: 334.1418 m/z.Benzyl ethyl(1-oxo-1-phenylbutan-2-yl)carbamate (25). Yield 83%; 1H-NMR : 8.10-7.83 ; 7.60-7.26 ; 5.62 ; 5.22  3.14 ; 2.07\u20131.72 ; 0.99 ; 13C-NMR : 198.8; 156.7; 136.7; 135.9; 133.3; 128.7; 128.7; 128.5; 128.5; 128.5; 128.5; 128.0; 127.6; 127.6; 67.5; 60.3; 38.3; 21.7; 15.1; 10.5; HR-MS (ESI): for C20H23NO3 [M+H]+ calculated: 326.1756 m/z, found: 326.1763 m/z.Benzyl methyl(1-oxo-1-phenylpentan-2-yl)carbamate (26). Yield 80%; 1H-NMR : 8.06-7.82 ; 7.56 ; 7.48\u20137.27 ; 5.60 ; 5.33-5.12 ; 2.78 ; 1.93-1.72 ; 1.43-1.30 ; 0.99 ; 13C-NMR : 199.0; 156.7; 136.7; 135.7; 133.3; 128.7; 128.7; 128.5; 128.5; 128.3; 128.3; 128.0; 127.6; 127.6; 67.5; 58.5; 29.7; 29.3; 19.1; 13.9; HR-MS (ESI): for C20H23NO3Na [M+Na]+ calculated: 348.1576 m/z, found: 348.1574 m/z.Benzyl methyl(1-oxo-1-(m-tolyl)propan-2-yl)carbamate (27). Yield 81%; 1H-NMR : 7.83\u20137.58 ; 7.41\u20137.18 ; 5.63 ; 5.29-5.10 ; 2.80 ; 2.36 ; 1.41 ; 13C-NMR : 199.4; 156.2; 138.5; 136.7; 135.4; 134.1; 128.9; 128.5; 128.5; 128.5; 128.5; 128.0; 127.6; 125.6; 67.4; 55.2; 29.4; 21.3; 13.5; HR-MS (ESI): for C19H21NO3Na [M+Na]+ calculated: 334.1419 m/z, found: 334.1419 m/z.Benzyl (1-(3-chlorophenyl)-1-oxopropan-2-yl)(methyl)carbamate (28). Yield 91%; 1H-NMR : 8.00\u20137.86 ; 7.84\u20137.63 ; 7.56\u20137.49 ; 7.47\u20137.44 ; 7.42\u20137.23 ; 5.55 ; 5.27\u20135.11 ; 2.79 ; 1.41 ; 13C-NMR : 198.0; 156.2; 136.9; 136.5; 136.1; 135.0; 133.2; 130.0; 128.4; 128.4; 128.7; 127.7; 127.7; 126.5; 67.6; 55.5; 29.5; 13.3; HR-MS (ESI): for C18H18ClNO3Na [M+Na]+ calculated: 354.0873 m/z, found: 354.0863 m/z.Benzyl (1-(4-methoxyphenyl)-1-oxopropan-2-yl)(methyl)carbamate (29). Yield 79%; m.p.: 48\u201350 \u00b0C; 1H-NMR : 7.91 ; 7.43\u20137.24 ; 6.85 ; 5.67 ; 5.36-5.08 ; 3.87 ; 2.77 ; 1.42-1.35 ; 13C-NMR : 197.3; 163.7; 156.2; 136.7; 130.9; 130.9; 128.5; 128.5; 128.2; 128.0 127.7; 127.7; 113.9; 113.9; 67.4; 55.4; 54.6; 29.2; 13.4; HR-MS (ESI): for C19H21NO4 [M+H]+ calculated: 328.1549 m/z, found: 328.1537 m/z.Benzyl methyl(1-oxo-1-(p-tolyl)propan-2-yl)carbamate (30). Yield 84%; m.p.: 62\u201363 \u00b0C; 1H-NMR : 7.89-7.74 ; 7.42-7.21 ; 5.60 ; 5.24-5.04 ; 2.86\u20132.76 ; 2.07 ; 1.43-1.33 ; 13C-NMR : 198.1; 155.7; 143.8; 137.3; 133.2; 129.2; 129.2; 128.4; 128.4; 128.3; 128.3; 127.8; 127.5; 127.5; 66.8; 55.6; 30.0; 20.7; 12.8; HR-MS (ESI): for C19H21NO3Na [M+Na]+ calculated: 334.1419 m/z, found: 334.1419 m/z.Benzyl (1-(4-chlroophenyl)-1-oxopropan-2-yl)(methyl)carbamate (31). Yield 90%; m.p.: 72\u201373 \u00b0C; 1H-NMR : 7.90 ; 7.47 ; 7.40\u20137.27 ; 5.53 ; 5.22\u20135.02 ; 2.87\u20132.76 ; 1.39 ; 13C-NMR : 197.6; 155.7; 138.5; 137.2; 134.6; 129.9; 129.9; 128.7; 128.7; 128.4 128.4; 127.9; 127.9; 127.5; 66.8; 56.3; 29.7; 12.5; HR MS (ESI): for C18H18 ClNO3Na [M+Na]+ calculated: 354.0873 m/z, found: 354.0864 m/z.Benzyl (1-(4-bromophenyl)-1-oxopropan-2-yl)(methyl)carbamate (32). Yield 92%; m.p.: 63\u201364 \u00b0C; 1H-NMR : 7.90\u20137.74 ; 7.63 ; 7.40\u20137.27 ; 5.57\u20135.45 ; 5.22\u20135.02 ; 2.82 ; 1.39 ; 13C-NMR : 197.8; 155.7; 137.2; 134.9; 131.7; 131.7; 129.9; 129.9; 128.4; 128.4; 127.8; 127.5; 127.5; 127.2; 66.8; 56.3; 29.8; 12.5; HR-MS (ESI): for C18H18BrNO3 Na [M+Na]+ calculated: 398.0368 m/z, found: 398.0367 m/z.Benzyl -1-oxopropan-2-yl)(methyl)carbamate (33). Yield 79%, 1H-NMR : 7.78\u20137.59 ; 7.41\u20137.28 ; 7.21 ; 5.61 ; 5.15  2.88-2.74 ; 2.29 ; 2.07  1.41-1.33 ; 13C-NMR : 198.2; 155.7; 142.5; 137.3; 136.9; 133.6; 129.7; 129.7; 129.3; 128.4; 128.4; 127.8; 127.5; 125.9; 66.7; 55.5; 30.0; 19.1; 18.9; 12.9; HR-MS (ESI): for C20H23NO3Na [M+Na]+ calculated: 348.1576 m/z, found: 348.1575 m/z.Benzyl -1-oxopropan-2-yl)(methyl)carbamate (34). Yield 79%; m.p.: 60\u201361.5 \u00b0C; 1H-NMR : 7.67 ; 7.51-7.26 ; 6.85-6.64 ; 6.06 ; 5.58 ; 5.33-5.12  2.78 ; 1.44-1.33 ; 13C-NMR : 197.0; 156.2; 152.0; 148.2; 136.6; 130.0; 128.5; 128.5; 128.0; 127.7; 127.7; 125.0; 108.3; 108.1; 101.8; 67.5; 54.6; 29.2; 13.6; HR MS (ESI): for C19H19NO5 [M+H]+ calculated: 342.1342 m/z, found: 342.1354 m/z.Methyl methyl-1-oxopropan-2-yl)carbamate (35). Yield 69%; 1H-NMR : 8.41 ; 8.00 ; 7.93\u20137.87 ; 7.63\u20137.46 ; 5.80-5.29 , 3.68 ; 2.87 ; 1.53-1.42 ; 13C-NMR : 203.7; 157.1; 134.2 134.0; 132.5; 130.5; 128.5; 127.8; 127.2; 126.4; 125.3; 124.5; 58.0; 52.9; 30.0; 13.6; HR-MS (ESI): for C16H17NO3Na [M+Na]+ calculated: 294.1106 m/z, found: 294.1107 m/z.Phenyl -1-oxopropan-2-yl)(methyl)carbamate (36). Yield 68%; 1H-NMR : 8.38 ; 7.94 ; 7.65-7.44 ; 7.41-7.22 ; 5.63 ; 2.95\u20132.82 ; 1.50\u20131.45 ; 13C-NMR : 203.5; 156.4; 136.6; 134.2; 134.0; 132.5; 130.6; 128.5; 128.5; 128.5; 128.5; 127.9; 127.8; 127.6; 127.2; 126.4; 125.3; 124.5; 58.2; 30.2; 13.6; HR-MS (ESI): for C21H19NO3 [M+H]+ calculated: 334.1443 m/z, found: 334.1450 m/z.Phenyl -1-oxopropan-2-yl)(methyl)carbamate (37). Yield 71%; m.p.: 85-87 \u00b0C; 1H-NMR : 8.50-8.35 ; 7.87 ; 7.72\u20137.58 ; 7.39\u20137.18 ; 7.06\u20136.92  5.66 ; 3.03 ; 1.54 ; 13C-NMR : 202.8; 155.2; 151.3; 136.7; 133.4; 131.7; 131.3; 129.3; 129.3; 128.6; 127.6; 127.0; 125.7; 125.5; 125.0; 124.8; 121.6; 121.6; 58.6; 30.9; 13.6; HR-MS (ESI): for C21H18ClNO3 [M+H]+ calculated: 368.1053 m/z, found: 368.1067 m/z.Phenyl -1-oxopropan-2-yl)(methyl)carbamate (38). Yield 72%; m.p.: 103\u2013104 \u00b0C; 1H-NMR : 8.46\u20138.30 ; 7.88-7.83 ; 7.72\u20137.56 ; 7.39\u20137.19 ; 7.06\u20136.91 ; 5.64 ; 3.03 ; 1.54 ; 13C-NMR : 203.0; 155.2; 151.2; 134.3; 132.5; 131.6; 129.3; 129.3; 128.8; 128.6; 127.9; 127.8; 127.1; 126.2; 125.7; 125.5; 121.6; 121.6; 58.7; 30.9; 13.6; HR MS (ESI): for C21H18BrNO3Na [M+Na]+ calculated: 434.0368 m/z, found: 434.0352 m/z.Benzyl methyl-1-oxopropan-2-yl)carbamate (39). Yield 68%; 1H-NMR : 8.48\u20138.27 ; 7,.; 7.65\u20137.45 ; 7.41\u20137.22 ; 5.63 ; 5.24\u20135.02 ; 2.89 ; 1.48 ; 13C-NMR : 203.6; 156.4; 136.6; 134.2; 134.0; 132.6; 130.5; 128.5; 128.5; 127.9; 127.8; 127.8; 127.6; 127.6; 127.2; 126.4; 125.3; 124.5; 67.4; 58.2; 30.2; 13.6; HR-MS (ESI): for C22H21NO3 [M+H]+ calculated: 348.1600 m/z, found: 348.1608 m/z.Benzyl -1-oxopropan-2-yl)(methyl)carbamate (40). Yield 72%; m.p.: 65\u201366 \u00b0C; 1H-NMR : 8.50\u20138.28 ; 7.87 ; 7.70\u20137.46 ; 7.43-7.19 ; 5.55 ; 5.26-4.98 ; 2.88 ; 1.48 ; 13C-NMR : 202.9; 156.3; 136.5; 136.4; 133.3; 131.7; 131.3; 128.7; 128.5; 128.5; 128.5; 128.5; 128.0; 127.6; 126.9; 126.2; 125.8; 124.9; 67.4; 58.3; 30.3; 13.4; HR-MS (ESI): for C22H20ClNO3Na [M+Na]+ calculated: 404.1029 m/z, found: 404.1014 m/z.Benzyl -1-oxopropan-2-yl)(methyl)carbamate (41). Yield 73%; m.p.: 59\u201360 \u00b0C; 1H-NMR : 8.47\u20138.22 ; 7.78 ; 7.70\u20137.51 ; 7.44\u20137.16 ; 5.54 ; 5.26-4.96 ; 2.88 ; 1.48 ; 13C-NMR : 203.0; 156.3; 136.5; 134.1; 132.5; 131.6; 128.7; 128.7; 128.5; 128.5; 128.5; 128.5; 128.0; 127.8; 127.7; 127.6; 127.0; 125.8; 67.4; 58.4; 30.4; 13.4; HR-MS (ESI): for C22H20BrNO3 Na [M+Na]+ calculated: 448.0524 m/z, found: 448.0512 m/z.Electrophorus electricus) and BChE from equine serum  was determined in vitro using a modified Ellman\u2019s method. The effectiveness of the inhibitors, which are expressed as the IC50 values, represent the concentration of an inhibitor that was required for reduction of enzyme activity (or reaction rate) to 50% . The Ellman\u2019s method is widely used for measuring cholinesterase activity and the effectivity of ChEIs [The ability of all the prepared compounds to inhibit AChE from electric eel , where v0 is the reaction rate of an uninhibited reaction and iv is the reaction rate of an inhibited reaction (for a given concentration of the inhibitor). First, v0 was determined. PBS , DTNB and ATCh were put into the cuvette. The enzymatic reaction was started by adding the enzyme. The dependence of absorbance (\u03bb = 412 nm) on time was observed for 70 s , and then the reaction rate (v0) was calculated (v = \u0394A/\u0394t). The measurement was performed at least in triplicate, and average v0 was determined. Then, vi (for a given concentration of the inhibitor) was determined. DTNB, ATCh and the selected volume of a suitably diluted inhibitor  and a certain volume of PBS  were put into the cuvette. The enzymatic reaction was started by adding the enzyme. The dependence of absorbance (\u03bb = 412 nm) on time was observed for 70 s (the reference solution was the same as for uninhibited reaction), and then the reaction rate (vi) was calculated. To determine the IC50 values, twelve different concentrations of the inhibitor were used and each measurement was performed at least in triplicate. Finally, the dependence of v0/vi on the concentration of the inhibitor was determined, and the IC50 values were calculated from the obtained equation of the regression curve for y = 2  [All of the examined compounds were dissolved in DMSO (concentration 0.01 M) and diluted in demineralized water (concentrations 0.001 M and 0.0001 M). The ability of the tested compounds to inhibit AChE (from electric eel) and BChE (from equine serum) was determined using a modified Ellman\u2019s method at 25 \u00b0C in the presence of phosphate buffered saline  in a glass cuvette with a 1 cm optical path. The enzyme activity in the total reaction mixture (2 mL) was 0.2 U/mL, the concentration of the substrate ATCh 40 \u03bcM and the concentration of DTNB 0.1 mM for all of the reactions. The IC0 value) . The obtx, y and z coordinates with a three-element weight vector that describes each neuron. The shape of a specific molecular surface (template) that is encoded in the weights of the trained Kohonen network can be used to process the signals coming from the surface of the other molecule(s) (counter-template), thereby producing a series of comparative SOM maps can be used to compare/contrast the superimposed molecular geometry. Self-organizing neural mapping (SOM) is regarded as being a nonlinear projection tool that decreases the dimensionality of the input object, e.g., converts 3D objects to 2D, while preserving the topological relationships between the input and output data. Moreover, a trained network can be used to project the specified molecular property (expressed as a vector) by generating a 2D color-coded clustering pattern that is called a feature map. Hence, the SOM algorithm was used to generate an electrostatic potential map in the form of a 2D topographic pattern produced from input signals (points) that were sampled randomly at the molecular surface . In thisApplying SOMs to compress/visualize/classify the structural data has been widely reported, e.g., for the 2D mapping of the electrostatic potential on 3D molecular surfaces or partial atomic charges for atomic molecular representation .y and the set of predictors X in a form that is represented by the following equation:b is the vector of the regression coefficients and e is the vector of the errors. Generally, PLS models are constructed for centered/autoscaled data, and their complexity is estimated using, e.g., the leave-one-out cross-validation (LOO-CV) procedure. The cross-validated obs is the observed value; pred is the predicted value; mean is the mean value of obs; and i refers to the object index, which ranges from 1 to m.The set of CoMSA shape/electronic descriptors is subsequently processed using the PLS method and expresses the relationship between variable n is the number of objects in a test set.The quality of the external predictions was measured using the standard deviation of error of the prediction (SDEP), mean(b)/s(b) ratio, where s(b) represents the standard deviation of the regression coefficient b [Redundant variables can impede the interpretation of a model by increasing its complexity; therefore, reducing the number of variables is advisable. The iterative variable elimination (IVE-PLS) procedure, which is a modification of the UVE-PLS algorithm  has been proposed, to analyze the stability of the regression coefficients, which are expressed as the icient b . GeneralA molecule might be encoded by an ensemble of structural (S) and physicochemical (P) properties that are organized in a vector, which represents an object in the chemical space (CS). The distribution of the empirically (FCS) and virtually (VCS) produced compounds can be visually scrutinized using, e.g., a linear projection procedure called Principal Component Analysis (PCA). PCA is regarded as being a classical method to explore data that permits the data dimensionality to be reduced, visualized and the relationships between the objects (molecules) and parameters (descriptors) to be interpreted. On the condition that the reduction of the data dimensionality is efficient, it is possible to capture interesting information about the data structure  to indicate the importance of the original data variables that contribute to the observed structure, and finally, to illustrate and interpret the relationships between the objects and the parameters in the X matrix  ,57.50) for the set of carbamate derivatives are listed in The same laboratory was employed to specify all of the pharmacological data to eliminate any potential data noise that might have been introduced by pooling the datasets from various sources. The in vitro AChE and BChE inhibition values  using the atom FIT method, which is based on matching the positions of the atoms between the corresponding atom pairs.The majority of the modeling studies were performed with a Sybyl-X 2.0/Certara software package running on a HP workstation with a Debian 6.0 operating system. The standard Tripos force field  with a 0.01 kcal/mol energy gradient convergence criterion and a distant dependent dielectric constant was used to optimize the initial geometry of each compound (MAXMIN2 module). For the electrostatic potential calculations, the Gasteiger\u2013H\u00fcckel method (implemented in Sybyl-X) was initially used to produce the partial atomic charges. One 13-ordered atom trial alignment on 32 was selected to form the template molecules. The output maps were subsequently transformed into a 400\u20132500-element vector, which was used by the PLS method implemented in the MATLAB programming environment. The SONNIA software was used in the CoMSA analysis to simulate 20 \u00d7 20 to 50 \u00d7 50 SOMs with a winning distance that varied within a range of 0.2\u20132.0. The Cartesian coordinates of the molecular surfaces for the superimposed molecules were produced by a SOM network to form a 2D map of the electrostatic potential. Pretty active against AChE and BChE The crystallographic structure of BChE, which contained one amino acid chain and rivastigmine analog molecule, was retrieved from the PDB repository (code 6eul). Only the 1,2-ethanediol molecule was retained, because it was specified as being valid in the enzyme active site AC2. The rest of the heteroatoms, including the crystallographic waters, were extracted prior to the calculations. The ligand/protein structures for the docking study were prepared in the pdbqt file format with the Gasteiger charges calculated. During the AutoDock simulation, various poses (default nine) were generated progressively from a single conformer (an energy-optimized molecule) by applying a collection of the preferred torsion angles to the rotatable bonds and were evaluated using the united-atom scoring function. All of the predicted binding 2D/3D modes, including the positions of the flexible side chains, were visualized using PyMol, Maestro and VMD molecular graphics viewers and the Protein-Ligand Interaction Profiler (PLIP).1H NMR spectroscopy and HRMS. All 41 of the tested compounds were evaluated for their in vitro ability to potentially inhibit AChE and BChE, respectively. The selectivity index of the individual molecules to cholinesterases was also specified. Roughly speaking, a rather moderate inhibitory effect against AChE was revealed; however, some of the compounds  proved to be very selective for the BChE enzyme. In fact, two compounds (23 and 28) had a very high selectivity index (SI = 13.93 and 15.31). Specifically, compound 28 had the lowest IC50 value revealing an approximately seven-fold higher inhibitory activity against BChE than RIV (IC50 = 5.51 vs. 38.40 \u03bcM), which corresponds quite well with GLT (IC50 = 2.77 \u03bcM). It was observed that the methyl-substituted carbamates generally exhibited a lower inhibitor ability compared to their phenyl or benzyl counterparts. Interestingly, compounds 17 and 29, which had a methoxy group in the same position, were weaker inhibitors\u2014the presence of the hydrophilic electron-donating -OCH3 substituent of the phenyl ring at the para-position decreased the potency of the compounds, especially against the BChE enzyme. On the other hand, the presence of methyl group(s) in the meta/para-position(s) of the phenyl ring  resulted in an improvement of the IC50 value for the BChE enzyme, thus suggesting the significance of a hydrophobic character for the interactions with the enzyme. Placing the electron-withdrawing chlorine substituent in the meta-position of the phenyl ring appeared to be strongly preferable, especially for the BChE inhibition activity (compounds 16 or 28). To partially explain the observed variations in the anti-AChE/BChE potential of the investigated carbamate series, comparative receptor-independent (RI) and receptor-dependent (RD) structure\u2013activity studies were conducted, respectively. The principal purpose of the ligand-based study was to comparatively analyze the molecular surface (CoMSA) to gain insight into the electronic and/or steric factors that govern the ability of the tested compounds to inhibit the AChE/BChE activities. The findings for the surface descriptors were compared with their force field counterparts (CoMFA) when modeling the inhibiting potency for multiple training/test subsets using the stochastic model validation (SMV) procedure. Moreover, a similarity analysis was performed using the PCA approach on the pool of Dragon descriptors. The spatial distribution of the potentially important steric and electrostatic factors on the BChE inhibitory potency was specified using the probability-guided pharmacophore mapping procedure based on the iterative variable elimination (IVE-PLS) method. Finally, a comprehensive scrutiny of the guest\u2013target interactions for the inhibitors that were comparatively active to rivastigmine BChE (IC50 < 30 \u03bcM) was conducted using the site-directed computer-assisted docking methodology. Planar (2D) and spatial (3D) maps of the host\u2013target interactions were created for all of the active compounds and compared with the marketed drug (GLT and RIV) molecules, which generally revealed two types of non-binding interactions\u2014hydrophobic and hydrogen bond formation, respectively. The hydroxyl substituent of Thr120 appeared to be crucial in forming the hydrogen bond with the ether or carbonyl oxygen (1 substituent) seemed to be valid for hydrophobic interactions with the Asp70 amino acid residue, which is in line with ligand-based findings. Regrettably, a clear explanation of the variations that the meta/para-positioned carbamate derivatives exerted on the BChE reaction site was not revealed; however, some regularities were observed in the ligand\u2013receptor interaction pattern. It appeared that the close proximity of the positively charged nitrogen atom of His438 may potentially be beneficial to the inhibition potential, especially the negatively charged chlorine- or bromine-based carbamates, which corresponds relatively well with the IVE-PLS CoMSA results. The electrostatic repulsion between the negatively charged atoms and the oxygen of Ser79 can partially explain the detrimental impact of the \u2013OCH3 or \u2013OCH2O- groups that were attached to the phenyl ring. On the other hand, the postulated hydrophobic interactions with Phe329 can favorably contribute to the inhibitory potential, as was observed for (di)methyl derivatives. To summarize, a series of novel benzene-based derivatives was designed, synthesized and characterized using l oxygen c. For thThe combination of consensus pharmacophore mapping with a systematic screening of the multifaceted guest\u2013host interactions using target-tailored approaches seems to be the path towards an intelligent drug design system."}
+{"text": "Bifurcaria bifurcata, due to their abundance in bioactive linear diterpenes. In this appraisal, a thorough review concerning the methodologies used in the extraction, fractionation, and identification of diterpenes from B. bifurcata is provided and discussed in detail. An exhaustive compilation of the mass spectra and nuclear magnetic resonance (NMR) data are also provided. The in vitro and in chemico assays already performed to assess different biological activities attributed to B. bifurcata diterpenes are also reviewed, emphasizing the use of isolated components, enriched fractions, or crude extracts. The associated major strengths and challenges for the exploitation of B. bifurcata diterpenes for high-value applications are critically discussed. Marine resources are considered as a very promising source of bioactive molecules, and macroalgae in particular have gained special attention, due to their structurally diverse composition. Particular interest has been devoted to the brown macroalga  Marine resources have been seen as a promising source of added value molecules, an alternative of finite fossil resources, allowing for the boost of concepts such as biorefinery, circular economy, and blue economy ,2,3. In Macroalgae are ecologically and commercially important, being significant primary producers in oceanic aquatic foods chains . These mSpecial attention has been devoted to brown macroalgae in particular, due to the presence of specific components, such as fucoidan, phlorotannins, or even fucoxanthin, for which promising bioactivities have been described, namely antitumor, antioxidant, and antihypertensive activities, among others ,11,12.Bifurcaria bifurcata species + at m/z 306 representing the chemical formula C20H34O2 and also other characteristic product ions, namely [M \u2212 H2O]+ at m/z 288 +) at m/z 327.23193, indicating the molecular formula C20H32O2 + at m/z 278, and also some major product ions at m/z 43 ([C3H7]+), 57 ([C4H9]+), 68, 82, 95 ([C7H11]+), 109, and 123 ([C9H15]+), were observed. In the same way, geranylgeraniol (39) mass spectrum presented the molecular ion [M]+ at m/z 290, and characteristic products ions at m/z 69 ([C5H9]+), 81 ([C6H9]+), 121 ([C11H19]+), 272 ([M \u2212 H2O]+)+).B. bifurcata is systematized in A more detailed discussion of the spectroscopic features of these compounds is beyond the scope of the present review, however its availability in a systematized way is undoubtedly important for the readers in the field. Therefore, an exhaustive compilation of NMR and MS data for linear diterpenes from B. bifurcata linear diterpenes-enriched extracts has increased in recent years due to the vast range of biological activities already associated with these compounds. Despite being very rare in nature, they are present in high amounts in B. bifurcata lipidic extracts, as shown above. An overview of the biological activities already reported for B. bifurcata linear diterpenes-enriched extracts or purified components is present in B. bifurcata and those involving B. bifurcata extracts previously found to be rich in linear diterpenes were considered).The interest in B. bifurcata linear diterpenes and extracts through in vitro or in chemico assays. Therefore, the future development of in vivo assays to corroborate these results may be crucial to their exploitation for high-value applications. Most of the studies have evaluated the biological activities of 2O extract of B. bifurcata showed antimicrobial activity against Mycobacterium smegmatis [15), which also inhibited the bacteria growth at 75 \u03bcg mL\u22121 ), as well as that of Bacillus subtilis (MIC = 2.5 mg mL\u22121), Mycobacterium aquae (MIC = 400 \u03bcg mL\u22121), Mycobacterium ranae (MIC = 100 \u03bcg mL\u22121), Mycobacterium xenoqui (MIC = 200 \u03bcg mL\u22121), and Mycobacterium avium (MIC = 100 \u03bcg mL\u22121) [9) and eleganolone (15) against a strain of Bacillus sp. (MIC = 8 \u03bcg mL\u22121) [Biard et al. firstly reported that the Etmegmatis  Subsequeui MIC = 0 \u03bcg mL\u22121\u03bcg mL\u22121) .15) derivatives, -16-hydroxy-2,10,14-trimethyl-6-methylenehexadeca-2,10,14-triene-4,7-dione (22) and -16-hydroxy-2,6,14-trimethyl-10-methylenehexadeca-2,6,14-triene-4,11-dione (25), against a gram-positive bacteria, namely Bacillus sp. (MIC = 8 \u03bcg mL\u22121), as well as against marine fungi, namely Corollospora maritima, Lulworthia sp., and Dendryphiella salina, MIC = 8 \u03bcg mL\u22121 [Furthermore, Hellio et al. reported the antimicrobial activity of two eleganolone ( \u03bcg mL\u22121 .B. bifurcata extract rich in linear diterpenes (48.29 mg g\u22121 of extract) exhibited antibacterial activity against both gram-positive  and ATCC\u00ae43300 (MIC = 2048 \u03bcg mL\u22121)) and gram-negative ) bacteria. In addition, this extract was shown to reinforce the antimicrobial activity of several antibiotics. The combination of the extract with gentamicin or tetracycline resulted in a severe decrease of the antibiotic MIC against the three strains. Rifampicin MIC also decreased about 87% and 50% in the presence of the extract toward Staphylococcus aureus ATCC\u00ae43300 and Escherichia coli ATCC\u00ae25922, respectively. Thus, this synergism of B. bifurcata extracts with antibiotics could be further exploited in a way to overcome the antibiotic-resistant bacteria strains, which is a serious public health problem [Santos et al. demonstrated that a DCM B. bifurcata. Both extracts were revealed to be active against Escherichia coli ATCC 10536 , while MeOH extract was also shown to inhibit Staphylococcus aureus ATCC 25923 (7.0 \u00b1 0 mm ZI) [Bacillus subtilis ATCC 6633 (6.7 \u00b1 0.6 mm ZI) and Escherichia coli ATCC 25922 (6.7 \u00b1 0.6 mm ZI) [B. bifurcata extracts was also demonstrated against another gram-negative bacteria, Pseudomonas aeruginosa ATCC 27853 , and toward Saccharomyces cerevisiae ATCC 9763, which was expressed in the inhibitory concentration of the extract required to decrease microbial concentration by 50% (IC50), IC50 = 26.68 \u03bcg mL\u22121 and IC50 = 17.06 \u03bcg mL\u22121, for MeOH and DCM extracts, respectively [Pseudomonas aeruginosa ATCC 27853 in the MeOH extract was observed while the DCM extract showed a higher antifungal activity against Saccharomyces cerevisiae ATCC 9763 [Alves et al. studied the antibacterial activity (expressed in zone of inhibition (ZI)) of MeOH and DCM extracts of 0 mm ZI) . The MeO6 mm ZI) . In factectively . AccordiTCC 9763 .B. bifurcata. Protozoal infections, in particular malaria, leishmaniosis, and sleeping sickness, among others, are fatal diseases [14), obtained after several purification steps [Plasmodium falciparum (IC50 = 0.65 \u00b1 0.05 \u03bcg mL\u22121), with a good selectivity, and protozoal activity against Trypanosoma brucei rhodesiense (IC50 = 11.8 \u00b1 0.01 \u03bcg mL\u22121), Trypanosoma cruzi (IC50 = 47.8 \u00b1 0.59 \u03bcg mL\u22121), and Leishmania donovani (IC50 = 18.8 \u00b1 0.12 \u03bcg mL\u22121) [15) was responsible for the antimalarial  activity of the extract toward Plasmodium falciparum (IC50 = 2.6 \u03bcg mL\u22121), with a good selectivity index (SI = 21.6). However, this component presented lower trypanocidal activity against Trypanosoma brucei rhodesiense (IC50 = 13.7 \u03bcg mL\u22121) and Trypanosoma cruzi (IC50 = 17.7 \u03bcg mL\u22121) than the crude extract, which exhibited a promising trypanocidal activity with a mild selectivity index  = 11.6) [B. bifurcata extracts enriched in linear diterpenes may be important considering the gaps associated with sleeping sickness (trypanosomiasis) therapy, namely the fact that available drugs are outdated, causing severe adverse reactions [Antiprotozoal activity has been one of the most studied biological activities for linear diterpenes from diseases . Recentlon steps . This lidex SI = .6. Howevdex SI = .6. HowevB. bifurcata. Antiprotozoal activity of EtOAc:MeOH soluble extract against Leishmania donovani (IC50 = 6.4 \u03bcg mL\u22121) was also shown [w/v) extract was shown to be the most active against amastigotes of this strain, presenting an IC50 of 3.8 \u03bcg mL\u22121 [Plasmodium falciparum (100% of growth inhibition (GI) at 9.7 \u03bcg mL\u22121) and Trypanosoma cruzi trypamastigostes (78% of GI at 9.7 \u03bcg mL\u22121). These activities seem to be induced by toxicity [B. bifurcata extract against Trypanosoma brucei rhodeisense (IC50 = 1.9 \u03bcg mL\u22121), Trypanosoma cruzi (IC50 = 34.7 \u03bcg mL\u22121), and Mycobacterium tuberculosis (MIC = 64.0 \u03bcg mL\u22121) (antitubercular activity) was also reported [Leishmaniosis, which is a common disease worldwide, affecting several mammal species, including humans , has beeso shown . Althoug \u03bcg mL\u22121 . Vonthrotoxicity . Antiproreported .B. bifurcata extracts with high contents of linear diterpenes. The exploitation of this capacity of B bifurcata diterpenes could be an environmentally safe alternative for modern marine engineering and shipping operations, for offshore structures, and for aquaculture equipment [Antifouling activity has been also proven in quipment ,36. In fquipment , with thquipment ,36. Bactquipment . 2O B. bifurcata extract, in which two linear diterpenes were identified, was also tested against Balanus amphitrite cyprid, and was shown to be highly active  \u03bcg mL\u22121), whereas eleganolone (15)  \u03bcg mL\u22121) and eleganediol (9)  \u03bcg mL\u22121) were shown to be moderate and low active, respectively. This study also proved that B. bifurcata extracts are toxic against Balanus amphitrite nauplius II after 24 h of treatment  = 0.64  \u03bcg mL\u22121)). Compounds 9  \u03bcg mL\u22121) and 15  \u03bcg mL\u22121) showed lower toxicity [9 and 15 were also studied against a strain of Polibacter sp.  and of Paracoccus sp. , with the component 15 being more active toward these two strains [The antifouling activity of an Et strains ,44.B. bifurcata linear diterpenes, considering the biofouling promoted by macroalgae and blue mussel, were also evaluated. The antifouling activity of eleganediol (9) was tested by Hellio et al., who proved that this linear diterpene is able to inhibit macroalgal spore and zygote development, particularly of Enteromorpha intestinalis (65.2% \u00b1 1.2% GI) and Sargassum muticum (81.5% \u00b1 3.4% GI), and to inhibit diatom growth (Amphora coffeaformis (37.3% \u00b1 1.9% GI), Phaeodartylum tricornutum (42.8% \u00b1 1.5% GI), and Cylindrotheca closterium (39.5% \u00b1 1.6% GI)) [B. bifurcata, such as 12-(S)-hydroxygeranylgeraniol (1) and geranylgeraniol (39) also showed antifouling activity toward macroalgal spores and zygotes. Compound 1 was shown to inhibit the development of Sargassum muticum (72.0% \u00b1 2.2% GI) spore and zygote, whereas compound 39 was active against Enteromorpha intestinalis (67.3% \u00b1 2.1% GI) and Ulva lactuca (83.1% \u00b1 3.4% GI), as well as inhibiting the blue mussel Mytilus edulis adhesion (77.0% \u00b1 2.8% inhibition of phenoloxidase activity (IPA)) [The antifouling activity of .6% GI)) . Other ly (IPA)) .B. bifurcata Et2O extracts  against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina (MIC: 12.2 \u00b1 0.4 to 26.3 \u00b1 1.3 \u03bcg mL\u22121) and Pseudoalteromonas haloplanktis (MIC: 6.5 \u00b1 0.6 to 15.5 \u00b1 0.8 \u03bcg mL\u22121) [Mar\u00e9chal et al. studied the seasonal variation on the antifouling activity of crude \u03bcg mL\u22121) .B. bifurcata from different sampling sites was also tested. An extract of B. bifurcata from Quiberon, France was found to be active against Ulva lactuca spore and zygote development (78.7% \u00b1 2.8% GI), whereas the Port Sall (France) and Oualidia (Morocco) B. bifurcata extracts inhibit the growth of diatoms (Amphora coffeaformis (83.9% \u00b1 4.2% GI), Phaeodartylum tricornutum (86.2% \u00b1 3.4% GI), Cylindrotheca closterium (69.2% \u00b1 2.4% GI)) and inhibit the adhesion of the blue mussel Mytilus edulis (80.6% \u00b1 2.3% IPA), respectively [Furthermore, the antifouling activity of ether extracts of ectively .E,10E,12R)-3,11,15-trimethyl-7-methylenehexadeca-2,10,14-triene-1,6,12-triol (6), and -3,7,11,15-tetramethylhexadeca-2,10,14-triene-1,7,12-triol (7) isolated from B. bifurcata were evaluated. These linear diterpenes were shown to be active against the NSCLC-N6 cell line , presenting IC50 values of 12.3 and 9.5 \u03bcg mL\u22121, respectively) [9) and bifurcane (10), isolated from a B. bifurcata extract, showed anti-proliferative activity at 100 \u03bcg mL\u22121, toward MDA-MB-231 tumor cells (mammary gland/breast adenocarcinoma), resulting in 1.8% and 2.9% of cell viability, respectively [The cytotoxic activity of two bifurcadiol derivatives ((2ctively) . Eleganeectively .B. bifurcata extracts. Zubia et al. tested the cytotoxic activity of DCM:MeOH extracts of this brown macroalga against three tumoral cell lines , which resulted in, approximately, 40% of cell viability reduction with Daudi and K562 and about 20% of cell viability reduction with Jurkat cell lines [B. bifurcata extracts. In this research, Alves et al. showed high cell proliferation inhibition of both DCM and MeOH extracts (at 1 mg mL\u22121) against these tumoral cell lines. The IC50 values obtained with MeOH extract was 437.1 (266.0 \u2013 718.1) \u03bcg mL\u22121 for Caco-2 and 252.0 (162.0 \u2212 392.2) \u03bcg mL\u22121 for HepG-2. Although, DCM extract exhibited promising cell proliferation inhibition and cytotoxicity, against Caco-2 (IC50 = 82.31 (54.7 \u2212 123.8) \u03bcg mL\u22121; IC50 = 90.09 (70.82 \u2212 114.6) \u03bcg mL\u22121) and HepG-2 (IC50 = 95.63 (69.66 \u2212 131.3) \u03bcg mL\u22121; IC50 = 123.9 (95.47 \u2212 160.8) \u03bcg mL\u22121), when compared with the results obtained for cisplatin and tamoxifen, which are two commercial drugs [Some studies also evaluated the in vitro antiproliferative activity of ll lines . Two caral drugs .B. bifurcata MeOH extract rich in diterpenes. This antiproliferative effect is a mechanism of action that overcomes the limited effectiveness of many cycle-dependent anticancer drugs on such slowly developing tumors. After 72 h of treatment with B. bifurcata extract (2.5 \u03bcg L hours\u22121), the cell growth in the G1 phase of the cell cycle was inhibited, and kinetic assays in pretreated cells proved that this growth arrest was irreversible [In addition, Moreau et al. showed irreversible arrest of well-differentiated pathologic cells (such as NSCLC-N6) proliferation induced by a versible .B. bifurcata linear diterpenes and extracts, bioaccessibility and bioavailability assays were not yet performed. In fact, most of the in vitro studies have not used gut cell lines, which allow consideration of the absorption and distribution stages. Thus, other in vitro assays are required in the future to complement these results.Besides these results regarding anti-proliferative activity of B. bifurcata extracts [The prospection of natural components from macroalgae with antioxidant activity has been object of several studies, since oxidative stress is known to be related with several diseases, such as cancer, chronic inflammation, atherosclerosis, and cardiovascular disorder, among others . Furtherextracts ,38,54,55B. bifurcata linear diterpenes and extracts has been evaluated by different assays, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2\u2019-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and oxygen radical absorbent capacity (ORAC). The results of DPPH and ABTS assays are commonly expressed as IC50 values, defined as the inhibitory concentration of the extract required to decrease by 50% the initial radical concentration. Nonetheless, the comparison of the different IC50 values between published studies is not always possible, since the IC50 value depends on the methodology used by each author, as well as on the standards used. For example, Santos et al. used ascorbic acid and 3,5-di-tert-4-butylhydroxytoluene (BHT) as positive control whereas Pinteus et al. used only BHT [The antioxidant potential of only BHT ,54. Desponly BHT ,55.B. bifurcata crude DCM:MeOH extract by three methods: DPPH , reducing activity (90.97% at 500 mg L\u22121), and \u03b2-carotene-linoleic acid system (76.13% of inhibition at 500 mg L\u22121) [B. bifurcata sample of Ria de Aveiro, Portugal, was evaluated by DPPH and ABTS assays, exhibiting IC50 values of 365.57 \u00b1 10.04 \u03bcg mL\u22121 and 116.25 \u00b1 2.54 \u03bcg mL\u22121 for DPPH and ABTS, respectively [50 = 58.82 (50.65 \u2212 68.31) \u03bcg mL\u22121) and DCM (IC50 = 344.70 (246.10 \u2212 482.80) \u03bcg mL\u22121) extracts of a B. bifurcata from Peniche Coast, Portugal, however with MeOH extract showing a higher antioxidant activity than DCM extract [50 value close to that obtained in a similar study [\u22121 extract (ext)) and DCM (589.98 \u00b1 7.33 \u03bcmol TE g\u22121 ext) extracts were determined [Zubia et al. determined the antioxidant activity of  mg L\u22121) . A DCM eectively . Alves e extract . Notwithar study ,54,55. IB. bifurcata extract enriched in linear diterpenes was described by Santos et al., who analyzed a DCM extract from a Portuguese sample [\u22121 [The anti-inflammatory activity of a e sample . The capmple [\u22121 .B. bifurcata samples, namely eleganediol (9) and eleganolone (15), were studied regarding antihypertensive and relaxing activities. These components were isolated from Cystoseira balearica. These pharmacological assays were performed on different guinea pig intestinal preparations, and allowed researchers to identify bioactive effects of compounds 9 and 15, such as blocking the isoprenaline inotropic activity and inhibiting the contractile activities of acetylcholine  and histamine  on ileum musculature. In addition, compounds 9 and 15 relaxed, in a dose-dependent manner, the same preparations precontracted with 300 mM BaCl2  or with 600 mM KCl  [Two components widely reported in ctively) ,64.B. bifurcata DCM extract also presents neuroprotective potential. Neuroprotective effects were highlighted in a neurotoxic model induced in a human neuroblastoma cell line (SH-SY5Y), whereas the neuroprotection mechanisms were evaluated by the determination of mitochondrial membrane potential, H2O2 production, among others. After fractionation, the cyclohexane-EtOAc (1:2) fraction was shown to have a promising neuroprotective performance, due to its ability to prevent changes in mitochondrial potential (218.10% \u00b1 14.87% of control), and to induce the reduction of H2O2 levels of production (204.50% \u00b1 15.12% of control), as well as to revert neurotoxic effect on cell viability (about 20%\u201325%). Considering these results, Silva et al. investigated the composition of this fraction, through purification steps, in order to isolate the potential bioactive molecules. Eleganolone (15) and eleganolal (23) were the two major compounds of this fraction. Then, their antioxidant activity was evaluated, demonstrating lower ability to reduce the DPPH radical, when compared to the fraction from which they were isolated. Although, taking into account the results of FRAP and ORAC assays, compounds 15  and 23  expressed antioxidant capacity, since these linear diterpenes exhibited a high potential in reducing peroxyl radicals and have a strong iron reduction capacity, compared to the BHT. In this sense, these components might be promising candidates for further neuroprotection studies [Silva et al. proved that a  studies .The evaluation of biological dose effects should be combined with the respective assessment of toxicity, since this is a crucial parameter to ensure that bioactive compounds and/or extracts are safe for humans.B. bifurcata sample, collected in Brittany, France, was evaluated using sea urchin eggs, which have been considered a model for studies on cell division and embryologic development and commonly used to obtain a general screening of cytotoxicity [Paracentrotus lividus was evaluated through a cytotoxicity activity test [S)-hydroxygeranylgeraniol (1), bifurcane (10), and bifurcanol (41) were active, with EC50 values of 18 \u03bcg mL\u22121, 12 \u03bcg mL\u22121, and 4 \u03bcg mL\u22121, respectively. Whereas, (S)-12-hydroxygeranylgeranic acid (2) and eleganediol (9) exhibited moderate antimitotic activity with EC50 values of 60 \u03bcg mL\u22121 and 36 \u03bcg mL\u22121, respectively [The cytotoxicity of linear diterpenes obtained from a toxicity . The abiity test ,43. The ectively ,43.B. bifurcata were tested by G\u00f6thel et al. [10), bifurcanone (18), eleganonic acid (31), and bibiolone (34) were shown to be inactive (when IC50 > 40 \u00b5g mL\u22121) against mouse fibroblast cell line (L929), whereas eleganolone (15) (IC50 = 22 \u03bcg mL\u22121), -16-hydroxy-2,6,10,14-tetramethylhexadeca-2,6,10,14-tetraen-4-one (21) (IC50 = 18 \u03bcg mL\u22121), eleganolonebutenolide (32) (IC50 = 27 \u03bcg mL\u22121), 14,15-dihydro-eleganonic acid (33) (IC50 = 20 \u03bcg mL\u22121), and bifurcanol (41) (IC50 = 24 \u03bcg mL\u22121) exhibited very low toxicity [15, for example, the cytotoxicity IC50 value was higher than the EC50 found for antifouling activity (against Trypanosoma brucei rhodesiense (IC50 = 13.7 \u00b5g mL\u22121), Trypanosoma cruzi (IC50 = 17.7 \u00b5g mL\u22121), Plasmodium falciparum (IC50 = 2.6 \u00b5g mL\u22121), and Balanus amphitrite cyprid  \u03bcg mL\u22121) [The cytotoxicity of nine linear diterpenes isolated from l et al. . Bifurcatoxicity . In the en-4-one  (IC50 = \u03bcg mL\u22121) . 14), showing that this linear diterpene had cytotoxicity against the L6 rat myoblast cell line, IC50 = 56.6 \u00b1 0.004 \u00b5g mL\u22121 [50 values observed for antimalarial and antiprotozoal activities, compromising, therefore, the use of this compound for such purposes. For instance, Smyrniotopoulos et al.  analyzedB. bifurcata extracts, with both expressed at \u00b5g mL\u22121 [In fact, other studies have shown biological activities and cytotoxicity of  \u00b5g mL\u22121 ,61. NotwIn vitro toxicity assays involving marine bacteria should also be considered in the future, since equations for interspecies dose correlations have already been established, allowing researchers to correlate the bacteria data with rat or mouse toxicity, which avoids mammalian laboratory tests ,66.Finally, the safe use of these components must be verified by additional studies, in particular in vivo assays, considering the different range of possible applications, in order to confirm that they are safe to humans.B. bifurcata linear diterpenes, different studies have already exploited specific applications for these components or enriched extracts. Miranda et al. studied the effect of a preliminary dipping treatment containing a B. bifurcata extract in the fish quality during chilled storage. The choice of this brown macroalga was due to its content on antioxidant and antimicrobial compounds, i.e., diterpenes, among others [B. bifurcata extract, had beneficial effects on fish quality. In fact, the quality during chilled storage is a major concern of the food industry. Wild marine species are often exposed to several handling and technological processes, which determine the quality of the final product [B. bifurcata extract may be a practical application for both on-board and in-land fish storage strategies [Taking into account the diverse biological activities already attributed to g others . Through product . A water product . Thus, trategies . B. bifurcata extract in the green synthesis of copper oxide nanoparticles (CONPs) was also reported. This strategy aimed to overcome some drawbacks of conventional processes , which limit the use of nanoparticles in applications related to clinical fields. The synthesis of CONPs using this marine brown macroalga was focused on \u201cgreen\u201d chemistry and bioprocess approaches [Enterobacter aerogenes) (ZI = 14 mm) and gram-positive (Staphylococcus aureus) (ZI = 16 mm) bacteria [The use of a proaches . In addibacteria .B. bifurcata, including the methodologies of extraction and structural elucidation and the biological activities already associated to these brown macroalga components. A detailed collection of NMR and MS data is provided, which will be of utmost importance in future studies of bioprospection of linear diterpenes from B. bifurcata.Macroalgae are one of the most promising sources of secondary metabolites, which have already been associated to several biological activities. For this reason, an increase in the investment and research on these marine resources has been observed. The focus of this review was the bioprospection of linear diterpenes from B. bifurcata has earned special emphasis, due to their special ability to biosynthesize linear diterpenes, which are rarely found in brown macroalgae species outside the Sargassaceae family. In fact, some studies have proven that these secondary metabolites are responsible for several biological activities, such as antibacterial, antiprotozoal, antifouling, and antitumoral, among others. Linear diterpenes from B. bifurcata, particularly eleganolone (15) and eleganediol (9), showed significant antifouling, antibacterial, and antihypertensive activities in the range of \u00b5g mL\u22121.Notwithstanding, extraction methodologies have been identified as one of the major challenges to develop viable and safe high-value applications, namely in food, nutraceutical, or pharmaceutical fields. The conventional extraction methodologies normally used to extract these compounds involve the use of organic solvents, often toxic to humans and environmentally hazardous. Furthermore, these extraction methodologies also raise other concerns, namely, the long operation times, consumption of high volumes of expensive solvents, and high energetic demand. Thus, new and eco-friendly methodologies should be applied, taking into account their overall feasibility. In particular, emergent extraction methodologies able to use small amounts of organic solvents, or even offering the possibility for their replacement by water, should be considered. B. bifurcata, although there are also studies reporting biological activities of B. bifurcata extracts without isolating the bioactive compounds. The isolation, or at least the fractionation, should be considered in order to enhance the bioactivity and also eliminate some antagonistic effects, for instance, bio-guided fractionation or a faster strategy that has already been tested, known as \u201cpharmacophoric deconvolution\u201d, should be further exploited. Another strategy could be the development and optimization of more selective extraction methodologies. Additionally, before any possible application, the cytotoxicity of the extracts, fractions, or isolated compounds should be evaluated. The purification and/or fractionation steps have been already applied by some authors, frequently for identification purposes, for B. bifurcata linear diterpenes in pharmaceutical, cosmetic, or nutraceutical applications. In addition, the incorporation of these extracts in biomaterials should be considered. Finally, for possible food industry applications, the legislation may be revised in order to evaluate B. bifurcata as an edible species.In vivo assays for the determination of dose effect and the consumer security evaluation may be also considered to confirm the potential of B. bifurcata, although the new advances in these fields should be undoubtably considered in future works. Actually, the increasing development of multi-omics techniques, including genomics, transcriptomics, proteomics, and metabolomics have enabled the creation of libraries and models that can predict the behavior of biological systems as well as allow the optimization of cellular processes in order to produce target compounds. This could be a valuable and key strategy in the exploitation of B. bifurcata as a source of linear diterpenes for high-value applications. Finally, and despite the use of metabolic engineering and synthetic biology in macroalgae\u2019s bioactive components, production is still in its infancy and largely unexploited in"}
+{"text": "A serious crisis has engulfed Brazil in recent years, threatening the access to theuniversal health system and setting back the achievements of the last decades. Thiseditorial aims to present relevant elements, necessary for the sustainability of auniversal system  that has adopted the universal system. The system operates with morethan 3.5 million workers, of which about 50% work in nursing. This category is presentin all Brazilian cities, with even greater prevalence in the public, philanthropic andprivate sectorsIn spite of the considerable number of nursing professionals , it still does not meet the Brazilian social needs and fallsbelow countries such as Canada (9.84), Sweden (11.87), United Kingdom (8.43) and Uruguay(12.49)This context motivated the World Health Organization (WHO) and the United Nations (UN) tolaunch the Nursing Now ProgramOn the situation of the nursing profession in Brazil, based on data collected fromDATASUSThe reality of Brazilian nursing is a reflection of recent policies of austerity, cuts,containment of public expenditures, provisional measures that allow foreign capitalflows, and the Pol\u00edtica Nacional da Aten\u00e7\u00e3o B\u00e1sica itself, which recognizes and financesother modalities of attention other than the Family Health Strategy. In addition, theprivate system and the market economy have repercussions on low salaries, long hours ofwork of the nurse  and occupational stress.We emphasize that, for the sustainability of the universal system, the nurse must be oneof its main allies, since this professional is the one that has most incorporated thepractice of caring. The values, vision and mission of this profession are intertwinedwith the exercise of care, which means giving attention, treating, respecting andattending the human being in all their needs.Several countries have movements to define the competencies expected for nurses,considering social determinants and inequities ,aging (there will be nearly 2 billion elderly people by 2050) and an increasinglydemanding, distrustful and unsatisfied population.The advanced nursing practice is a relevant mechanism for supporting the leading role ofnurses in health production in Latin America and the Caribbean. However, there are otherreferences and even complementary ones that not only address the increase of the actionscovered by the nurse in quantitative terms, but redesign the profession in thequalitative aspects, defining the essential core of the profession and its horizons inthe contemporary world, mediated by equity, integrity, justice, law and ethics.Nurse of the future nursing core competencies,produced by the Massachusetts Department of Higher Education Nursing Initiative, whichemphasizes the art and science of careThus we bring the These guidelines have influenced the training and practice of nurses in the United Statesof America and today serve as an interesting basis for thinking about nurses\u2019 curriculaand practice in universal systems. Evidently considering the economic, political,ideological and cultural differences and the distinctions between the logics oforganization of the health system in our country and in the US (free market), we find inthis reference the perspective of a bold nursing, with the breadth and perspectiveexpected.We do not intend to close with a single reference, but to open up new possibilities thatconsider the nurse as a necessary professional now and in the future, support politicaldecisions, guaranteed by the Welfare State and human dignity.It is imperative and strategic to think about nursing today. Based on these reflectionswe can find forms of health production consistent with the fundamentaltheoretical-philosophical, legal and ethical basis of health systems. Among theprofessions, it is the one that most assimilates the values of universal systems.Therefore political commitment, along with critical mass and social capital, arenecessary to defend this proposal and this profession."}
+{"text": "The outcomes for neuropathic and ischemic DFUs were limb salvage , healing , healing time , major amputation , death  respectively. Revascularization failure and ESRD were independent predictors of major amputation, while heart failure and number of co-morbidities (\u22655) were independent predictors of death. Ischemic DFUs patients showed more severe clinical and ulcers features as well worse outcomes than neuropathic DFUs patients.This study aims to evaluate clinical and ulcer characteristics as well the outcomes of patients with diabetic foot ulcers (DFUs). The study group was composed of DFUs patients managed by a limb salvage protocol according to guidance. Clinical and ulcers findings were described, and 1-year outcomes defined as limb salvage, healing, healing time, major amputation and death were compared between neuropathic and ischemic DFUs. One thousand, one hundred and ninety-eight subjects were included; 386 (32.2%) neuropathic and 812 (67.8%) ischemic DFUs. Neuropathic patients were younger (69.5 \u00b1 11.5 vs. 74.5 \u00b1 11.5,  Diabetic foot disease (DFD) is the most severe consequence of two diabetes related long-term complications: peripheral neuropathy (PN) and peripheral arterial disease (PAD). Foot ulceration is usually the main clinical expression of DFD . DiabetiPatients with DFD are often very fragile and foot ulceration may be just a part of an extremely complex clinical condition in which specific long-term complications (PN and PAD) and concomitant co-diseases affect the general health of patients. A 5-year mortality rate was reported following new-onset of DFU, which is between 25% and 60% ,5 higherWithin this framework, it is necessary to consider two patterns of DFUs in patients with or without peripheral arterial disease (PAD), termed respectively as neuro-ischemic/ischemic ulcers and neuropathic ulcers. It has been reported that until now, in developed countries, the rate of PAD in patients with foot ulceration is approximately 50% ,9, whilePAD increases the risk of non-healing and major amputation ,13,14, aBased on their daily experience, the authors retain that a deep understanding of the patients DFU history, management and outcomes could lead to an improvement in our strategies.This study aims to evaluate the pattern of diabetes-related complications and co-morbidities in patients with DFUs, comparing neuropathic and ischemic patients. Furthermore, the characteristics of neuropathic and ischemic/neuro-ischemic DFUs will be reported and compared, as well as the long-term outcomes.Consecutive patients who were referred to our diabetic foot unit for a new diabetic foot problem between January 2010 and December 2018 were considered for this study. Patients included were those attending the clinic for a new foot ulceration, including both neuropathic and ischemic/neuro-ischemic DFUs. Subjects referred with an unsalvageable foot condition requiring major amputation, those with a life expectancy of less than 6 months and those who lost to follow-up during the first 12 months were excluded.All patients included were managed through a pre-set limb salvage protocol including revascularization in the case of ischemic/neuro-ischemic ulcers, antibiotic therapy and surgical treatment for infected wounds, dedicated off-loading, appropriate wound care, and management of diabetes and comorbidities according to guidance ,18.Data were collected in a local database and retrospectively analyzed. Baseline demographic, clinical and ulcer findings were recorded.The study has been done and approved according to local ethics committee policy. At admission, patients provided their verbal consent.Diabetic retinopathy, neuropathy, and nephropathy were conditions reported. Retinopathy was considered in the case of proliferative or not proliferative retinopathy; peripheral neuropathy was considered in the case of loss of peripheral sensitivity detected through vibration perception (128 Hz tuning fork) or Semmes-Weinstein 10-g monofilament ,18; nephIschemic heart disease (IHD) was considered in the case of previous acute coronary syndrome or coronary revascularization, evidence of angina, significant changes on electrocardiography . Cerebrovascular disease was considered in the case of previous cerebrovascular ischemia, previous carotid revascularization, or significant carotid artery disease (occlusion >70%).Hypertension was considered in the case of blood pressure >130/80 mmHg persistently or current antihypertensive therapy ; hyperchBaseline ulcer characteristics  reported at the first assessment were recorded. Infection was considered in the case of clinical signs according to International Working Group on the Diabetic Foot (IWGDF) ,18. UlceNeuropathic ulcers were considered in the case of patients with PN without PAD; ischemic ulcers were considered in the case of patients with PAD, regardless of the presence or not of PN. PAD was considered in the case of the absent pulses and ankle-brachial index of <0.9 or TcPO2 < 50 mmHg, in addition to evident stenosis and/or an obstruction at duplex ultra-sound ,19,20.Micro and macrovascular diabetes-related complications, co-morbidities, ulcers characteristics and outcomes in patients with neuropathic and ischemic DFUs were reported and compared.Limb salvage, healing, healing time, amputation, and mortality after 1-year of follow-up were the primary outcomes considered. Limb salvage was considered in the case of healing or incomplete healing in patients with preserved limb function; healing was considered in the case of complete epithelialization of previous ulceration during the follow-up; healing time was considered as the time reported in weeks which occurred from the first assessment and the complete epithelialization; amputation was considered as any amputation above-the-ankle and included below and above-the-knee.As secondary endpoint, the association between the number of comorbidities and limb salvage in the whole population of neuropathic and ischemic subjects was reported.All potential predictors of major amputation and death where evaluated.t test (frequency data) or ANOVA (continuous data). Univariable logistic regression analysis was performed with all potential predictor variable with the outcome of interest (major amputation and death). Then, all positive predictors were entered simultaneously in a multivariate logistic regression model. These models yielded a set of variables that best predict the outcome of interest. p < 0.5 was considered statistically significant.Statistical analysis was performed by SAS  for personal computer. Data are expressed as means \u00b1 SD. Comparison between groups were reported by the Student\u2019s One thousand, one hundred and ninety-eight patients were included. The mean age was 73 \u00b1 12 years; 758/1198 (63.3%) were males, 1130/1198 (94.3%) had type 2 diabetes and the mean HbA1c was 62 \u00b1 24 mmol/mol .Three hundred eighty-seven (387) (33.2%) patients had neuropathic DFUs while 812 (67.8%) patients had ischemic/neuro-ischemic DFUs. Ischemic DF patients were older, had a longer duration of diabetes and higher baseline HbA1c values when compared to neuropathic DF subjects .They were characterized by the high presence of long-term diabetes-related complications, mainly peripheral neuropathy (92%), retinopathy (51%), nephropathy (34%), PAD (68%) and ischemic heart disease (32%). Regarding concomitant co-morbidities, they frequently reported hypertension (79%), dyslipidemia (40%), and often ESRD requiring dialysis (22%) and heart failure (20%) .Ischemic DF patients had more diabetic nephropathy and less peripheral reduced sensitivity than neuropathic DF subjects .Ischemic DF patients showed a higher rate of ischemic heart disease and cerebrovascular disease than neuropathic subjects .Ischemic DF patients showed more cases of HF, ESRD and anemia than neuropathic persons. No difference was recorded in terms of hypertension, dyslipidemia and smoking between the two groups .Overall, 16.5% of patients reported 4 co-morbidities concurring with diabetes and 10.8% had 5 or more. More patients with ischemic DF presented with 4 or more concomitant co-morbidities than patients with neuropathic DF .Ischemic DF patients showed more cases of multiple lesions in comparison to neuropathic DF patients, while neuropathic DF subjects showed more cases of forefoot localization than ischemic DF patients. Ischemic DFUs were larger, more infected and deep to the bone in more cases than neuropathic DFUs .One thousand and fifty (1050) (87.7%) patients had limb salvage after 1-year of follow-up, 1022 (85.3%) patients healed in an average time of 35.4 weeks (range 31.9\u201339.1), 55 (4.6%) were amputated (major amputation), and 93 (7.7%) died .p < 0.0001), healing , average healing time , amputation , and death   .In the whole population, the rate of limb salvage gradually decreased as the number of concomitant co-morbidities increased; furthermore, the rate of limb salvage in persons with ischemia was less than those with neuropathy, despite having a similar number of coexisting diseases .At the multivariate analysis of all predictors found at univariate analysis, revascularization failure and ESRD were independent predictors of major amputation, while heart failure and the presence of five or more concomitant co-diseases were independent predictors of mortality .This study offers a complete overview on ulceration findings, clinical characteristics, and long-term outcomes in a large cohort of diabetic foot patients including those with both neuropathic and neuro-ischemic/ischemic DFUs.Overall, 87.7% had limb salvage and 85.3% of patients healed in an average time of 35.4 weeks, while 4.6% had major amputation and 7.7% died.On the one hand, the reported data are very encouraging because through a specialized multi-disciplinary team approach, we achieved a great rate of limb salvage and healing, mainly in neuropathic subjects. On the other hand, we observed a significant increase in ischemic DFUs, which were approximately two times higher than neuropathic DFUs and, among those reported, showed a higher rate of amputation (6.6 vs. 0.5%) and death (11 vs. 1.1%), and a lower rate of healing (79.6 vs. 97.3%) in comparison to neuropathic subjects.These data are partially similar to those reported by the Eurodiale study  which shThe ulcer size and the presence of infection could increase the risk of non-healing . AdditioPatients included in our study group were elderly (mean age >70 years), with a long diabetes duration (approximately 24 years) and poor glycemic control. They were characterized by the high presence of microvascular and macrovascular complication, and several concomitant co-morbidities, such as hypertension, dyslipidemia, ESRD and heart failure.Patients with ischemic ulcers were older than those with neuropathic ulcers and showed higher rates of microvascular (40% with nephropathy) and macrovascular (37% with ischemic heart disease and 17% with carotid artery disease) complications, and concomitant co-morbidities  in comparison to patients with neuropathic ulcers.In the whole population, approximately 16% of patients reported at least four concomitant co-morbidities, and 10.8% had five or more. Patients with ischemic DF were more likely to present with four or more concomitant co-morbidities than patients with neuropathic DF (32 vs. 16%). It is also very interesting to highlight the rate of limb salvage decreases with the increase in the number of co-morbidities. However, the limb salvage rate was 100% and 90%, respectively, in subjects without and with three concomitant co-diseases or fewer. Nonetheless, the rate of limb salvage is significantly lower in those with ischemic DF than neuropathic DF despite the similar number of co-morbidities. These data are already evident in the presence of one co-disease  and much more evident in the presence of three or more co-diseases, suggesting that co-morbidities in patients with PAD reduce the possibility of limb salvage, which is probably due to the impact of PAD perse.The role of co-morbidities is reinforced by the multivariate analysis which revealed that ESRD was an independent predictor of major amputation and heart failure, and, additionally, the number of concomitant co-diseases (\u22655) were independent predictor of mortality.The role of co-morbidities in DFD is often underestimated, even though it is well known that they can significantly influence outcomes for patients with DFUs. It is maintained that ulcer-related outcomes may underestimate the morbidity and mortality associated with DFD , and recOur data highlight that DF should be considered as a marker of multi-organ disease in ischemic subjects, with a significant impact on outcomes. Many papers have reported that co-morbidities, mainly dialysis and heart failure, increase the risk of major amputation and outcomes ,27.Patients with DFUs, mainly ischemic, are extremely difficult to treat, and foot injury is often just a part of a very complex clinical condition. Ischemic patients often require hospitalization to be treated for revascularization and due to the presence of severe co-morbidities, in some cases, a fast limb salvage protocol may not only address limb salvage, but may also save the patient\u2019s life.Similarly, the Eurodiale study showed that heart failure and ERSD had a greater incidence in patients with PAD, and in addition, ESRD was an independent predictor of non-healing .Gershater et al., in a prospective study on 1148 hundred and eighty patients, reported that the absence of uremia and heart disease were clinical factors related to primary healing in the whole population and in survivors. Conversely, diabetic nephropathy and uremia were predictors of non-healing in survivor patients with ischemic/neuro-ischemic ulcers, and uremia was related to major amputation in ischemic ulcers. Furthermore, deceased patients showed more ischemic ulcers and more co-morbidities than the other groups .Alpeqvist et al. showed that Creatinine values <130 \u03bcmol/L and the absence of congestive heart failure were independent predictors of primary healing in a population composed of 1115 patients with ischemic DFUs .Faglia et al. showed that ischemic heart disease was the leading cause of death; dialysis and a history of cardiac disease were independent predictors of death; dialysis was an independent predictor of major amputation in 554 patients with DFUs and CLI when treated using a limb salvage protocol, which includes revascularization .In our previous experience, we observed that dialyzed patients had higher risk of non-healing and major amputation than subjects with preserved renal function , and thaTherefore, co-morbidities, such as heart disease, including both coronary artery disease and heart failure, and ESRD not only reduce the chance of healing, but they are also independent predictors of mortality.In the current study, we confirm that ischemic heart disease is quite typical in diabetic foot patients with PAD. It has already been reported that approximately 50% of diabetic patients with PAD have a concomitant ischemic heart disease . Heart iTherefore, patients with DFUs, mainly ischemic DFUs, should be considered as subjects with a high risk of heart disease\u2014mainly heart failure secondary to ischemic heart disease.It is noteworthy that micro/macrovascular diabetes-related complications were more frequent in patients with ischemic DF than neuropathic DF, although a similar diabetes duration was reported. Therefore, it may be evident that age, concomitant co-morbidities , poor glycemic control and individual susceptibility could increase the risk of developing PAD and the abovementioned complications.It is also necessary to highlight that revascularization failure was an independent predictor of major amputation. Revascularization failure was considered as technical recanalization failure of occluded vessels (defined as the impossibility to overcome the obstruction) and/or absence of arterial flow to the foot. On the one hand this data suggests that the severity of PAD could negatively influence the revascularization procedure and outcome, but on the other hand also confirms that failed revascularization is a predictor of major amputation as already reported by Faglia et al.  and our Therefore, DFD is a complex clinical condition characterized by foot injury, severe patterns of PAD, and several concomitant co-morbidities which could influence management and outcomes. The presence of a foot ulceration in diabetics should always be considered a strong risk factor for early and long-term mortality, mainly in ischemic subjects which have shown 5-year-mortality, of approximately 60% . It is eThis study gives a complete overview on clinical and ulcers features, and the long-term outcomes in a very large cohort of patients with DFUs, which proves to identify specific characteristics in the current pattern of neuropathic and ischemic subjects. To the best of our knowledge it is the first study after the Eurodiale study to evaluate and compare the prevalence, characteristics and outcomes of neuropathic and ischemic DF patients in a very large cohort of patients; furthermore, it is the first to highlight that the number of comorbidities could influence the possibility of limb salvage, in addition to the fact that concomitant co-diseases may have a major influence on ischemic rather than neuropathic DFUs.The current study is a retrospective study and data were collected from one single diabetic foot center and, accordingly, outcomes are related to our comprehensive limb salvage protocol, performed by an expert multidisciplinary diabetic foot team. Future research may be useful to identify if outcomes are influenced only by the number of comorbidities or by a comorbidity score, whereby each concomitant disease has a specific burden.Our data illustrate that among current patients affected by DFD, there is a prevalence of ischemic DFUs in comparison to neuropathic DFUs, and there also seems to have been an increase in ischemic subjects over the last years. Patients with DFUs are complex subjects, who in addition to foot injury, are often older in age and display several diabetes-related complications and comorbidities\u2014mainly cardiovascular. This study reinforces the concept that patients with ischemic DFUs report more severe wound and clinical features than those with neuropathic DFUs, and that there is a lower rate of limb salvage in spite of the similar number of concomitant co-diseases. Furthermore, co-morbidities appear to play a key role in the outcomes of patients with DFD.While aiming to reduce amputation and mortality, DFD should be considered a multi-organ disease, which means that patients need an intensive global and multidisciplinary treatment plan with close control of cardiovascular risk factors."}
+{"text": "The Thermodynamic Formalism provides a rigorous mathematical framework for studying quantitative and qualitative aspects of dynamical systems. At its core, there is a variational principle that corresponds, in its simplest form, to the Maximum Entropy principle. It is used as a statistical inference procedure to represent, by specific probability measures (Gibbs measures), the collective behaviour of complex systems. This framework has found applications in different domains of science. In particular, it has been fruitful and influential in neurosciences. In this article, we review how the Thermodynamic Formalism can be exploited in the field of theoretical neuroscience, as a conceptual and operational tool, in order to link the dynamics of interacting neurons and the statistics of action potentials from either experimental data or mathematical models. We comment on perspectives and open problems in theoretical neuroscience that could be addressed within this formalism. Initiated by Boltzmann ,2, the gAlthough this program is, even nowadays, far from being completed ,5, the wThe introduction of Thermodynamic Formalism that occurred in the 1970s was primarily due to Yakov Sinai, David Ruelle, and Rufus Bowen ,9,10. ThIn the context of dynamical systems, Sinai, Ruelle, and Bowen were able to connect the theory of hyperbolic (Anosov) dynamical systems to results in statistical mechanics. Indeed, Sinai found an unexpected link between the equilibrium statistical mechanics of spin systems and the ergodic theory of Anosov systems by a codification using Markov partitions , althouThe rest of the article is organised, as follows. In Our goal in this section is to present the basic tools and ideas of Thermodynamic Formalism to the unfamiliar reader . This cn matrix .B. These distributions are the set of shift-invariant probability measures denoted by A invariant under the dynamics, i.e., We now define the measurable sets on er 1, in ). We aren sites based on the sequence The analogy with the spin systems in statistical mechanics at the root of the terminology \u201cThermodynamic Formalism\u201d, goes, as follows. Let We define the measures that assign probability to the cylinder sets based on the potential function, with the so called \u201cGibbs property\u201d. There exist constants  , is callA, .p and q on the same probability space X, the KL divergence is:Another important quantity is the Kullback\u2013Leibler (KL) divergence, which quantifies the difference between probability distributions. Consider two probability distributions p is absolutely continuous with respect to q, and it is only zero if This divergence is finite whenever Following the analogy with systems in statistical mechanics, an equilibrium state is defined as the measure that satisfies the so-called variational principle, namely:suph(\u03bd)+\u222bEquation . The equsuph(\u03bd)+\u222bobservablef is a function i, An K observables U for the potential, instead of An important observable, considered in the following sections, is the potential:f in a sample of size n, that is,Now, we denote This quantity is important for empirical purposes. If ics, see .n byLet us consider a pair of observables f at time n,One also might be interested in the auto-correlation (or auto-covariance) of Observe that these quantities might decay fast. The mixing property implies that the correlations go to zero with n with respect to From the pressure , importansidered :(7)\u22022F. In contrast, when they do not converge fast enough , the series diverges, leading to a divergence of the second derivative of the pressure, corresponding to a second-order phase transition. Here, follow the Ehrenfest classification of phase transitions  or  or \u03bbls t measure  for up tR and a finite number of neurons provided l in  and syn a spike . The conR is the membrane resistance and the term In its simplest form, for a single isolated neuron, this equation takes the form:CdVdt=\u22121Rhase see B, bottomWhile this is a simple artificial way to generate spikes, there is a huge price to pay on mathematical grounds because the threshold introduces a singularity set in the phase space where the dynamic is not differentiable. We develop this aspect below.N neurons is straightforward. Adding the contribution of synaptic currents, k, R is assumed to be the same for all neurons. In the synaptic current,j to the post-synaptic neuron k k26) look a priori to an uncountable set. There are two alternatives. The first one, briefly explored in this section, consists of discretising time as done e.g., in .Here, we present a second example, where the Gibbs potential can be computed. This model is known as the Galves\u2013L\u00f6cherbach model introduced by Antonio Galves and Eva L\u00f6cherbach in  hi and gjN that affect the cardinality of the alphabet considered and the range R of the potential (memory in transition probabilities) associated to the equilibrium measure characterising the system. This is represented in In this review, we introduce different ideas and tools from Thermodynamical Formalism and show how they can be applied in theoretical neuroscience. As a summary, we grouped these approaches depending on two main characteristics. The first one is the number of neurons While there have been interesting applications of Thermodynamic Formalism to neuroscience, there are interesting ideas and developments still to come. In this concluding section we raise questions, challenges and new avenues for the application of Thermodynamic Formalism to theoretical neurosciences.possibly uncountable set of potential spike trains. The question is whether we are dealing here with a realistic property of biological neuronal networks or with an artifact created by the instantaneous reset. Real spikes have a duration (a few ms) and a refractory period , so, for a fixed initial condition, spike trains produced by a continuous-time neuronal network are countable. Now, it might be that the set of spike trains depend continuously on the initial condition, so that we are still left with an uncountable set of spikes.In this review, we have considered rather academic models of neuronal networks, where, especially, time is considered to be discrete. There are good reasons for that. As we remarked at the beginning of It is out of the scope of this review to discuss from a biological perspective, whether or not neuronal networks have the cardinality of the continuum . Accordingly, the trick is the following. Fix x becomes at most countable.Spiking variables are therefore time-discrete events with a time resolution volution  until thThis trick can be used to generalise the Integrate-and-Fire model into a conductance-based Integrate-and-Fire model, which was introduced by Rudolph and Destexhe in  and mathNow, would Thermodynamic Formalism apply to more realistic neuronal models, like Hodgkin\u2013Huxley , FitzHugA natural question in this context is what is the link between spike train statistics and the underlying dynamical model, with \u201chidden\u201d dynamical variables, such as membrane potential, but also, e.g., activation/inactivation variables? If we think in terms of spike coding, the alternative is the following. Either spike trains contain all the necessary information to characterise the dynamics, e.g., the spike response to a stimulus, and then characterising  is suffiWhat Thermodynamic Formalism brings to the analysis of these models is twofold: (1) a way to rigorously handle probabilistic representations of spikes ; and, 2, where aSeveral studies have shown that the population of vertebrate retinal ganglion cells responding to naturalistic stimulus is poised near a \u201ccritical state\u201d ,74. Fromibutions .T. This quantity can be obtained as the second derivative of the pressure, Equation  of the energy Equation , which izes, see .T plot, for maximum entropy models of Ising type obtained from the recording of retinal ganglion cells responding to naturalistic stimuli are shown in U) when The form of  regime\u201d ).The behavior of the specific heat that is observed in This interesting approach leads, nevertheless, to several questions in the context of Thermodynamic Formalism.Does this signature of criticality extend to Gibbs distributions with potentials of range  How does it depend on R? We are not aware of any experimental results addressing this issue. This question is related to the following:What is this signature of criticality from the point of view of Thermodynamic Formalism? The occurrence of a second-order phase transition mathematically means that the pressure is R tends to infinity or the number of neurons N tends to infinity. These two limits could also be addressed simultaneously and they do not necessarily commute. For potentials associated to finite R and N the Perron-Frobenius theorem guarantees the existence and uniqueness of the Gibbs measure and the analyticity of the pressure can also be proved, preventing phase transitions. When R or N are infinite, the properties of the RPF operator  ,143,144.Can we relate known examples of dynamical systems exhibiting phase transitions to models in neuroscience? Another possible example to be interpreted in neuroscience is the Dyson model [on model , in whicon model . An inteon model  presentsWhat could the dynamical or mechanistic origins of a second-order phase transition be in a spiking neuronal network model? Handling experimental data is of course important, but for long experiments with living neuronal tissue, one cannot control the size of the sample, the stationarity of data, and so on. Accordingly, assume that we have been able to find an example of a Gibbs distribution exhibiting a second-order phase transition when R and N, which has this Gibbs distribution in the limit R, or N, he model  can arisJ that together predict with high accuracy the spiking behaviour of the neuron i? Mathematically can be written in this way J is a subset of neurons in the population of spiking neurons. Other questions related to the neural coding and dimensionality reduction can be addressed studying the energy landscape From the perspective of the maximum entropy distributions built from experimental data of spiking neurons, there have been interesting applications of the Gibbs distributions that were obtained to answer questions related to the retinal code that are not related to criticality . From thU\u03bb(x) of . For exaThe relationship between mathematics and physics has been historically symbiotic and Thermodynamic Formalism is an interesting example of how ideas from physics may help to solve problems and introduce ideas into the field of mathematics. The history of Thermodynamic Formalism also shows how purely mathematical results can be obtained as corollaries of physical laws, inverting the frequently assumed relationship between physics and mathematics .However, in the case considered in this review\u2014the link between Thermodynamic Formalism and neuroscience\u2014the mathematical problem is motivated by biology, not by physics. While Eugene Wigner argues in favour of the \u201cThe unreasonable effectiveness of mathematics in the natural sciences\u201d , Israel Especially, we have described how Thermodynamic Formalism: (1) provides a conceptual and operational  framewoHere, we would like to propose some other extensions, not yet explored so staying at the level of ideas, all based on the power of Thermodynamic Formalism to make explicit and operational links between dynamics, statistics and symbolic coding.Geometry of the state space. A prominent aspect of Thermodynamic Formalism, that we haven\u2019t discussed yet in this review, is its link to the characterisation of the geometry of attractors and, especially, fractal sets [tal sets ,166. Fortal sets  generatital sets . It woulTransitions between attractors. The concept of attractor is actually central in describing brain dynamics [dynamics ,169. Espdynamics  and refedynamics ,173,174.dynamics , which iNon-stationarity and link with generating functional formalism. As we mentioned, Thermodynamic Formalism is constructed from a variational approach based on entropy and, thus, requiring time translation invariance. We have briefly described how we can depart from this constraint while using linear response theory. It would be interesting to explore beyond this point and consider general types of response to stimuli . For this, one would have to construct a Thermodynamic Formalism based on the optimisation of a quantity, which is not the entropy. This is somehow what generating functional approaches like the dynamic mean-field theory does (see introduction), although using other constraining hypotheses . It would be interesting to try to close the gap between these two approaches .One of the biggest challenges in science of the XXI century is to understand the brain functions within a conceptual framework that are capable of unifying the multi-scale dynamics that take place in the brain. This framework should also make sense in the light of the overwhelming amount of experimental data capable of predicting macroscopic phenomena, such as motor behaviour or visual experience from the activity of billions of neurons.Physicists have been able to make a deep connection between mechanics, statistical physics, and thermodynamics. A similar quest is presumably guiding the research of (some) theoretical and experimental neuroscientists. While there is still a long way to go before achieving this goal (as some argue we are still searching for principles ), duringWe hope that theoretical tools and ideas from Thermodynamic Formalism and its current application in neuronal dynamics and spike train statistics will lead to a better and unified understanding of the neural phenomena. We also hope that the present review may serve as an encouragement for the mathematical community that is interested in applications of Thermodynamic Formalism in order to study these interesting and important problems."}
+{"text": "Here, we demonstrated that the multikinase framework including ERK/AKT and CDK2 promotes the proliferation and invasion of lung adenocarcinoma cell lines through activating DRP1. Our results further uncovered the prognostic significance of DRP1 in early\u2010stage lung adenocarcinoma, showing that the expression and activation of DRP1 are both significantly associated with an increased risk of postoperative recurrence. DNM1L gene, in regulating the growth of cancer cells of various origins. However, the regulation, function, and clinical significance of DRP1 remain undetermined in lung adenocarcinoma. Our study shows that the expression and activation of DRP1 are significantly correlated with proliferation and disease extent, as well as an increased risk of postoperative recurrence in stage I to stage IIIA lung adenocarcinoma. Loss of DRP1 in lung adenocarcinoma cell lines leads to an altered mitochondrial morphology, fewer copies of mitochondrial DNA, decreased respiratory complexes, and impaired oxidative phosphorylation. Additionally, the proliferation and invasion are both suppressed in DRP1\u2010depleted lung adenocarcinoma cell lines. Our data further revealed that DRP1 activation through serine 616 phosphorylation is regulated by ERK/AKT and CDK2 in lung adenocarcinoma cell lines. Collectively, we propose the multikinase framework in activating DRP1 in lung adenocarcinoma to promote the malignant properties. Biomarkers related to mitochondrial reprogramming, such as DRP1, can be used to evaluate the risk of postoperative recurrence in early\u2010stage lung adenocarcinoma.Recent studies revealed the role of dynamin\u2010related protein 1 (DRP1), encoded by the  AKTprotein kinase BALKanaplastic lymphoma kinaseAMPK5\u2032\u2010adenosine monophosphate\u2010activated protein kinaseATPadenosine triphosphateATF4activating transcription factorCRISPRclustered regularly interspaced short palindromic repeatsCas9CRISPR\u2010associated protein 9CDKcyclin\u2010dependent kinaseDNAdeoxyribonucleic acidDRP1dynamin\u2010related protein 1DNM1Ldynamin 1\u2010likeECARextracellular acidification rateEGFRepidermal growth factor receptorEMTepithelial\u2010to\u2010mesenchymal transformationERKextracellular signal\u2010regulated kinaseGTPaseguanosine triphosphatasesH\u2010scorehistological scoreIHCimmunohistochemistryKOknockoutMFN1mitofusin 1MFN2mitofusin 2mtDNAmitochondrial DNAmRNAmessenger RNANRF2nuclear factor erythroid 2\u2010related factor 2NTUHNational Taiwan University HospitalOPA1optic atrophy 1OSoverall survivalOXPHOSoxidative phosphorylationPETpolyesterPFSprogression\u2010free survivalPCK2phosphoenolpyruvate carboxykinase 2PHGDHphosphoglycerate dehydrogenasePSAT1phosphoserine aminotransferase 1PSPHphosphoserine phosphataseP(S616)\u2010DRP1phosphorylated DRP1 at serine 616qPCRquantitative polymerase chain reactionRNAribonucleic acidTCGA\u2010LUADthe Cancer Genome Atlas Lung AdenocarcinomasiRNAsmall\u2010interfering RNA1DNM1L gene, regulates mitochondrial fission through interactions with various adaptors at the outer mitochondrial membrane [DNM1L mutations are related to various neurologic diseases in humans [Mitochondrial dynamics, consisting of morphological changes associated with mitochondrial fission and fusion, are fundamental for metabolic reprogramming in response to various environmental stimuli . Dynaminmembrane . Mitochomembrane , 4 and mmembrane . DNM1L mn humans , neurolon humans , and macn humans  in murinConsidering the role of mitochondrial dynamics in orchestrating cell metabolism, the cancer\u2010specific regulation of mitochondrial dynamics might be required to support proliferation, invasion, and metastasis , 9. StudIn this study, we aimed to clarify the function, regulation, and biological importance of DRP1 in lung adenocarcinoma, and the clinical significance of DRP1 expression and activation was extensively explored.22.1The study protocol regarding human samples was approved by the Institutional Review Board at National Taiwan University Hospital  and conformed to the standards set by the Declaration of Helsinki. Consecutive adult patients with lung adenocarcinoma diagnosed from January through December 2013 were identified using the database of the Cancer Registry, Medical Information Management Office of NTUH. The disease staging was evaluated according to the 7th edition of the American Joint Committee on Cancer. The study population consisted of patients who received definitive surgical treatment for stage I to stage IIIA lung adenocarcinoma. Patients were excluded if they had combined tumor histology, such as adenosquamous carcinoma. The written consent was obtained from each subject before the operation. We followed the study population to evaluate postoperative recurrence until 5\u00a0years after the surgery, and recurrence\u2010free survival was determined from the operation date until the first objective sign of recurrence.2.2Formalin\u2010fixed paraffin\u2010embedded tissue sections in 4\u00a0\u03bcm thickness were used for Immunohistochemistry (IHC) staining. The tissue sections were deparaffinized with xylene and then rehydrated with graded ethanol. The staining was performed using a Leica Biosystems BOND\u2010MAX autostainer or a Roche VENTENA BenchMark autostainer. The information on primary antibodies is summarized in Table\u00a02.3https://www.cancer.gov/tcga) and were obtained using the R package TCGAbiolinks. To evaluate the association between DNM1L expression and prognosis, including progression\u2010free survival (PFS) and overall survival (OS), we categorized the tumors based on the median and the tertiles of DNM1L expression. The analyses were performed in the R environment (v3.6.1).The Cancer Genome Atlas Lung Adenocarcinoma (TCGA\u2010LUAD) clinical information and gene expression profiles were generated by the TCGA Research Network  1640 medium  containing 10% fetal bovine serum  and 1% penicillin/streptomycin . CRISPR/Cas9 DNM1L and CDK2 knockout (KO) cell lines were generated through lentiviral transduction, using the lentiCRISPRv2 vector  [Human lung adenocarcinoma cell line CL1\u20100 was established as previously described , 25. LunNJ, USA) . The seqNJ, USA) , 29, wasNJ, USA) , and theNJ, USA) , double 2.5imagej software . Invasion assay was performed using a 24\u2010well plate and cell culture inserts with 8.0\u2010\u00b5m transparent polyester membrane . The Matrigel  was diluted to 2\u00a0mg/mL using RPMI medium and was used to coat the cell culture inserts. Cells and serum\u2010free RPMI medium were added to the cell culture insert and complete RPMI medium to the outer chamber. After culture for 16\u00a0h, the invading cells on the membrane were fixed and stained using Hoechst 33342. We obtained 8 images from each cell insert using the 10X objective, and the cell number was counted using FIJI running imagej software (version 1.51).Colony formation assay was performed using a 6\u2010well culture plate, and 500 cells were added to each well. After culture for 10 or 14\u00a0days, the cells were fixed and stained with crystal violet. The area fraction in each well covered by the cells was measured using microscopic images and FIJI running 2.6DNM1L\u2010KO CL1\u20100 xenograft implantation [6 cells were prepared in serum\u2010free RPMI1640 medium and mixed with Matrigel (Corning) in 1:1 ratio on ice. The cell suspension with a final volume of 100\u00a0\u03bcL was injected subcutaneously at the flank. The tumor volume was recorded weekly after implantation and was calculated using the following equation: tumor volume (mm3) = length (mm)\u00a0\u00d7\u00a0(width (mm))2/2. The mice were euthanized 5\u00a0weeks after tumor implantation, and the tumor weight was measured after dissection.All animal experiments were approved by the Institutional Animal Care and Use Committee at Medical College, National Taiwan University Hospital. Nude mice were purchased from the National Laboratory of Animal Center. Male nude mice at 6\u00a0weeks of age were used for control or antation . For eac2.7Protein extraction was performed as previously published , and pro2.8Mitochondrial and nuclear staining was performed as previously published , and the2.9The oxygen consumption rate and extracellular acidification rate (ECAR) were measured with a Seahorse XFe24 Analyzer . The assays were performed using the Mito Stress Test Kit , based on the manufacturer\u2019s instructions. The data were normalized using the CyQUANT Cell Proliferation Assay Kit  and analyzed using Wave software (version 2.6.1.38).2.10\u2212\u0394\u0394Ct method was used to calculate RNA expression relative to TBP, and mtDNA copy number relative to genomic DNA copy number.Real\u2010time qPCR was applied to measure mRNA expression and mitochondrial DNA (mtDNA) quantification. The primer sequences for qPCR are summarized in Table\u00a02.11A FACSCalibur flow cytometer  was used for cell cycle analysis. The cells were stained using the Propidium Iodide Flow Cytometry Kit , based on the manufacturer\u2019s instructions. Mitophagy detection was performed on a FACSAria III (BD Biosciences) in the Flow Cytometric Analyzing and Sorting Core Facility at NTUH, using the cells expressing pH\u2010sensitive mtKeima fluorescence. Excitation using 405\u2010nm and 561\u2010nm laser was used to detect mtKeima at pH 7.0 and at pH 4.0, respectively . Identic2.12p value less than 0.05 was considered statistically significant. All analyses were performed using SPSS  or GraphPad Prism .For differences in continuous variables, Student\u2019s t\u2010test or Mann\u2013Whitney U\u2010test was used for comparisons between two groups as appropriate, whereas one\u2010way ANOVA with a Bonferroni post hoc test was used for multigroup comparisons. Categorical variables were compared by Pearson\u2019s chi\u2010square test or Fisher\u2019s exact test, as appropriate. The dichotomization of the study population was performed based on the H\u2010score median or the optimized H\u2010score cutoff determined by receiver operating characteristic curves and the Youden index. For evaluating the correlation between two continuous variables, the correlation coefficient was calculated using both the linear regression analysis and the Spearman correlation analysis. Kaplan\u2013Meier curves were plotted for recurrence\u2010free survival, progression\u2010free survival, or overall survival, and survival differences were compared using log\u2010rank tests. Multivariate Cox proportional hazard models were used to calculate the hazard ratio (HR) for postoperative recurrence. A two\u2010sided 33.1DNM1L expression above the median were significantly associated with worse PFS  or P(S616)\u2010DRP1  H\u2010score was independently significantly associated with 5\u2010year postoperative recurrence \u2010DRP1 in CL1\u20100 and PC9 cells \u2010DRP1 in CDK2\u2010KO CL1\u20100 \u2010DRP1 in CDK2\u2010KO CL1\u20100 and A549 cells  or with resistant T790M EGFR mutation (H1975). H1975 cell is sensitive to the 3rd\u2010generation EGFR tyrosine kinase inhibitor, osimertinib. We confirmed that osimertinib effectively decreased DRP1 phosphorylation in H1975 cells. EGF\u2010driven EGFR signaling also increased DRP1 phosphorylation in CL1\u20100 cells. Therefore, we herein unveiled the critical role of EGFR signaling in regulating mitochondrial fission through a multikinase framework. Several driving mechanisms other than EGFR, such as ignaling . The mitignaling . HoweverImbalances in mitochondrial dynamics are associated human neuromuscular diseases and can result in dysfunctions in various organ systems in murine models . We founWe surmised that the upregulation of mitochondrial fission occurs early in the development of lung adenocarcinoma and investigated the clinical significance of DRP1 specifically in lung adenocarcinoma of operable stages. Whether DRP1 expression is associated with worse survival in patients with stage IV lung adenocarcinoma remains unclear and requires further examination. Screening through low\u2010dose computed tomography has proven effective to detect early\u2010stage lung cancer in the high\u2010risk population and to reduce lung cancer\u2010related mortality , 53. Bio5In conclusion, we present the functional role of DRP1 in enhancing the proliferation and invasiveness of lung adenocarcinoma cell lines and further show that multikinase regulatory molecules, including ERK, AKT, and CDK2, secure the activation of DRP1. Our findings reveal that the expression and activation of DRP1 in early\u2010stage lung adenocarcinoma are important features, suggesting an increased risk of postoperative recurrence. Further studies will broaden our understanding of mitochondrial functions and regulation in lung adenocarcinoma.KPC and CJY conceived and designed the study. KPC, YLH, YJC, and YHJ performed the experiments. YLH and YLC provided technical support for pathologic sample processing, IHC staining, and the interpretation of IHC results. KPC, CL, and YTH performed the data analyses, and KPC, KN, MWL, SGW, JYS, YLC, and CJY provided critical discussions and comments for data interpretation. KPC wrote the manuscript, and coauthors reviewed and approved the final manuscript.The authors declare no conflict of interest.Fig S1. . Analysis of data from TCGA\u2010LUAD to evaluate the prognostic significance of DNM1L expression in lung adenocarcinoma.Fig S2. . DRP1 expression and activation are associated post\u2010operative recurrence in early stage lung adenocarcinoma.Fig S3. . DRP1 expression and activation are significantly associated with post\u2010operative recurrence of lung adenocarcinoma.Fig S4. . DRP1 depletion increases mitophagy at baseline and after mitochondrial damage in lung adenocarcinoma cell lines.Fig S5. . DRP1 expression and activation are associated with proliferation and disease extent of lung adenocarcinoma.Fig S6. . The effects of oxidative phosphorylation inhibition to proliferation and invasion of lung adenocarcinoma.Fig S7. . Gefitinib decreases DRP1 phosphorylation in sensitive lung adenocarcinoma cell lines.Fig S8. . Transduction of the lentiCRISPRv2 vector did not alter the cell cycle progression.Fig S9. . The effects of various CDK inhibitors to cell cycle progression.Fig S10. . CDK2 regulates DRP1 phosphorylation during cell cycle.Fig S11. . CDK2 regulates DRP1 phosphorylation during cell cycle.Click here for additional data file.Table S1. Information of the reagents and antibodies.Click here for additional data file.Table S2. Nucleotide sequences for qPCR primer pairs and guide RNA for CRISPR/Cas9 knockout.Click here for additional data file.Table S3. Clinical characteristics of the study population.Click here for additional data file.Table S4. Multi\u2010variate Cox proportional hazard ratio model for 5\u2010year post\u2010operative recurrence.Click here for additional data file."}
+{"text": "The study aim was to develop a linguistic-cultural adaptation of the KEZKAK questionnaire to be completed during the practicum of podiatric medical students in Spain, to validate the questionnaire and to evaluate its psychometric properties.The cross-sectional study was carried out in two stages: 1. Cross-cultural adaptation; 2. Clinimetric validation based on assessments of interobserver reliability, test-retest reliability and internal consistency. The participants were podiatric medical students at the universities of Malaga and Miguel Hernandez, Alicante (Spain) and were recruited during the period February\u2013October 2019. The following inclusion criteria were applied: aged at least 18 years, studying the third or fourth year of a university degree in Podiatry. All gave signed informed consent and completed the State-Trait Anxiety Inventory and the Podiatry version of the KEZKAK questionnaire. No sampling was performed and thus the entire eligible population was included in the study.The analysis was based on 205 participants , with a mean age of 23.05 (SD 5.37) years. Internal consistency was excellent, with a Cronbach\u2019s alpha of 0.95. This version of the questionnaire had five factorial structures (61.18%). No floor/ceiling effect was observed in any item. The KEZKAK presented high test-retest reliability after 21 days, with an overall ICC of 0.95 (95% CI [0.93\u20130.98]).For university students of podiatry in Spain, the KEZKAK Podiatry version questionnaire is a valid, reliable instrument for measuring stressors during the practicum. Stress is a non-specific response of the organism to various stimuli. It is an adaptive, emergency process, and essential to survival . It is tIn general, stress is positive at low levels, which predispose us to act against the demands presented by the environment. However, when it is moderate or extreme, stress demands greater attention, because the reactions it provokes in our ways of thinking and feeling may lead us to respond inappropriately .The clinical practicum is an element of vital importance in the curriculum of Health Sciences students, giving them the opportunity to perform techniques, acquire knowledge, develop skills and learn attitudes, within a clinical health environment that was previously unfamiliar and which is sometimes not as welcoming as they expect. Preparation and training prior to the student\u2019s departure from academia to the real world is one of the great challenges of teaching, but not one to be addressed solely by the graduate student; undergraduates, too, must complete their theoretical-practical training in real environments. The presence of stress in university students is a reality. In healthcare programmes in which internships are carried out with patients in clinical situations and real environments, the stress inherent to the situation is aggravated by the psychological tension faced .Studies have shown that, in general, health sciences students are exposed to higher levels of stress than are other students and the population in general. This stress is associated with the requirement to carry out clinical practices with real patients and in authentic clinical settings. However, most previous research in this field has focused on students of medicine and nursing . AccordiIn this line of stress measurement, a recent study evaluated the reliability and repeatability of findings obtained by the Stress Assessment in Nursing Students (ASNS) scale, modified for podiatry students, concluding that this instrument presented good psychometric properties .In this respect, too, the KEZKAK questionnaire was developed by Zuripia to identify stressors experienced by nursing students in their practicum . This quIn another investigation, the KEZKAK and other questionnaires were considered as the basis for an integrative evaluation of general self-efficacy, perceived competence, resilience and stress among nursing students, thus facilitating training in professional competencies, in line with the European guidelines of higher education. The authors suggested it would be interesting to follow up this research with comparative studies, considering different clinical contexts .Accordingly, the aims of the present study are to develop a linguistic-cultural adaptation of KEZKAK for Spanish university students of podiatry, to be employed for measuring stressors during their practicum, to validate this adaptation and to evaluate its psychometric properties.This study was approved by the Medical Research Ethics Committee of the University of Malaga (CEUMA 88-2019-H) and of Miguel Hernandez University, San Juan de Alicante (DCC. ECL.01.19 PROV). The study was conducted in accordance with the Declaration of Helsinki and with all applicable ethical standards for human experimentation. Written informed consent from all participants (students) was obtained.This observational, cross-sectional study was conducted to validate the adaptation of the KEZKAK questionnaire for Spanish university students of podiatry, for measuring stressors during their practicum.All participants were university students of podiatry, at the University of Malaga (Spain) or the Miguel Hernandez University, Alicante (Spain), and were recruited from February to October 2019. A convenience sample was obtained of 223 students who met the criteria for inclusion: aged at least 18\u00a0years and currently studying the third or fourth year of a Bachelor\u2019s Degree in Podiatry. International exchange students were excluded because they were not native speakers of Spanish. First and second-year students were excluded because they were considered to have insufficient theoretical clinical knowledge. No sampling was performed and thus the entire eligible population was included in the study.Before undertaking the psychometric validation of this questionnaire for podiatry students, permission was obtained from the author (X. Zuripia Gorostedi).The linguistic-cultural adaptation of the original KEZKAK into the Spanish-language version for university students of Podiatry was performed by two Spanish podiatrists with nursing qualifications and university professor with PhDs, working independently . The oriIn the initial questionnaire, based on 62 ideas, 55 statements were presented, based on items initially suspected of generating stress in nursing practice students. These statements were subsequently analysed and streamlined to combine those which were similar and to eliminate items not directly relevant to the project aims, resulting in a final version with 41 items. The fundamental aim of the questionnaire is to detect, according to the nursing students consulted, the most stressful aspects of their clinical practice. Among the questionnaire items, those concerning the nurses\u2019 relationships with patients, the contact with human suffering and the tasks involved in patient care were the most representative of nurses\u2019 concerns, rather than those concerning nurses\u2019 relations with co-workers or the organisation of responsibilities. The students were instructed to choose one of four response options: No stress = 0, Some stress = 1, Quite a lot of stress = 2, A great deal of stress = 3. The questionnaire addresses a single main construct, but includes nine subscales referring to different types of stress. Factor 1 involves items related to fear of harm (to oneself or the patient) or of being unable to help the patient. Factor 2 refers to situations of contact with suffering; Factor 3, to the relationship with tutors and peers; Factor 4, to feelings of helplessness and uncertainty; Factor 5, to the lack of control over relationships with patients; Factor 6, to emotional involvement, both with the patient and with professional responsibilities; Factor 7, to ill-treatment or lack of consideration by the patient, and the resulting malaise; Factor 8, to behaviour suggesting the patient is seeking an intimate relationship with the student; finally, factor 9 refers to situations of work overload.\u03b1\u00a0=\u00a00.95), considerable reliability (0.72 at two months and 0.68 at six months), and acceptable concurrent validity (0.39 with anxiety- feature). The factor analysis yields nine factors that have a high internal consistency and explain 64.4% of the variance.The KEZKAK questionnaire presents high internal consistency  and completed the State-Trait Anxiety Inventory (STAI) questionnaire and the Podiatry version of the KEZKAK questionnaire.The STAI questionnaire is a generic instrument that is used to measure anxiety, one of the psychological disorders that is most prevalent among the general population, including university students . STAI coAll statistical analyses were performed with IBM SPSS Statistics for Windows, Version 25.0. . Data cleansing was performed by item analysis, applying a corrected item-total correlation of <0.5, unless the item referred expressly to the context of podiatry. Descriptive statistics of the variables were obtained, and the normality of their distribution was confirmed by the Kolmogorov\u2013Smirnov test. Spearman\u2019s test correlations were determined, according to normality. All calculations were performed assuming a 95% confidence interval (95%CI). A test reading was performed, using the Flesch Kincaid Grade Level and the Flesch Reading Ease tests, to assess the readability of the questionnaire . ContentFor the clinimetric validation, interobserver reliability and test-retest reliability were evaluated using Pearson correlation coefficients and intraclass correlation coefficients (ICC). These tests were administered twice by the same observer, at an interval of 21days, and the results obtained were classed as >0.7 \u201cexcellent\u201d, 0.60\u20130.74 \u201cgood\u201d, 0.40\u20130.59 \u201cfair\u201d and <0.40 \u201cpoor\u201d. InternaA total of 223 students were initially recruited to this study. After application of the inclusion and exclusion criteria, 205 third and fourth-year students from the universities concerned finally completed the questionnaires and were included in the analysis. Of these students, 33.5% were male and 66.5% female, with an average age of 23.05 (SD 5.37) years. Only 22.3% were economically active, and their work activity was not related to health care .A linguistic-cultural adaptation of the KEZKAK questionnaire was developed to enable its Spanish-language use with university students of Podiatry. The participants all understood the items in the final questionnaire without requiring any kind of interpretation or assistance. The data obtained were digitised and then cleansed by application of the following threshold for item elimination: total/corrected item correlation <0.4. As a result of this exercise, items K1, K2 and K32, corresponding to \u201cNot feeling part of the work team\u201d, \u201cDoing my job incorrectly and harming the patient\u201d and \u201cReceiving contradictory orders\u201d, respectively, were eliminated from the questionnaire. The Flesch Reading Ease and Flesch Kincaid Grade Level tests were then applied, producing results of 57.4 and 7.9, respectively.The adapted questionnaire obtained excellent internal consistency, with a Cronbach\u2019s alpha of 0.953. The inter-item correlation matrices are shown in No ceiling/floor effect was observed for any item. The KEZKAK questionnaire presented high test-retest reliability after 21 days, with an overall ICC of 0.95 (95% CI [0.93\u20130.98]).Factor analysis was conducted by application of the Kaiser\u2013Meyer-Olkin and Bartlett tests. The adapted questionnaire was found to have five factorial structures . Accordip\u00a0<\u00a00.05)  .The aim of this study was to develop a cultural-linguistic adaptation of the KEZKAK questionnaire to be completed by Spanish university students of podiatry, as a means of measuring stressors during their practicum. We then evaluated this questionnaire and its psychometric properties. The results obtained show that the instrument tested is valid and reliable for this target population.The study population were all volunteers and homogeneous in terms of age, although by sex there was a predominance of female students (66.5%), presumably due to their greater interest in university studies related to the health sciences . This geFive of the initial items were unrelated to the context of podiatry students  and so were excluded from the questionnaire. After filtering the items, the instrument finally obtained consisted of a self-administered questionnaire with 33 items, 5 factors, internal consistency, test re-test reliability, item-total and inter-item correlation and convergent validity to the STAI.The Podiatry questionnaire presented five factors, in contrast to the nine of the original version, However, factorial analysis of the former showed that these five factors accounted for 61.185% of the total variance, a very similar level to that of the original, which had nine factors . These findings support the assumption of construct validity and show that the adapted questionnaire can safely be used to assess the presence and impact of stressors during the practicum of podiatry students. The first factor (17.22%) was composed of items related to feelings of powerlessness in the relationship with the patient. The second (32.99%) concerned the student\u2019s perceived lack of professional skills. The third (44.51%) was related to emotional involvement and uncertainty, the fourth (55.96%) to relationships with tutors and peers, and the fifth (61.18%), to the student\u2019s ability to relate to patients when they were given bad news or were suffering.The difference in factorial structure between the adapted questionnaire and the original version may have arisen from inherent differences in the two branches of university studies (Nursing vs. Podiatry). Although both correspond to the field of health sciences, they differ regarding the clinical practices required and the relationship and degree of involvement with the patient during daily practice . In this\u03b1\u00a0=\u00a00.95 and \u03b1\u00a0=\u00a00.935, respectively). No floor or ceiling effects were observed. The Podiatry version of KEZKAK presented convergent validity, with weak correlation for the STAI, which is in line with the original version and indicates that this questionnaire is capable of identifying stressors encountered during the practicum of podiatry students. In other words, it does not measure the degree of stress experienced by the student, but identifies potentially stressful situations , but falls short of the excellent internal consistency we obtained (\u03b1\u00a0=\u00a00.95). The ASNS instrument consists of 30 items and six domains, in contrast to the Spanish-language version of KEZKAK, adapted for podiatric students, which consists of five factors and explains 61.185% of the total variance. No factor analysis was performed in the ASNS adaptation, and so the number of domains might have been modified. Neither was the respondent population fully identified , and so it is unclear the extent to which these students had previously had contact with the clinical practices of podiatry.In the field of podiatry, the only measurement instrument comparable with ours is the Spanish-language version of the ASNS instrument for podiatric students. However, this is used to mediate stress factors, not the situations that may provoke stress, such as clinical practices with real patients during the practicum. The ASNS instrument presents good reliability  age; job ; K (number of questionnaire questionnaire); SAE (number of STAI questionnaire); FAC Click here for additional data file.10.7717/peerj.10439/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10439/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10439/supp-4Supplemental Information 4Click here for additional data file."}
+{"text": "Background and Purpose: Acute ischemic stroke (AIS) with large artery occlusion (LAO) may lead to severe disability or death if not promptly treated. To determine the source of cerebral artery occlusion thrombosis, we studied the pathological components of cerebral artery thrombosis with different etiological classifications to guide clinical formulation of preventive treatment.Materials and Methods: Eighty-eight thrombi from AIS patients with LAO, 12 atrial thrombi from patients with valvular heart disease (VHD), and 11 plaques obtained by carotid endarterectomy (CEA) from patients with carotid artery stenosis were included in this retrospective study. The hematoxylin and eosin\u2013stained specimens were quantitatively analyzed for erythrocytes, white blood cells (WBCs) and fibrin; platelets were shown by immunohistochemistry for CD31.Results: The thrombi of VHD showed the highest percentage of fibrin, followed by those of cardioembolism (CE) and stroke of undetermined etiology (SUE), and these values were higher than those of the other groups. Plaques obtained by CEA showed the highest erythrocyte number, followed by the large artery atherosclerosis (LAA) thrombi, and showed significantly noticeable differences between other stroke subtypes. The proportions of fibrin and erythrocytes in the thrombi of CE and SUE were most similar to those in the thrombi of VHD, and the LAA thrombi were the closest to those obtained by CEA. CE thrombi and CEA plaques had a higher percentage of WBCs than thrombi of other stroke thrombus subtypes and VHD.Conclusions: CE and most cryptogenic thrombi may originate from the heart, and the formation of carotid atherosclerotic plaques may be related to atherosclerotic cerebral embolism. Inflammation may be involved in their formation. Acute cerebral large artery occlusion (LAO) may lead to severe disability or even death if the patient does not have access to prompt treatment. In recent years, the benefit of endovascular therapy has been demonstrated in patients. Embolic clots of the cerebral large arteries may come from deciduous cardiac valvular thrombi or intracranial/extracranial large artery plaques. Thus, it is important to identify the etiology of LAO in clinical treatment . AlthougStaessens et al. found that all thrombi were composed of platelet-rich regions and erythrocyte-rich regions by analyzing the composition and internal structure of AIS thrombi retrieved from endovascular therapy; platelet-rich areas are composed of fibrin, von Willebrand's factor (vWF) and platelets. They also suggested that platelet-rich thrombi based on vWF and DNA as well as dense fibrin were the main reason for the failure of intravenous thrombolysis, and the histological characteristics of platelets, vWF and fibrin networks in thrombi were observed by fluorescence microscopy . Recent (1) This retrospective study comprised 263 Chinese patients with LAO from January 2017 to March 2019. This study was conducted according to the recommendations of guidelines from the Institutional Review Board of the First Affiliated Hospital of Jinan University. All protocols and procedures of our research were carried out in conformity to the Helsinki Declaration. All patients who were treated in our hospital signed informed consent for medical research of their images and specimens.The inclusion criteria included patients over 18 years old; all patients who had been treated by intra-arterial mechanical thrombectomy with thrombus were retrieved for histopathological analysis, and LAO patients who did not have visible thrombi for analysis were excluded from this study. Data regarding patient demographics, clinical presentation, treatment strategies, outcome, imaging findings, and stroke pathogenesis were collected. The stroke subtypes were classified using the Trial of Org 10172 in Acute Stroke Treatment classifications . Drinkin(2) Valvular heart disease (VHD) patients with a left atrial thrombus removed during cardiac valvectomy or replacement were enrolled retrospectively at the same time. The inclusion criteria were age >18 years old, patients who underwent valvular surgery for valve lesions such as mitral stenosis or aortic stenosis, and patients who had thrombus material for histopathological analysis.(3) Patients who underwent carotid endarterectomy (CEA) and had carotid atherosclerotic plaque specimens were included retrospectively at the same time. Patients with carotid atherosclerotic plaque stenosis over 75% and >18 years of age were included.Atrial thrombi were obtained during cardiac surgery, carotid atherosclerotic plaques were obtained from carotid endarterectomy, and emboli were retrieved during mechanical thrombectomy (stent-retriever and contact aspiration). All samples were flushed with 0.9% saline, gently placed on sterile gauze wipes, and then fixed in 10% buffer formalin for 2\u20138 h. The volume of the formaldehyde was approximately 10 times the size of the thrombus. After the sections were treated with solvents and embedded in paraffin, the thrombus organization was identified in the largest section of 6 serial sections that were each 4 \u03bcm; these sections were stained with hematoxylin and eosin. Another section was immunohistochemically stained for CD31  by stepwise procedures. All thrombus slices were dried in a 60\u00b0C oven for 30 min, dewaxed in xylene and rehydrated in decreasing ethanol grades. A 6% solution of hydrogen peroxide in water and the biotin-blocking reagent were used to block endogenous peroxidase and endogenous biotin successively. Primary antibodies against CD31 were added and incubated for 22 min at normal temperature, followed by streptavidin-peroxidase conjugation and incubation for 22 min. The sections were counterstained with hematoxylin, dehydrated in increasing grades of ethanol, cleared in xylene and mounted.For the H&E staining images, red represents red blood cells; pink regions represent fibrin; and blue dots represent WBCs. For CD31 immunohistochemical staining, dark brown represents platelets, and other components are not colored . HistoloThe data were assessed using ANOVA or the Kruskal-Wallis method, depending on the type of data. The correlation analysis between basic information about the participants and the structure of thrombosis was calculated by Spearman's method. All the analyses above were performed by SPSS (Version 23).n = 12) had valvular heart disease, and nine patients had atrial fibrillation. All patients in the CEA group (n = 11) had no atrial fibrillation. There were statistically noticeable differences among the VHD, CEA, and LAO groups in the proportion of patients with atherosclerotic coronary heart disease and atrial fibrillation. There were significantly more VHD patients with atherosclerotic coronary heart disease and atrial fibrillation in the VHD group than in the other two groups (n = 46), (2) large artery atherosclerosis , and (3) stroke of other determined etiology , including patients with arterial dissection, (4) stroke of undetermined etiology , (5) small-artery occlusion . LAA is defined as artery-to-artery embolism in our study, excluding in-situ atherothrombosis. Among the LAO subtypes, the proportion of coronary atherosclerotic heart disease and atrial fibrillation in the CE group was significantly higher than that in the other three groups. The distribution of the National Institutes of Health Stroke Scale (NIHSS) score and Alberta Stroke Program Early CT Score (ASPECTS) before thrombectomy and the modified Rankin Scale (mRS) at 3 months after discharge were the same in each group. There was no significant difference among the subtypes . The quantification of fibrin, red blood cells and platelets was used to measure the area covered by various components in the image [%], and the WBCs were used to automatically measure the particle number of the threshold set .VHD thrombi showed the highest percentage of fibrin, the lowest percentage of red blood cells and the lowest WBC count. The percentage of CEA was the lowest, and CEA thrombi showed the highest numbers of red blood cells and WBCs. The percentage of LAO was between them and showed statistically noticeable differences.The percentages of fibrin in thrombi of the CE and SUE subtypes were higher than those in thrombi of the LAA and SOE subtypes, showing statistically noticeable differences between other stroke subtypes. LAA thrombi showed the highest red blood cell count among stroke subtypes but less fibrin . ThrombiIn this study, we analyzed the histological characteristics of thrombi from patients with acute stroke of LAO and compared the histological composition with atrial thrombi and carotid atherosclerotic plaques for the first time, which showed a stronger reference. The sample size in the present study was larger than those in many previous studies.In our cases, there were significantly more VHD patients with atherosclerotic coronary heart disease and atrial fibrillation than in the other two groups, suggesting that coronary heart disease and atrial fibrillation are both important risk factors for cardiac thrombosis. The age of the patients, sex, hypertension, diabetes, hyperlipidemia, smoking, alcohol consumption, history of stroke or transient ischemic attack, and long-term bedridden status were not different among the LAO, VHD, and CEA groups. All included patients with different stroke subtypes had no significant differences in baseline data, preoperative NIHSS scores, ASPECT scores or mRS scores 3 days after the operation. The proportion of patients with CE thrombi with coronary atherosclerotic heart disease and atrial fibrillation was significantly higher than that among the other three subtypes, which was related to the TOAST classification criteria.It is worth noting that thrombus composition plays a crucial role in recanalization therapy in LAO patients . We founWe found that the level of WBCs in the CE thrombus of LAO was significantly higher than that in the VHD group and higher than that in the other subtypes of acute LAO. Boeckh-Behrens et al. studied the histopathology of 34 patients with acute anterior circulative stroke and found that the WBC content of CE thrombi was significantly higher than that in thrombi of other causes of acute stroke , which wOur findings suggested that thrombi in the CEA group had the highest proportion of red blood cells, followed by those in the LAA group, whose red blood cell content was higher than that in thrombi of the other LAO subtypes. Niesten et al.  studied Our study found that the plaques of the CEA group were rich in WBCs. Many studies have shown that inflammation plays many key roles in the development and progression of atherosclerosis , 44 and Currently, the SUE thrombi showed histological characteristics and compositions similar to those of cardiogenic thrombi seen in several studies \u201310, suggThe SOE group in our study included patients with arterial dissection. These thrombi were characterized by a higher proportion of red blood cells and significantly less fibrin content than atrial thrombi of VHD, suggesting that such thrombi might be emboli caused by the formation and shedding of a hematoma in the arterial wall. In a previous study of three patients with arterial dissection, thrombi of mixed but predominantly red blood cells were observed , and thrIn conclusion, the proportions of fibrin and red blood cell content of CE and SUE thrombi were the closest to those of atrial thrombi in VHD. CE thrombi originated from cardiac thrombi, suggesting that most SUE thrombi may also originate from the heart. The proportions of red blood cells and fibrin in thrombi in the LAA group were close to those in thrombi in the CEA group, suggesting that the formation of carotid atherosclerotic plaques may be related to stroke of LAA. These results provide a reference for the study of embolus sources. There were significantly more WBCs in the CE group and plaque in the CEA group than in the atrial thrombus group, suggesting that in addition to traditional thrombolysis and thrombectomy, anti-inflammatory therapy may improve the success rate of recanalization in the treatment of CE stroke. For patients with carotid artery plaque and stenosis, early anti-inflammatory therapy may have a certain inhibitory effect on plaque rupture or carotid artery occlusion, which provides a theoretical basis for clinical treatment.A limitation of this study is that it is a preliminary and retrospective study. Second, the composition statistics of thrombi or plaques in each group were averages, which cannot exclude the existence of the same type of thrombus in different subtypes of a cerebral thrombus. For example, red thrombi were found in both the LAA and CE groups; thus, it is not simply considered that red thrombi are from strokes of the LAA or CE. The results of this study provide only a reference for the etiological source of stroke. Furthermore, intravenous thrombolysis may also affect the composition of the thrombus, but the proportion of such cases is small. However, the samples we selected were all previously treated with intravascular therapy after thrombolysis failure, and the proportion of thrombolysis was small; thus, this effect was excluded.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/All patients who were treated in our hospital signed informed consent for medical research of their images and specimens. This study was conducted according to the recommendations of guidelines from the Institutional Review Board of the First Affiliated Hospital of Jinan University. All protocols and procedures of our research were carried out in conformity to the Helsinki Declaration.YL and MG performed all the pathological experiments, performed data analyses, and wrote the manuscript. DL and SH performed the patients' TOAST classification. YS and JL performed data analyses. XZ, XX, and DY wrote and edited the manuscript. LH and HQ contributed to the conception and design of the study and wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "O-glucoside (C3G)-rich haskap (Lonicera caerulea L.) berry extracts can attenuate the carcinogen-induced DNA damage in normal lung epithelial cells in vitro. Here, the efficacy of lyophilized powder of whole haskap berry (C3G-HB) in lowering tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)-induced lung tumorigenesis in A/JCr mice was investigated. Three weeks after daily oral administration of C3G-HB (6 mg of C3G in 0.2 g of C3G-HB/mouse/day), lung tumors were initiated by a single intraperitoneal injection of NNK. Dietary C3G-HB supplementation was continued, and 22 weeks later, mice were euthanized. Lung tumors were visualized through positron emission tomography (PET) and magnetic resonance imaging (MRI) 19 weeks after NNK injection. Dietary supplementation of C3G-HB significantly reduced the NNK-induced lung tumor multiplicity and tumor area but did not affect tumor incidence. Immunohistochemical analysis showed reduced expression of proliferative cell nuclear antigen (PCNA) and Ki-67 in lung tissues. Therefore, C3G-HB has the potential to reduce the lung tumorigenesis, and to be used as a source for developing dietary supplements or nutraceuticals for reducing the risk of lung cancer among high-risk populations.In our previous study, we demonstrated that cyanidin-3- Lung cancer is the most commonly diagnosed cancer , and the leading cause of cancer deaths  among both men and women worldwide. Among western populations, over 80% of lung cancer incidence is attributed to tobacco smoking ,3. In thin vitro, in vivo, and epidemiological studies, have reported the benefits of flavonoids and flavonoid-rich plant extracts in preventing or curing cancer, including lung cancer [Lonicera caerulea L.), also known as blue honeysuckle, is a berry fruit with abundant anthocyanin, particularly cyanidin-3-O-glucoside (C3G). Haskap berry has a higher antioxidant capacity than other common fruits [in vitro [in vivo.Numerous g cancer ,9. For eg cancer . Anthocyg cancer ,12,13. Hn fruits ,15. Recen fruits ,17, antin fruits , antioben fruits , and antn fruits  propertiin vitro . The objTwo mice from groups C3G-HB supplemented diet continuously before and after NNK-injection (conti.-C3G-HB) and C3G-HB supplemented diet only after NNK-injection (post-C3G-HB) were euthanized due to weight loss and eliminated from the study. Observations for symptoms of stress, i.e., changes in fur color or texture, food consumption, and behavioral abnormalities such as hunched posture, fast movements, and vocalization, were performed daily.The nutritional composition of the C3G-HB cv. Tundra is presented in t-test, p < 0.0001) in comparison to the control and no-C3G-HB groups. For instance, at the termination, the body weight of mice given C3G-HB supplement and NNK was reduced by 3.5% (naive vs. control) and 7.6%, respectively (naive vs. no-C3G-HB). Conversely, long-term C3G-HB supplementation significantly (p < 0.001) increased the body weight of NNK-injected mice by 2% (no-C3G-HB vs. conti.-C3G-HB), and 3.2% (no-C3G-HB vs. post-C3G-HB), respectively  (Dietary supplementation of C3G-HB and NNK injection affects the body weight of mice A. Body wectively A. In fac 0.0035) B.p > 0.05) from no-C3G-HB group.PET/MRI images confirmed the presence of tumors in the lungs of NNK-injected mice A. The efThe tumor incidence was not affected by the consumption of the C3G-HB dietary supplement. The tumor incidence of NNK-injected mice was 100% (10/10 and 9/9). Untreated mice  in naive group; 2 out of 5 mice (0.4 \u00b1 0.2) and control; 1 out of 5 mice (0.2 \u00b1 0.2) showed one \u201cspontaneous\u201d tumor on their lungs.p < 0.0001) tumor area in the no-C3G-HB group that received NNK and the control diet. The tumor area in each section was calculated using ImageJ software. The tumor burden in NNK-injected mice was 21.6 \u00b1 4.1. Tumor area was significantly reduced in NNK-treated mice that received the C3G-HB-supplemented diet; 7.6 \u00b1 2.8 (pre-C3G-HB), 7.1 \u00b1 0.6 (conti.-C3G-HB), and 6.9 \u00b1 0.6 (post-C3G-HB), and accordingly reduced by 64.7%, 67.3%, and 68.1%, respectively.The lung tumor area was measured in three consecutive lung sections, representing three depths  of the lungs . The H ap < 0.0001) in the lungs of NNK-injected mice  relative to the saline-injected control mice (naive and control groups). The expression of PCNA was significantly higher compared to that of Ki-67. The level of PCNA and Ki-67 was significantly (p < 0.0001) reduced in the lungs of NNK-injected mice that were fed C3G-HB. As a percentage, the expression of PCNA was decreased by 41% to 64%  reduced the NNK-induced lung tumor multiplicity, the most sensitive indicator of potency [w/v C3G-rich pomegranate juice [C3G-HB (6 mg of C3G in 0.2 g of lyophilized whole haskap berry power/mouse/day) significantly  inhibits cell proliferation through deactivating MAPK and PI3K/AKT signaling pathways, which are activated by NNK at cancer progression [PCNA is necessary for DNA synthesis (a processivity factor of DNA polymerases) and DNA repair (involved in nucleotide excision repair and base excision repair). PCNA is highly expressed during the active cell cycle; Gll cycle . Similarll cycle ,4,5, NNKgression ,13,32. Tin vivo.Regardless of the feed intake, the C3G-HB supplement reduced the body weight of the mice by 3.5% comparison to the regular diet (control vs. naive). C3G enhances energy metabolism by upregulating brown adipose tissue mitochondrial function . IngestiIn humans, C3G metabolism generates various metabolites such as C3G glucuronides, methylates of the C3G, i.e., peonidin-3-glucoside, and simple phenolic acids including protocatechuic acid, ploroglucinaldehyde, and hippuric acid ,38,39. EThis study was performed at Dalhousie University\u2019s animal care facility, following the approval of the University Committee on Laboratory Animals (protocol 15\u2013106). The carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone  was purchased from Toronto Research Chemicals Inc., Toronto, ON, Canada. Frozen haskap berry cv. Tundra was obtained from LaHave Natural Farm, Blockhouse, NS, Canada. Female A/JCr albino mice at 3\u20134 weeks age (n = 50) were purchased from Charles River Laboratories, Inc., Montreal, QC, Canada.Frozen haskap berries were lyophilized, ground to a fine powder, and stored at \u221280 \u00b0C. A representative sample was analyzed to determine the nutrient composition . C3G was quantified by high-performance liquid chromatography and mass spectrophotometry  after extraction (1 mg/mL) using methanol containing 1% acetic acid and filtered through a 0.22 \u00b5m nylon filter . 2). The Km factors of mouse and adult human are 3 and 37, respectively [Ingestion of 1.5 g polyphenols/day for a healthy adult of 70 kg body weight is considered to be a health-promoting dose ,43. A heectively .\u00ae. C3G-HB powder was mixed thoroughly for 20 min to obtain a homogeneous preparation for use in making pellets. Pellets were prepared every two days and stored in sealed containers in the dark at 4 \u00b0C.Accordingly, the experimental diet/mouse/day consisted of 0.2 g C3G-HB  and 5% Splenda\u00ae mixed into regular mouse chow  and formed into a 2 g (dry weight) pellet. The control diet consisted of regular mouse chow containing 5% Splenda\u00ae RMH 3000 diet and distilled water were provided ad libitum. The C3G-HB or control pellets were given daily as a dietary supplement. Briefly, mice in naive and no-C3G-HB groups were given control pellets, while the diet of control, pre-C3G-HB, conti.-C3G-HB and post-C3G-HB groups was supplemented with C3G-HB  at the IWK Children\u2019s Hospital, Halifax, NS, Canada. Briefly, three mice from naive and no-C3G-HB groups were randomly selected and fasted for six hours and then injected with v/v) acetate-buffered formalin. Peripheral lung tumors were enumerated using a dissecting microscope. Lungs were embedded in paraffin, and paraffin-embedded tissues were stored for histopathological examination. Twenty-two weeks after NNK treatment, mice were anesthetized with isoflurane. Blood samples were collected by cardiac puncture, and a higher dose of isoflurane was used to sacrifice mice. Dissected lungs were perfused and washed in phosphate-buffered saline (PBS) before being fixed in 10% (Paraffin-embedded lungs were cut into 5 \u00b5m thick sections (50 tissue sections/lung) using a microtome . Three sections representing three areas of the lungs were selected at predetermined depths and stained with hematoxylin and eosin (H and E). The H and E-stained lung sections were imaged under bright field microscopy , and lung tumor area was quantified using ImageJ software . 2O2 in Tris-buffered saline (TBS) for 10 min. Non-specific binding sites were blocked by incubation with rodent block (M) from Biocare Medical, Pacheco, CA, USA. The sections were incubated overnight at room temperature in a humid chamber with monoclonal antibodies against PCNA (1:6000 dilution) or Ki-67 (1:50 dilution). After several washes with TBS, the slides were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody  for 30 min, then washed three times with TBS and incubated with chromogen 3-diaminobenzidine  for 3 min. The slides were carefully rinsed under running tap water and counterstained with hematoxylin. The slides were observed under bright field microscopy  at 200\u00d7 magnification.The expression of proliferating cell nuclear antigen (PCNA) and Ki-67 was evaluated by immunohistochemistry (IHC). Briefly, paraffin sections were deparaffinized in xylene, rehydrated through an ethanol solution gradient and washed carefully in running tap water. Antigen retrieval was carried out by heating sections in 0.01 M citrate buffer (pH 6.0) for 30 min in the decloaking chamber. Endogenous peroxidase activity was quenched by incubating the section in 3% HThe observed differences in the tumor multiplicity, tumor area, PCNA and Ki-67 expression were tested for statistical significance using one-way Analysis of Variance (ANOVA). Tukey\u2019s pairwise comparison and Dunnett\u2019s test with a 95% confidence interval was used for comparisons among multiple groups. Minitab statistical software was used for data analysis.In summary, we have demonstrated that dietary supplementation of C3G-HB can inhibit the NNK-induced lung tumorigenesis in A/JCr mice. C3G-HB may be a promising dietary supplement to suppress lung cancer development among high-risk populations such as smokers, possibly via effects on critical cellular signaling pathways that regulate cell proliferation. Future studies of the effects of C3G-HB on phase I and phase II metabolic enzymes and cell signaling pathways will elucidate the mode of action of C3G-HB against lung carcinogenesis."}
+{"text": "The aim of this study was to examine the efficacy and the shortfalls of the Birmingham Eye Trauma Terminology classification system for ocular trauma in predicting the visual outcome.The records of 256 eyes of 246 patients with a diagnosis of mechanical ocular trauma admitted to the Osman Gazi University Hospital ophthalmology department between 1995 and 2000 were retrospectively reviewed. The zone, type, grade, and pupil status of the injuries were determined according to the Birmingham classification system. Injuries with a good prognosis were defined as injuries that resulted in vision of equal to or better than counting fingers at 1 meter. Fischer\u2019s exact test was used to determine the statistical significance of relationships between the final visual acuity and the initial clinical findings.Open eye injuries restricted to zone I, those with no afferent pupillary defect, and those graded as 3 or better or classed as type B were significantly associated with a better visual outcome (p<0.05). Open eye injuries that extended to zone III, had an afferent pupillary defect, or were graded as 4 or worse were significantly associated with a poorer visual outcome (p<0.05). Closed eye injuries classified as type B or grade 4 were significantly associated with a poor visual outcome (p<0.05).The Birmingham classification system for mechanical ocular trauma offers a standardized method for both open and closed eye injuries, however, adding subclasses to type C (injuries with foreign body involvement) could enhance the classification method and help to understand the influence of foreign body properties and sizes on the outcome. Eye injuries are serious ocular incidents that constitute 10\u201315% of all ophthalmic diseases with a worldwide incidence of more than 55 million/year . In ScotThe initial clinical findings and the final visual outcomes of 256 eyes of 246 patients who had eye injuries between June 1995 and June 2000 were retrospectively evaluated.\u2022All eye injuries attended Osmangazi University Hospital ophthalmology department between 1995 and 2000The following criteria were excluded from the study:\u2022Patients with incomplete or missing clinical notes\u2022Patients who had parts of their treatment continued in other units\u2022Patients who had follow up periods of <14 days.The following data were collected from the notes: Date of surgery, type of injury, and laterality, whether the injury was open or closed, mechanism of injury (blunt or sharp), presence of intraocular foreign body, the results of ancillary examinations including computed tomography scans, presence of afferent pupillary defect, the extent of the injury noted during clinical examination, the extent of the injury noted during surgery, the duration of follow-up, and visual acuity at the time of initial presentation and at the final follow-up visit were recorded using decimal values. Cases with a follow-up period of a minimum of 15 days and a maximum of 4 years were included in the study. Kuhn\u2019s terminology and Birmingham classification were applied after the completion of data collection .Eye injuries were first divided into open and closed eye injuries depending on the presence or the absence of a full-thickness defect in the eye wall. Although, posterior to the limbus the eye wall consists of three layers, clinically, the term eye wall was used only to denote the rigid structures of the cornea and the sclera. Eye trauma with full-thickness wounds in the eye wall  was classified as open eye injuries. Injuries without full-thickness wounds were called closed eye injuries.Open eye injuries were then divided into four types according to the mechanism of injury. Full-thickness injuries caused by a blunt object were classified as type A (rupture). Single and full-thickness injuries caused by sharp objects in the eye wall were classified as type B (penetrating injury). Injuries with a foreign body in the eye were classified as type C (intraocular foreign body). Double full-thickness injuries in the eye wall that constituted an entry and an exit site were classified as type D (perforating injury).Both open and closed eye injuries were then divided into five grades according to their visual acuity at the time of initial presentation. Injuries presenting with a visual acuity of 0.5 or better were classified as Grade 1, injuries presenting with visual acuities ranging between 0.4 and 0.2 were classified as Grade 2, injuries presented with visual acuities ranging between 0.1 and counting fingers at 1 m were classified as Grade 3, and injuries with visual acuities ranging between counting fingers at less than 1 m and light perception were classified as Grade 4. Injuries with presented with visual acuities of no light perception were classified as Grade 5.The injuries were then classified into pupil positive and pupil negative depending on the presence and the absence of relative afferent pupillary defects. The presence of a relative afferent pupillary defect in the injured eye was evaluated as pupil positive, while the absence of relative afferent pupillary defect in the injured eye was considered as pupil negative.Open eye injuries were also classified into three zones according to the anatomical structures involved. Injuries limited to the cornea  were classified as zone I, injuries involving areas between the corneoscleral limbus and 5 mm posterior to the corneoscleral limbus were classified as zone II, and injuries extending beyond the anterior 5 mm of the sclera were evaluated as zone III. In eyes with multiple open corneoscleral injuries, the zone was defined by the most posterior injury. In perforating injuries, the zone was defined by the most posterior eye wall defect. However, the zone of the injury could be determined at the time of the presentation depending on the initial clinical findings. In our study, the precise zones of the injuries were determined according to the surgical findings as this would have provided more accurate estimation for the zone.Similar to open eye injuries, closed eye injuries were also divided into types according to the mechanism of the injury. Closed eye injuries caused by blunt objects were classified as type A (contusion). Closed eye injuries caused by sharp edged objects were classified as type B (lamellar laceration), closed eye injuries caused by projectile objects with a foreign body embedded in the conjunctiva and/or the eye wall  in the absence of full-thickness defect were classified as type C , and last, injuries caused by several mechanisms were classified as type D (mixed).Closed eye injuries were also classified into pupil positive and pupil negative in the same way as open eye injuries. Closed eye injuries were also divided into three zones depending on the anatomical structures involved. Zone I injuries were limited to the external bulbar conjunctiva (sclera and cornea) and classified as zone I. Injuries falling behind the cornea and involving any of the structures within the anterior segment including pars plica but not the pars plana were considered zone II. Injuries involving structures posterior to the posterior lens capsule were classified as zone III injuries.Finally, both closed and open eye injuries were classified into injuries with good prognosis and injuries with poor prognosis, depending on the visual outcome of the injury at the final follow-up visit. Injuries with a final visual outcome of counting fingers at 1 or better vision were classified as injuries with good prognosis and injuries with a final visual outcome of worse than counting fingers at 1 m were classified as injuries with good visual outcome .Two-sided Fisher\u2019s exact test was computed using SPSS statistical software package version 19 . This determines the P value of the association of each category of the classification with the final visual outcome category. P<0.05 was regarded as statistically significant.A total of 205 male and 51 female patients were admitted to Osmangazi University Hospital with the diagnosis of an eye injury between June 1995 and June 2000. The mean age of males was 30 years (SD 16.5) (range 1\u201375). The mean age of females was 19.9 years (SD 17.9) (range 1\u201362). About 47.2% of the patients had injuries to the right eye, and 48.8% had injuries to the left eye. The injuries were bilateral in 4.1% of the patients. The grade of the injury was not determined in eight eyes with open eye injuries and in three with closed eye injuries due to poor cooperation. Open eye injuries that were classed as zone I, pupil (-), Grade 3 or better, or type B showed statistically significant association with good visual outcome (p< 0.05). While open eye injuries that were classed as zone III, pupil (+), Grades 4 and 5 showed statistically significant association with poor visual outcome (p<0.05). Closed eye injuries that were classed as type B and Grade 4 showed statistically significant association with poor visual outcome (p<0.05). Type E open eye injuries which are caused by multiple mechanisms were associated with the highest rate of poor visual outcome followed by Type D penetrating open eye injuries which were caused with an entry and an exit site. This was in line with other studies that reported bad prognostic outcomes in such types of eye injuries -11. SimiOpen eye injuries with intraocular foreign bodies which were classified as type C in our study were found to have better prognosis than types A, D, and E. Such reasonable outcomes after well-managed intraocular foreign body (IOFB) have been reported in the previous studies also , 12, 14.In line with other studies, open eye injuries that had good visual acuities at the time of presentation did better than those who presented with poor initial visual acuities. Open eye injuries graded as Grades 1, 2, and 3 in our study, had statistically significant better outcome compared to those graded as 4 and 5 (p<0.05). The importance of good visual acuity at the time of presentation in open eye injuries has been reported in the previous studies as well .Relative afferent pupillary defect reflects the function of the optic nerve and ganglion cell layer . In our The zone of the injury gives an insight into the number of the anatomical structures involved. Our study showed that open eye injuries not extending beyond zone I, had significantly better final visual outcome compared to those that extended to zone II, which, in turn, had significantly better prognosis than those extended to zone III (p<0.05). The previous studies also showed that open eye injuries restricted to the cornea had better final visual outcomes, with only 27\u201352% of such injuries having a final visual acuity of worse than 0.1, while 30\u201359.4% of those that extended to zone II had a final visual acuity of worse than 0.5. In comparison, 94% of open eye injuries extending beyond 5 mm of the corneoscleral limbus had a final vision of worse than 0.3 .In relation to closed eye injuries, similar to the previous studies, our study also showed that closed eye injuries caused by blunt objects, had a better final visual outcome. This compared to open eye injuries cause by similar objects, with only 6.1\u201320.8% of closed eye injuries caused by blunt objects had a final vision of worse than 0.7 -30. A coHyphema, traumatic iritis, iris sphincter rupture, angle recession, iridodialysis, iridodonesis, traumatic cataract, and phacodonesis are common findings in type A closed eye injuries that have extended to zone II. Temporary vision loss is reported in such injuries in 4.3\u201350% -30. It iVitreous base detachment, intravitreal hemorrhage, retinal hemorrhage, commotio retinae, choroidal rupture, and retinal pigment epithelial edema were the main findings in type A closed eye injuries that have extended to zone III. Final visual acuities were reported to be worse than 0.7 in 4.3\u201353.2% of such injuries .Type B closed eye injuries mainly included lamellar lacerations in the conjunctiva and cornea caused by sharp objects while Type C injuries included superficial foreign bodies. Such injuries are usually treated within outpatients. Therefore, the number of patients that fall into these categories in our study did not reflect the actual total numbers presented to the eye clinic with this kind of injuries. Among those who fell into these categories and at the same time required hospital admissions, were patients who had worse fellow eye injuries, mental health issues. In one case, a patient developed corneal stromal infiltration with hypopyon and the reason for admission was to commence intensive topical treatment.One of the shortfalls of the study was its retrospective design, however, every effort was made to ensure meticulous collection of data from the notes to enhance the accuracy of the study. Another limitation of the study was its relative short follow-up period of a maximum of 4 years, nevertheless, the authors believe that a 4-year period was long enough to ascertain the final visual acuity in most ocular trauma taking into consideration that some of the consequences, for example ,secondary glaucoma and cataracts could take longer to establish.Birmingham classification for mechanical ocular trauma offers a standardized method for both open and closed eye injuries, however, adding subclasses to type C (injuries with foreign body involvement) could enhance the classification method and help to understand the influence of foreign body properties and sizes on the outcome. In open eye injuries, the final outcome is significantly dependent on the zone, pupil status, and the grade of the injury, while in closed eye injuries, the final outcome is significantly related to the type of the injury.Authors would like to thank Sister Vivienne Padfield RGN, RM, BSC honors, MA for her contribution, read proofing, and correcting grammar and punctuation errors in the paper.Osman Gazi University, applied as requirement for MD Thesis, number 650428, date 27/03/2001.Externally peer-reviewed.None declared.Involved in design and conduct of the study ; preparation and review of the study (SKE)."}
+{"text": "Pemphigoid (Pg) diseases are a group of potentially fatal autoimmune mucocutaneous diseases. They have different clinical phenotypes, involving only the skin or multiple mucous membranes. They occur globally and frequently affect the elderly. The common marker among all variants is the presence of autoantibodies targeting the dermal-epidermal or mucosal-submucosal junctions, or basement membrane zone (BMZ). Four target antigens in the BMZ were studied. These included BPAG1, BPAG2 and subunits of \u03b16 and \u03b24 human integrins. Our objective was to find a molecular basis for the global incidence of Pg diseases and a mechanism that will explain the vast differences in clinical phenotypes and outcomes. All the variants of Pg that were analyzed had a statistically significant association with HLA-DQ\u03b21*03:01 in ten countries on four continents. This explains the reason for global incidence. Prediction models discovered multiple peptides in each of the four antigens that serve as T cell epitopes. These T cell epitopes were shown to bind to HLA-DQ\u03b21*03:01. In addition, structure modelling demonstrated the peptide-HLA complex bound to the T cell receptor. These autoreactive T cells would stimulate B cells to produce specific anti-BMZ autoantibodies. Anti-BMZ autoantibodies with different specificities will produce different phenotypes, which will account for involvement of different tissues and organs in different molecules. The contribution this study makes is that it provides a molecular basis of why a similar disease occurs in different racial groups. Furthermore, it provides the basis for the production of autoantibodies with different specificities, which resultantly produces different phenotypes. Pemphigoid (Pg) is a group of autoimmune blistering diseases that can affect the skin and multiple mucous membranes . They caThe lesions in bullous pemphigoid (BP) are vesicles and bullae of variable sizes, in an acral or generalized distribution . In someMucous membrane pemphigoid (MMP) predominantly affects multiple mucosae and not infrequently the skin. As the blisters heal, they cause scarring, which is the reason for its former name, cicatricial pemphigoid. In most patients, this scarring produces sequelae that are catastrophic and severely impact the quality of life , 6. Of sin vitro, they bind to the BMZ, when studied by histology and immunopathology. When injected into laboratory animals, they produce blisters in vivo  in the perilesional tissues . This is in vivo , 10. SucThe pathognomonic and unique feature of MMP is that it causes scarring as it heals, except in the oral cavity . ScarrinIn BP, the anti-BMZ autoantibodies are directed against BP antigen 1 (BPAG1 or BP230) and BP antigen 2 (BPAG2 or BP180) , 15. In Earlier studies from our group demonstrated that patients with BP, MMP, and its subsets had a strong correlation with the HLA-DQ\u03b21*03:01 allele, in spite of strikingly different clinical presentations, courses and prognoses \u201322.Recently, a new group of drugs has been added to the treatment of type II diabetes mellitus, known as dipeptidyl peptidase-4 inhibitors (DPP4-is). Many patients treated with these drugs have been reported to develop pemphigoid .The purpose of this study was to determine the global presence of pemphigoid diseases. Furthermore, to investigate the molecular basis for the global presence and simultaneously to study what mechanisms might explain such striking differences in clinical profiles and clinical outcomes, and the reasons for production of autoantibodies to diverse proteins in the BMZ.PubMed, Embase, and Medline searches were conducted using the following key words: bullous pemphigoid, mucous membrane pemphigoid, cicatricial pemphigoid, oral pemphigoid, BMZ proteins, anti-BMZ antibodies, and HLA genotyping.Inclusion criteria included (i) the presence of the location of the study, (ii) the ethnicity or race of the patients and the control group being identical, (iii) data on HLA genotyping, (iv) clinical profile of patients to confirm the clinical diagnosis, and confirmation of diagnosis by histology and immunopathology.Exclusion criteria included (i) inadequate or incomplete clinical description of the patients or the control group and/or absence of histology and immunopathology and (ii) presence of inappropriate (age and sex matched) control group.The data available on HLA typing results in patients with BP, MMP, and patients with diabetes mellitus treated with DPP4-i drugs who developed pemphigoid, formed the database for this study. In each study, presence of data on adequate controls was carefully analyzed.via written communication by the author.HLA studies were conducted at different centers worldwide. The results were used in the database as provided by the authors. In two studies regarding MMP, data was reported in the studies as allele frequencies and was converted into patient frequencies , 24. In Statistical significance of the difference in frequency of HLA DQ\u03b21*03:01 between patients and controls was estimated by Chi-square test and Yates\u2019 correction (SPSS 27). A p value of less than 0.05 was considered significant.http://imed.med.ucm.es/tools/rankpep.html). T cell epitopes within BP180, BP230, human \u03b24 integrin, and \u03b16 integrin that are restricted by HLA-DQ7 were predicted. The \u03b2 chain of HLA-DQ7 is DQ\u03b21*03:01. For the prediction of the peptide-HLA binding, RANKPEP relies on Position Specific Scoring Matrices (PSSM), which are derived from peptides that are known to bind to HLA molecules. Only those peptides that had a score higher than the Binding Threshold were considered as potential candidates for binding to DQ\u03b21*03:01., as described earlier (Autoreactive T cells recognize self-peptide antigens bound to HLA class II (HLA II) molecules. Hence T cell epitopes may be deduced by predicting peptide binding to HLA II molecules . In this earlier \u201328.The tertiary structure of DQ7 (DQA1*03:01/DQ\u03b21*03:01) in complex with 15-mer peptide antigens FNWLPPGKPMGYRV, DNVIRKYGDPGSLFG, PAKAIAAVKSGGAVL, and LERIRRSILPYGDSM from integrin \u03b24 (AC: NP_000204), integrin \u03b16 isoform a (AC: NP_001073286), dystonin-1 (BPAG1) (AC: NP_899236), and alpha 1 type XII collagen (BPAG2 or BP180) (AC: NP_000485), respectively, were generated by homology modeling after two known tertiary structures of DQ8 (PDB IDs: 1JK8 & 2NNA) using a standalone version of MODELLER . TertiarNineteen studies published between 1990 and 2020 were included in this study.Nine reports that included data on 904 patients with BP were included in this analysis. The studies were from the following countries: Iran , Brazil When the frequency of HLA DQ\u03b21*03:01 was collectively compared between BP patients and controls, it was highly statistically significant in the BP patients (p<0.00001).In eight reports, MHC class II genes were studied in 335 patients with MMP from the US , 22, 38,In examining data on DPP4i-associated BP, only two studies, one from Japan  and one These data clearly demonstrate that in many countries that are ethnically different from each other, the clinical profiles are similar. The MHC class II genes are identical, demonstrating the pivotal role of DQ\u03b21*03:01 being central to the pathogenesis of BP and MMP. Despite this remarkable similarity, patients develop two very distinct clinical profiles with different clinical outcomes. As described below, different peptides within each antigen may be involved in different racial or ethnic groups. Similarly, the presence of different portions of the peptide binding to DQ\u03b21*03:01 may produce a different clinical profile, accounting for involvement of different tissues or organs.Molecular analysis of possible antigen binding sites on HLA-DQ7 (DQ\u03b21*03:01) were done by a computer model. The computer model used identified peptides within the four antigens known to be pathogenic in pemphigoid diseases, that potentially could be T cell epitopes. Identified were 14 sites within BP180, 30 sites within BP230 14 sites within \u03b24 integrin, and 7 sites within \u03b16 integrin Figure\u00a01T cell epitopes (generated by computer modeling) within pemphigoid antigens binding to DQ\u03b21*03:01 were modeled for binding to T cell receptor. The graphic representation of this binding is presented in In this study, we have demonstrated that the association of BP with DQ\u03b21*03:01 has been observed in several countries spanning four continents Figure\u00a04One of the most recent advances in the treatment of type II diabetes mellitus is the use of DPP-4i drugs. The incidence of BP in these patients is significant . There aThe HLA DQ\u03b21*03:01 allele was not associated with pemphigoid diseases in two studies from Japan and China, conducted in 2000 and 2002 respectively , 37. How-3)  .Autoreactive T cell responses to BP180 have been investigated in BP patients. With regards to the DQ\u03b21*03:01 allele, Budinger et\u00a0al. observed proliferative T cell responses to BP180 were DQ\u03b21*03:01 restricted .The HLA DQ\u03b21*03:01 allele has been associated with other conditions. Statistically significant associations between the DQ\u03b21*03:01 allele have been observed in patients with erythema multiforme, cutaneous melanoma, gastric adenocarcinoma, and cervical cancer \u201349. BP hPreliminary data presented in this study demonstrates that many of those patients with diabetes mellitus treated with DPP-4i drugs who developed BP were carrying the HLA-DQ\u03b21*03:01 allele of MHC class II genes. It is entirely possible that this provided enhanced susceptibility to develop BP. These early observations warrant further detailed studies.Pemphigoid gestationis (HG) a rare autoimmune blistering skin disease that is associated only with pregnancy . HG was in vitro models  or organ(s).Using a computer-based model, we demonstrate a representation of how peptides from BP180, BP230, \u03b24 integrin, and \u03b16 integrin can be T cell epitopes presented by DQ7 Figure\u00a02In our hypothesis, we modeled the tertiary structure of DQ7 in complex with selected peptides. Selected antigens were those with the largest binding scores. HLA molecules contain \u03b1 and \u03b2 chains. The \u03b2 chain of DQ7 is HLA DQ\u03b21*03:01, which pairs with different \u03b1 chains. In the model we chose HLA DQA1*03:01 because it is the one that pairs most frequently with DQ\u03b21*03:01 .These computer models, in part, provide the molecular basis for two sets of clinical observation. First, the worldwide occurrence of an autoimmune autoantibody mediated disease with similar clinical presentation. The available data on MHC class II genes, molecular analysis of the four antigens, and the computer model used for this analysis. Second, the selective involvement of specific tissues or organs in the different subsets of pemphigoid diseases. These could in part be due to tissue or organ specific anti-BMZ autoantibodies.The epidemiology of several autoimmune diseases have been studied and indicate their global occurrence. Some of these are rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, myasthenia gravis, and immune thrombocytopenic purpura (ITP) \u201363. The in vitro studies and clinical correlation. Among other advances in immunopathogenesis, these models could assist in producing disease specific, race specific, targeted therapies.In this study, we do not provide data to demonstrate which peptide could be used in which ethnic or racial group or which peptide is associated with which organ or tissue involvement. Nonetheless, identification of potential T cell epitopes in these four antigens associated with BP, MMP, and its clinical variants, could be potentially very beneficial for The original concept and design of the study was made by AA. The potential peptides in the four antigens that can serve as molecules presented to T cell receptors was done by PR. The latest computer analysis of binding of peptides to the T cell receptor was done by PR. SA performed a detailed literature search for all the studies on the MHC Class II genes in pemphigoid diseases. The manuscript was read and edited by all three authors.This study in part was supported by an unrestricted research grant from the Dysimmune Diseases Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Background: Several pre-clinical and clinical reports suggest that HIV-1 protease inhibitors, in addition to the antiretroviral properties, possess pleiotropic pharmacological effects including anticancer action. Therefore, we investigated the pro-apoptotic activity in tumor cells of two molecules, RDD-19 and RDD-142, which are hydroxyethylamine derivatives\u2019 precursors of darunavir and several HIV-1 protease inhibitors. Methods: Three hepatoma cell lines and one non-pathological cell line were treated with RDD-19 and RDD-142, and cell viability was assessed. The expression levels of several markers for ER stress, autophagy, cellular ubiquitination, and Akt activation were quantified in HepG2 cells treated with RDD-19 and RDD-142 to evaluate apoptotic and non-apoptotic cell death. Results: RDD-19 and RDD-142 showed a greater dose-dependent cytotoxicity towards the hepatic tumor cell line HepG2 compared to the non-pathological hepatic cell line IHH. Both molecules caused two types of cell death, a caspase-dependent apoptosis, which was ascertained by a series of biochemical and morphological assays, and a caspase-independent death that was characterized by the induction of ER stress and autophagy. The strong increase of ubiquitinated proteins inside the cells suggested that the target of these molecules could be the proteasome and in silico molecular docking analysis that was used to support the plausibility of this hypothesis. Furthermore, cells treated with the two compounds displayed decreased levels of p-AKT, which interferes with cell survival and proliferation. Conclusions: These findings demonstrate that two compounds, RDD-19 and RDD-142, have pleiotropic effects and that they may represent promising anticancer candidates. FDA-approved human immunodeficiency virus 1 protease inhibitors (HIV-PI) are used in highly active antiretroviral therapy (HAART) that significantly improves the clinical conditions of HIV patients . To dateN--3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-methoxybenzenesulfonamide and N- -3-amino-2-hydroxy-4-phenylbutyl)-N-benzyl-4-methoxybenzenesulfonamide, indicated respectively with the abbreviations RDD-19 and RDD-142, were synthetized, purified, and finally characterized by NMR  colorimetric assay. Experimentally, HepG2, JHH6, HuH7, and IHH cells were seeded in 96-well plates (2 \u00d7 105 cells per well) and cultured overnight. They were treated for 24 h with different concentrations of RDD-19 and RDD-142 , using untreated cells and cells treated with DMSO as negative controls. The cellular morphology was observed using phase contrast microscopy (Nikon Eclipse).HepG2 cells were seeded into 12-well plates (2 \u00d7 105 cells per well) in a 12-well plate, incubated at 37 \u00b0C with a 5% CO2 atmosphere and then treated with various concentrations of the molecules . Untreated cells and cells treated only with DMSO (vehicle) were used as negative controls. After 24-h incubation, cells were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 20 min at room temperature, then washed with pre-chilled PBS three times, and stained with approximately 10 \u00b5g/mL of HOECHST 33258 at room temperature in the dark for 10 min. The images were acquired using a fluorescence microscope .To underline the possible morphological changes of cells, such as cell fragmentation and chromatin condensation, cell cultures were labeled using the cell-permeable, DNA-specific fluorescent dye HOECHST 33258 , according to . Briefly5 cells per well HepG2 cells were seeded in a six-well plate. As a positive control, cells were treated with 4.3 \u00b5M Doxorubicin. Untreated cells and cells treated with DMSO represented negative controls. After 24 h of treatment, cells were harvested, washed twice with cold PBS, and resuspended in Binding Buffer . Then, 100 \u00b5L of the cell suspensions were mixed with 5 \u00b5L of Annexin V-FITC and 5 \u00b5L of propidium iodide (PI) and incubated in the dark for 15 min at room temperature. Then, 400 \u00b5L of Binding Buffer was added to each tube and the analysis was carried out by FACS  with the Kaluza 2.1 analysis program .To assess the extent of apoptosis induction after 24 h of treatment with different concentrations  of RDD-19 and RDD-142, 5 \u00d7 105 cells per well and treated for 24 h at the concentrations of 75 \u00b5M, 50 \u00b5M, and 37.5 \u00b5M of the RDD 19 and RDD 142 inhibitors; then they were collected and lysed for 30 min in ice-cold RIPA buffer , supplemented with a mixture of PIC 100X (Phosphatase Inhibitor Cocktail\u2014CST) and 1M PMSF (Phenylmethanesulfonyl Fluoride\u2014CST). All the samples were also sonicated for 30 sec at 37% power with the BANDELIN SonoPuls sonicator and then centrifuged at 13,000 rpm for 20 min at 4 \u00b0C to remove insoluble cell debris. Protein content was quantified using the Bradford Reagent from Sigma. Equal amounts of protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked for 1 h with 5% nonfat dry milk in PBS and then overnight probed with the primary antibodies against BiP/GRP78 (1:1000), CHOP (1:1000), IRE1\u03b1 (1:1000), sXBP1 (1:1000), ATF4 (1:1000), ATF6 (1:1000), PARP-1/cPARP-1 (1:1000), Beclin-1 (1:1000), LC3 A/B (1:1000), pAKT(Ser473) (1:1000), AKT (1:1000), ubiquitin (1:1000), p-PERK (1:1000), actin (1:1000), and tubulin (1:1000). The membranes were then washed three times with wash buffer (PBST), incubated with the appropriate horseradish peroxidase-linked secondary antibody , and visualized with the enhanced chemiluminescent detection system . The chemiluminescent signal was detected through the Chemidoc XRS detection system  with the ImageLab software and quantified by densitometric analysis of the bands using the ImageJ software 1.52a . In. In28]. The introduction of HAART significantly decreased the incidence of several forms of cancer, previously frequent in HIV-positive patients . The effThe effects of HIV-PIs on liver cancer are less characterized than other types of cancer, despite that hepatocellular carcinoma (HCC) is one of the most widespread and growing diseases in the world and it is the most common form of liver cancer, accounting for 90% of the cases . Its comCancer cells, compared to non-tumorigenic cells, are characterized by fast metabolism and proliferation, which are accompanied by an aberrant production, folding, degradation, and turnover of proteins. In this regard, the Unfolded Protein Response (UPR) triggered in the ER and the protein degradation mechanisms, mediated by the ubiquitin\u2013proteasome pathway, play a key role in the regulation of cell fate . TherefoSeveral molecules have been developed and tested (and some of these have been approved by the FDA) as modulators of the UPR/degradation machinery and they have been shown to be particularly effective on hematological malignancies ,39,40. AIn the present study, the antitumor activity of two synthetic intermediates of darunavir analogs, RDD-19 and RDD-142, was evaluated. We considered a panel of three liver tumor cell lines, characterized by different degrees of differentiation , and an immortalized non-pathological liver cell line IHH. Our results showed a phenotype-dependent cytotoxicity, being greater for the more differentiated cell lines HepG2 and HuH7 than for JHH6, as already reported in the literature for another proteasome inhibitor . This leThe IRE1a branch was also activated, as confirmed by the upregulation of sXBP1, a transcription factor responsible for the expression of chaperones and genes involved in the ERAD system, which is capable of providing for the disposal of unfolded and misfolded proteins from the ER. Due to the high quantity of ubiquitinated proteins present after treatment with both compounds, we would like to hypothesize that an inhibition at the level of the proteasome occurred, which hindered the regular disposal of the proteins coming from the ER and thus creating a proteotoxic stress.Several studies ,48,49 haThe molecular docking studies of RDD-19 and RDD-142 in the human proteasome demonstrated the potential of the two molecules to bind in two of the binding pockets for known proteasome inhibitors. It is noteworthy that the two compounds, which are structurally very similar, both bind to each of the two binding sites in a similar position and conformation, which gives credibility to the docking solutions. It is also interesting that, whereas the two molecules bind in a similar way to the known proteasome inhibitors in the interface between the \u03b26\u2013\u03b27 subunits, in the \u03b21\u2013\u03b25 interface they are binding in a totally different way. It should be mentioned that another HIV protease inhibitor, ritonavir, which is structurally quite different from darunavir, has been found to inhibit the proteasome and, based on docking studies, that it is binding one of the inhibitor binding pockets of the yeast proteasome , in a siN-isobutyl or N-benzyl p-methoxyphenylsulfonamide and a free primary amine. Our results suggest a plausible mechanism of action that is consistent with the literature present for other HIV-PIs. Considering that FDA-approved proteasome inhibitors have several side effects in patients [Darunavir is a second-generation HIV-PI, highly active against the HIV-1 protease but with less frequent adverse effects. Some pleiotropic effects of darunavir are known, e.g., being able to prevent kidney injury via HIV-independent mechanisms  or showipatients  and show"}
+{"text": "Anxiety symptoms in people living with dementia (PLWD) are the most distressing symptoms for caregivers. While caregiving is bidirectional relationship, little is known how caregivers can influence anxiety in PLWD. The purpose of this study was to examine the relationship between caregiver mastery and anxiety symptoms in PLWD. Secondary data analysis was conducted using baseline data from Healthy Patterns Study. The conceptual model of Factors Associated with Behavioral and Psychological Symptoms of Dementia guided this study. Among the 169 study PLWD, 23.1% (n=39) reported having anxiety symptoms. In a multivariate logistic regression, adjusting for age, dementia stage, sleep, and depression, better caregiver mastery was significantly related to lower odds of having anxiety in PLWD . These results suggest that interventions aimed at improving caregiver mastery may improve anxiety symptoms in PLWD."}
+{"text": "Background: Misdiagnosis and delayed diagnosis of acute aortic dissection (AAD) significantly increase mortality. Lysophosphatidic acid (LPA) is a biomarker related to coagulation cascade and cardiovascular-injury. The extent of LPA elevation in AAD and whether it can discriminate sudden-onset of acute chest pain are currently unclear.Methods: We measured the plasma concentration of LPA in a cohort of 174 patients with suspected AAD chest pain and 30 healthy participants. Measures to discriminate AAD from other acute-onset thoracalgia were compared and calculated.Results: LPA was significantly higher in AAD than in the AMI, PE, and the healthy  within 48 h of symptom onset. LPA level peaked at 12 h after symptom onset, then gradually decreased from 12 to 48 h in AAD. LPA had an AUC of 0.85 (0.80\u20130.90), diagnosis threshold of 298.98 mg/dl, a sensitivity of 0.81, specificity of 0.77, and the negative predictive value of 0.85. The ROC curve of LPA is better than D-dimer . The decision curve showed that LPA had excellent standardized net benefits.Conclusion: LPA showed superior overall diagnostic performance to D-dimer in early AAD diagnosis may be a potential biomarker, but additional studies are needed to determine the rapid and cost-effective diagnostic tests in the emergency department. Aortic dissection is a life-threatening cardiovascular disease that causes ~10,000 deaths in the United States each year  \u20136. Not oSeveral researches have investigated AAD potential biomarkers for faster and more accurate clinical treatment, such as smooth muscle myosin , calciumLysophosphatidic acid (LPA) is a small and simple glycerophospholipid (1-acyl-2-hydroxy-3-phosphoglycerol structure) , which iThis is a single-center retrospective cohort including patients with suspected AAD within 48 h of onset and healthy participants, who came from the emergency department and medical examination center of the Second Xiangya Hospital of Central South University  between May 2020 and January 2021. All suspected AAD patients were examined for medical imaging and D-dimer for the final diagnosis , 21. TheAbout 3\u20135 ml of whole blood was taken from the brachial vein and placed in a sodium citrate anticoagulant tube immediately after hospital admission. The samples were centrifuged at 1000 r/min for 15 min to process into plasma, and stored at \u221280\u00b0C. All sample processing methods are similar.The Ethics Committee of the Second Xiangya Hospital of Central South University approved this study. Informed consent was obtained from all patients. However, consent was obtained from a family member in a case of sudden death after admission or during autopsy.All patients with AAD, characterized by symptoms onset-time within 48 h, had image information from aortic computed tomography to confirm the final diagnosis. AMI diagnosis criteria were: (1) chest pain lasting >20 min, (2) Serial ECG changes with new pathological Q waves or ST-segment and T-wave changes, and (3) a plasma creatine kinase-myocardial band elevation  level > 0.1 ng/ml). A positive pulmonary artery computed tomography scan was for PE diagnosis.The processed serum was measured by the human lysophosphatidic acid kit , and D-dimer was detected by the TOP700 automatic coagulation analyzer.T-test and Mann-Whitney U-test were used as a post-hoc analysis. Categorical variables were expressed as frequencies and compared using Fisher's precision probability test or Chi-square analysis. Logistic regression analysis was also used. P = 0.05 was considered statistically significant. Pearson correlation and delong test (Continuous variables were expressed as mean \u00b1 standard deviation or median (IQR). ANOVA and Kruskal-Wallis test were used for parametric and non-parametric data in multiple groups, ong test  were useong test , 24.https://www.r-project.org/), The R Foundation, and EmpowerStats (http://www.empowerstats.com), X&Y Solutions Inc, Boston, MA were used for all statistical analyses.The R (P < 0.001) , were selected . The pat< 0.001) .p < 0.05) . There was no significant correlation in Normal . The number of chest pain patients at different symptoms onset-time was not statistically significant (P = 0.148)  .P = 0.041, Delong test) , diagnosis threshold of 1.87 ug/ml, a sensitivity of 0.90, specificity of 0.55, and the negative predictive value of 0.91. LPA had an AUC of 0.85 (0.80\u20130.90), diagnosis threshold of 298.98 mg/dl, a sensitivity of 0.81, specificity of 0.77, and the negative predictive value of 0.85 . The ROCng test) . The decng test) .This study, which included 174 suspected AAD patients and 30 healthy participants, found that LPA may be used for the clinical diagnosis of AAD. LPA, mainly produced by activated platelets, may be an early biomarker of the initiation of thrombosis and coagulation. Researches have shown that coagulation cascade activates platelets to release a large amount of lysophospholipids and autotaxin with phospholipase D activity stored in alpha particles at the same time. The two substances undergo a biochemical reaction to produce LPA, which increases the plasma concentration , 25, 26.We found that the magnitude of elevated LPA can distinguish patients with AAD from patients with AMI, PE, and the healthy within 48 h after symptom onset. AAD is a disease with high mortality due to severe damage to the aortic structure. There are also some biomarkers showing clinical prognosis including CRP , NT-pro Our results suggested that the degree of the elevation of LPA levels is associated with the different magnitudes of vascular injury among AAD, AMI, and PE. Although LPA level in PE and AMI patients was higher than the healthy, there were still lower than AAD in our study. Substantial aortic hemodynamic changes significantly increased the circulating LPA level in the aorta (largest artery) compared with the small and medium blood vessels of pulmonary embolism and acute coronary syndrome. Studies have shown that LPA level is associated with the release location, releasing LPA directly and quickly to the aortic circulation may be another reason for the higher degree of AAD patients .P < 0.05, Delong test). This means that as a marker of platelet activation, LPA \u2265 300 mg/dl indicates a high risk of AAD, and may be earlier than D-dimer. However, D-dimer has higher sensitivity and negative predictive value compared to LPA. As a fibrin degradation product in the circulation after thrombolytic fibrinolysis, D-dimer has been found to increase similarly in many diseases, including PE and AAD. In fact, D-dimer aids clinical diagnosis for PE only as a rule-out tool when the test result is negative   can alsoLPA showed superior overall diagnostic performance to D-dimer in early AAD diagnosis may be a potential biomarker, but additional studies are needed to determine the rapid and cost-effective diagnostic tests in the emergency department.188212324@csu.edu.cn.The data analyzed in this study is subject to the following licenses/restrictions: We are deeply sorry for this, because there is still some research in progress for the time being. In order not to affect the following research, it is not convenient to disclose the data. Requests to access these datasets should be directed to The hospital institutional review board of the Second Xiangya Hospital approved the study. The data collection and analysis followed the Ethics Committee of the institution and the Declaration of Helsinki. The Ethics Committee of the institution reviewed the patient consents, and the data were used only for research purposes.XP and XC: drafted, revised, and reviewed the article. YZ and GY: conducted statistical analysis. ZH, HZ, ZP, WP, and ND: reviewed and revised the manuscripts. TG and MZ: organized the database. All authors significantly contributed to the conception, study design, execution, data acquisition, analysis, interpretation, approved the final version, agreed on the journal, and are responsible for this study.The Key Research and Development Program of Hunan Province; Hunan Health and Family Planning Commission Project; Hunan Provincial Natural Science Foundation Project  supported this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The rapid development of computer network technology in today's society has also brought new opportunities for the transformation of the teaching management mode of colleges and universities. Nowadays, most colleges and universities are gradually improving the construction of campus informatization projects, further integrating school teaching resources, centralizing the work related to the work process, and carrying out platform management to improve the daily work efficiency of various departments of the school, which is also prominent from the user experience. Humanized management out of school. This paper designs and develops a set of online teaching management system suitable for colleges and universities, realizes the informatization of college teaching management, and solves the traditional teaching methods, such as the inability of offline correspondence students to take classes in time and the limited teaching venues. Through comparison and analysis, in order to ensure that the system has better portability, this article will mainly adopt the MVC architecture model to develop and design the entire school hybrid teaching management system. The MVC architecture model is mainly developed based on the abstract design model. Strong reusability, combined with the system architecture, the main choice for database selection is development based on Oracle database technology. In the specific development process, the detailed business requirements and functional requirements of the entire school online teaching management system will be analyzed in detail. Based on this, by adopting an object-oriented approach, the system management included in the school's hybrid teaching management system will be further analyzed. Detailed analysis and design of the specific functional structure, work flow, work sequence diagram and class diagram of the online learning management and article management functional modules, and finally complete the implementation and testing of the various functional modules of the system. At present, computer technology and Internet technology have been integrated into all corners of society, greatly affecting and changing our production and lifestyles, and providing people with great convenience. From the time when the Internet was underdeveloped to the present, the convenience of Internet technology has affected all walks of life. Among them, education is a national priority and key development business, and informatization has also been applied early in education. Educational information The concept of chemistry has also begun to be widely recognized and valued. In recent years, with the continuous improvement of my country's education level, some schools have begun to use information management systems in order to provide their own management and teaching levels. According to their own teaching management characteristics, they have designed and developed a series of education information management platforms to promote teaching Management work is developing in the direction of informatization. The emergence of a networked hybrid teaching management system has brought major reforms to the hybrid teaching management, informatization construction, sharing and release of teaching resources, networked educational administration and comprehensive management of student information in colleges and universities, and many colleges and universities have burst out The new teaching management ideas are all benefited from the rapid development of networking and informatization. As a brand-new management method, the hybrid teaching management system is an important application of computer technology in the field of education. It strengthens the comprehensive management of schools, improves the efficiency of teachers, and advances the process of university management. At present, from a technical perspective, the current teaching management system used by private training institutions is mainly based on the C/S architecture. This architecture is simple in structure and easy to develop. The internal staff of the training institution can complete the development or commission small software The development work is done on behalf of the development, generally the cost is low, and it is widely used in small training institutions. For colleges and universities, C/S architecture is usually difficult to meet their needs, and they mostly use teaching management systems based on B/S architecture. The B/S architecture system is more difficult to develop than the C/S architecture, but it has more powerful functions and is more suitable for school use scenarios. The B/S structure system is easy to maintain, which is conducive to the expansion of the school. On the other hand, the B/S architecture adopts a modular system architecture, and the internal modules of the system are loosely coupled, which facilitates the expansion of the department and the increase of system functions, and is more suitable for changes in the teaching management business of colleges and universities. According to the school's mixed teaching management business needs and the needs of information construction, it is very necessary to design and develop an information management system suitable for the school's own development. Through the design and implementation of this system, it can not only improve daily work and student management Efficiency, but also can reduce costs, improve their own competitiveness, and better provide students with a good learning environment and conditions.The whole university hybrid teaching management system mainly adopts the object-oriented design method for development and design. Through comparison and analysis, the system architecture design selection is based on the MVC (Model View Controller) architecture method. Through the use of MVC The architecture system can effectively improve the scalability and maintainability of the system. At the same time, the code reusability can be further improved in the system development process, which is convenient to reduce the system development cycle and the development cost can also be reduced. The specific situation of the relationship between the layers of the entire MVC architecture is shown in The entire MVC architecture is mainly composed of three parts: view, model, and controller. Among them, the view part is mainly able to provide system users with a human-computer interaction interface, which can collect user specific operations, and can also display the content processed through the model layer. Normally, the view part is usually made of HTML elements. With the continuous development of computer technology, this module can be used to query the status of the model part and check the status change of the controller module. With the continuous development of computer technology, in order to be able to continuously enrich the machine interaction operation, provide users with more convenient and efficient interaction Experience, XML and Flash and many other elements are being applied. The model part is used to process various different types of data according to certain logic. This module can be used to query the status of the view part. In order to effectively improve the processing capabilities of various types of data, usually one model can be combined with multiple view blocks. Correspondence and processing. The controller part is the most core part of the entire MVC architecture. It can coordinate the information interaction between the view part and the model part, and process the user time information fed back by the view module to ensure the stable and reliable operation of the system. Combined with the MVC architecture diagram, it can be seen that after the user sends an operation request by using the model module in the MVC architecture, the system can call the user's specific operation request by using the view module, and at the same time feed back user event information to the controller. The controller will further retrieve the processing task data in the model module, and display the feedback and specific content information retrieved to the system user through the view module \u20136 It canThe basic genetic algorithm consists of 8-tuples in the following formula, namely:In formula :\u0421-represents the individual coding method (binary), which is the basic factor in genetic algorithm. A combination formed by the connection of genes and chromosomes is a solution for curriculum arrangement;\u0415\u2014\u2014 represents the fitness evaluation function of the individual. Use this function value to distinguish whether the individual meets the requirements; P0-represents the initial group, a combination of courses randomly formed by genetic algorithm, and subsequent optimization of genetic algorithm will be carried out around the initial group;\u041c\u2014\u2014represents the size of the group;\u0424\u2014\u2014represents the selection operator, and the calculation method is filtered by the fitness value. Generally, the larger the value, the easier it is to be selected;\u0433\u2014\u2014represents the crossover operator. The new individual is generated by the random exchange of genes from the parent individual, and the single point crossover method is adopted;\u03c8-represents the mutation operator, the parent individual randomly changes the individual's own gene value, and adopts a single point mutation of the gene;\u0422\u2014\u2014represents the termination condition of the algorithm. When performing genetic iteration, we generally set a certain parameter value, and the operation is terminated when the parameter is iterated algebraically [raically .Choose an appropriate way to encode system parameters, and convert the relatively optimized solution set into chromosomes for representation;Determine the initial group of the system;Define the fitness function;Genetic operation of the design system;Control the setting of parameters such as the size of the population and the probability of genetic manipulation;Judge whether the generated group meets a specified index, if it is satisfied, exit the calculation, if it is not satisfied, return to the operation of calculating the fitness function value . The speWhen using genetic algorithms to develop a hybrid teaching system, follow the steps below:In order to facilitate the system to have stronger scalability and maintainability, in the process of architecture selection, the MVC architecture is finally selected as the system architecture of the college online teaching management system, combined with specific design, the entire college online hybrid teaching management system The detailed design of the overall architecture is shown in The overall architecture of this hybrid teaching management system is mainly composed of the presentation layer, the system service layer and the data layer. The presentation layer is mainly used to provide access interfaces for system users of the university hybrid teaching management system. Through the front-end operation interface of this layer, users can provide interface services for various business operations in the system service layer .The system service layer is mainly composed of core business and business support parts. The core business part specifically includes the operation of system management, online learning management and other business functions. At the same time, in order to provide strong support for the main core business operations, in this layer The business support part has designed user authority configuration and information query and information exchange services.The data layer is mainly used to provide services for the retrieval and storage of all data information in the hybrid teaching management system of colleges and universities, and to provide data support for the smooth progress of various core businesses in the system service layer. The business functions to be realized mainly include a variety of data information such as online course information database and video-on-demand course information database , 11.By combining the overall architecture of the system, it can be seen that the entire university hybrid teaching management system mainly adopts the MVC three-tier architecture. In order to meet the characteristics of the scattered users of the system combined with the characteristics of the system architecture, the specific network topology structure diagram of the university hybrid teaching management system The design is shown in It can be clearly found that in the course of business operation of the college hybrid teaching management system, all users in the system can use the external network and the college intranet for system login access, and the terminal devices used are computers. The network architecture can be divided into the core area and the gatekeeper isolation area. For the core area, its firewall can effectively guarantee the security performance of the system. The main method is the connection between the load balancing server and the gatekeeper isolation area, thereby providing access to the database in this area. Manage and control servers and application servers \u201314.Online learning management is an important module of the entire university's hybrid teaching management system. It is mainly used by teachers to create, review and publish online courses. College students can also use this function for online course inquiry and registration. Combined with actual needs, the design of the functional structure diagram of the specific online learning management module is shown in The online course management sub-module is composed of two main functions: online course creation and online course review and release. The entire course management sub-module is composed of three main functions: online course inquiry, online course registration, and my course inquiry. By combining the actual needs and target requirements of the college, students can use this module to query relevant course information and combine The training goal requires course application and registration, and at the same time, it is possible to inquire in detail about the course status declared by the individual. The online learning sub-module is composed of three main functions: course study material management, course video on demand, and online interaction. In combination with the online learning function requirements, the detailed online learning data management work sequence diagram is shown in Combined with the working sequence diagram of the hybrid teaching management system, it can be known that the system operator clicks \u201conline learning management\u201d on the operation homepage, then enters \u201conline learning,\u201d and then completes the specific creation of the course video materials according to actual needs, and completes the course video Upload and edit on-demand resource information. After the operation is completed, after the course video on-demand resource information database is updated, college students can search for course video-on-demand resources. After entering the required query on-demand resource keywords, the system will combine the input keywords The matching information is screened in the database, and then the qualified resource information is displayed. In the process of watching the on-demand video, the college students can enter the interactive information of the course.The database logical structure design is the design of the overall logical structure of the database. The design methods mainly include top-down, bottom-up and one-by-one Three kinds of expansion. Among them, the top-down is mainly through the design of the overall data structure, and then gradually refines until the design of the entire data structure is completed; bottom-up merges the data structure according to the logical relationship between the data, until the overall data structure is completed the design of. Combining the detailed design of the overall E_R diagram of the university hybrid teaching management system and the specific data logic structure rules, the design of the main data information tables such as online course information, test paper information and basic learning file information included in the system is explained.For a detailed description of the online course information table, see Development end hardware and software configurationOn the development side, in order to facilitate the debugging of the entire college online teaching management system, in the process of selecting the terminal on the development side, the main terminal processor selected is i7-9750H, six-core CPU, memory capacity of 32GB, and hard disk type SSD. The size of the solid state drive is 1TB, and the main choice for software development is Eclipse 5.0.Server hardware and software configurationOn the server side, combined with the requirements of system development, the server side supports RDIMM/LRDIMM, with a minimum memory of 64GB, with data protection functions such as encrypted signature firmware, secure boot, system lock, and secure erasure. Oracle is selected in the selection of database software 11g version database.After completing the design of the functional modules and database of the hybrid teaching management system in universities, it is necessary to combine the detailed design of the system to design and implement the operation interface of each functional module of the system, combine the detailed design of the system, and control the system through the front-end operation interface. The specific implementation is fully demonstrated. In the implementation process, the corresponding hardware and software are required to support. In order to meet the stable and reliable operation of the implementation interface, the hardware and software configuration of the development side and the server side required for the system implementation are required:Combined with the actual function design, the entire blended learning management module mainly includes three sub-functions: online course management, my course management, and online learning materials. The detailed design of the class diagram of the online learning management module is shown in Combined with the specific design, the implementation of the operation interface of the online course management sub-functions in the blended learning management module is shown in The realization of the core code of the personal information management operation part in the management module of the entire hybrid teaching management system is shown in In the process of testing the online learning management module, detailed and comprehensive tests were mainly conducted on the related operations of online course management, my course management, and online learning. The specific online learning management module test case descriptions are shown in In the process of testing the performance of the entire university hybrid teaching management system, the author mainly conducts the performance of concurrent users of the entire system. The detailed test situation is shown in A detailed analysis of the business requirements for the application of the hybrid teaching management system in colleges and universities is carried out, and the business functions required by the hybrid teaching management system in colleges and universities as well as the specific functional requirements and performance requirements of the system are proposed.According to the specific design and implementation, the system function test and system performance test are completed by equivalence class division and boundary value analysis methods, and the test results can meet the expected index requirements.Based on the current situation of mixed teaching management, this paper attempts to design and develop a set of mixed teaching management system suitable for colleges and universities, realizes the informationization of college daily management, and solves the problems of low efficiency and high error rate of traditional manual management mode. By using the MVC architecture and Oracle database and other major technologies, the author has mainly completed the following aspects: \u201324.A detThe college hybrid teaching management system designed in this article still needs to be improved. For example, although the online learning management module already has the interactive function of online learning, it only realizes simple text communication. It is not possible to directly communicate with the instructor for the time being. Group discussions, etc., still need to be further improved."}
+{"text": "To better understand the patient's heterogeneity in fatty liver disease (FLD), metabolic dysfunction\u2013associated fatty liver disease (MAFLD) was proposed by international experts as a new nomenclature for nonalcoholic fatty liver disease (NAFLD). We aimed to evaluate the cardiovascular risk, assessed through coronary artery calcium (CAC) and epicardial adipose tissue (EAT), of patients without FLD and patients with FLD and its different subtypes.Cross sectional study of 370 patients. Patients with FLD were divided into 4 groups: FLD without metabolic dysfunction (non-MD FLD), MAFLD and the presence of overweight/obesity (MAFLD-OW), MAFLD and the presence of two metabolic abnormalities (MAFLD-MD) and MAFLD and the presence of T2D (MAFLD-T2D). MAFLD-OW included two subgroups: metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUHO). The patients without FLD were divided into 2 groups: patients without FLD and without MD  and patients without FLD but with MD (non-FLD with MD). EAT and CAC (measured through the Agatston Score) were determined by computed tomography.Compared with the reference group (non-FLD nor MD), regarding EAT, patients with MAFLD-T2D and MAFLD-MUHO had the highest risk for CVD , patients with MAFLD-MHO were also at risk for CVD , and patients with non-MD FLD did not have a significantly increased risk . Regarding CAC, patients with MAFLD-T2D had an increased risk for CVD . Patients with MAFLD-MUHO, MAFLD-MHO and non-MD FLD did not have a significantly increased risk compared with the reference group .MAFLD\u2013T2D and MAFLD\u2013OW phenotypes had a significant risk for CVD. MAFLD new criteria reinforced the importance of identifying metabolic phenotypes in populations as it may help to identify patients with higher CVD risk and offer a personalized therapeutic management in a primary prevention setting. In 2020, metabolic dysfunction\u2013associated fatty liver disease (MAFLD) was proposed by a panel of international experts as a new nomenclature for non-alcoholic fatty liver disease (NAFLD) considering the metabolic overload of each patient independently of the presence or not of other liver diseases . ExpertsThe association between NAFLD and cardiovascular disease (CVD) has been broadly described in the literature . EvidencThe study protocol was approved by the ethics committee of the Cl\u00ednica Universidad de Navarra (Protocol Number 2019.080). In this retrospective study, we reviewed the records of subjects who underwent routine health checkups, had a computed tomography whole body scan (CT-WBS) and blood test in the same visit at Cl\u00ednica Universidad de Navarra in Pamplona, Spain from July 1, 2003 to December 31, 2006. In our Center, CT-WBS and laboratory tests are routinely performed on the same day (or within a few days) of the initial visit. Exclusion criteria included ischemic heart diseases, heart failure, atrial fibrillation, pericarditis or valvular disease; personal history of cerebral vascular diseases (including transient ischemic attack); excessive alcohol consumption; advanced liver disease of other etiologies and malignant disease . Alcohol2). Weight categories were classified as follows: normal weight (18.5\u201324.9 kg/m2), overweight (25.0\u201329.9 kg/m2), obesity class 1 (30.0\u201334.9 kg/m2), obesity class 2 (35.0\u201339.9 kg/m2) and obesity class 3 (\u226540.0 kg/m2). The presence of metabolic dysregulation (MD) among normal weight individuals with FLD who did not have T2D was defined as the presence of two or more of the following metabolic abnormalities: (1) waist circumference \u2265 102 cm in men and 88 cm in women, (2) blood pressure \u2265 130/85 mmHg or specific drug treatment, (3) serum triglycerides (TG) \u2265 150 mg/dl (1.70 mmol/l) or specific drug treatment, (4) high-density lipoprotein (HDL) cholesterol <40 mg/dl (<1.0 mmol/l) for men and <50 mg/dl (<1.3 mmol/l) for women, (5) prediabetes , (6) HOMA-IR score \u2265 2.5, and (7) a plasma C-reactive protein level >2 mg/L.Extensive demographic, clinical , laboratory and radiological information were obtained from patient records. FLD was defined by evidence of hepatic steatosis on CT-WBS. MAFLD was defined as FLD in addition to one of the following three criteria: overweight/obesity, presence of T2D, or evidence of metabolic abnormalities. Body mass index (BMI) was calculated using the following formula: weight (in kilograms)/height (in metersPatients with FLD were divided into 4 groups: FLD without metabolic dysfunction (non-MD FLD), MAFLD and the presence of overweight/obesity (MAFLD-OW), MAFLD and the presence of two metabolic abnormalities (MAFLD-MD) and MAFLD and the presence of T2D (MAFLD-T2D). MAFLD-OW included two subgroups: metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUHO). MUHO was defined as having overweight/obesity and at least two of the following cardiometabolic abnormalities: (1) blood pressure \u2265 130/85 mmHg or specific drug treatment, (2) serum TG \u2265 150 mg/dl (1.70 mmol/l) or specific drug treatment, (3) HDL cholesterol <40 mg/dl (<1.0 mmol/l) for men and <50 mg/dl (<1.3 mmol/l) for women, (4) prediabetes/diabetes  (2 (1 point), age \u226550 years (1 point), ALT \u22652 times the normal upper value (1 point), and TG \u2265150 mg/dl (1.70 mmol/l) (1 point). A BAAT score \u2264 1 points is considered as low likelihood of liver fibrosis and a BAAT score \u22654 points have a high likelihood of liver fibrosis. A score between 2 and 3 points is considered as an indeterminant score.We used the BAAT Score  as the nCardiac function was assessed through echocardiographic study  which was performed in left lateral decubitus. Images were taken in the parasternal long- and short-axis views, two- and four- chamber apical views, and subxiphoid view.All CT-WBS were performed using a sixty-four-row multidetector CT . All images were stored in picture archiving and communication system (PACS). The protocol of CT-WBS includes low-dose chest CT (120 kV and 40 mA/s) without contrast material, CAC measurement through Agatston Score (120 kV and 138 mA/s), abdominopelvic CT (120 kV and 180 mA/s) performed after intravenous injection of 120-ml iodinated contrast medium at 2 ml/s ; portal phase was acquired at 65 seconds. CAC through Agatston Score was categorized as 4 categories according to the degree of calcification .3/m2 (0.725 x weight (kg)0.425) to calculate the body surface area  and visceral adipose tissue (VAT) by two radiologists, blinded to clinical data. EAT, VAT and SCAT were semiautomatically quantified in a research prototype software . EAT was defined as all cardiac adipose tissue, including the epi- and pericardial fat. EAT was semi-automatically quantified including voxels with attenuation values between\u221245 and\u2212190 Hounsfield units (HU). Adjusting for body surface area, indexed epicardial adipose tissue (EATi) was also calculated; the upper normal limit of EATi was 68.1 cmace area . The oveace area .P <0.05 was considered statistically significant.Demographic and clinical characteristics of patients were summarized using mean and standard deviation (SD) for continuous variables and percentages for categorical variables. The Kolmogorov-Smirnov test was used to assess the normal distribution of quantitative variables. Multiple group comparisons were done by analysis of variance (ANOVA) for normally distributed data. We used the chi-square test or Fisher's exact test for categorical variables. We used the analysis of covariance (ANCOVA) to calculate age- and sex-adjusted means and 95% confidence intervals (95% CI). Correlations were evaluated with the estimation of the product-moment correlation coefficient (r). The logistic regression was used to estimate age- and sex-adjusted odds ratios (OR) and 95% confidence intervals (95% CI). All analyses were performed with Stata 14 . A total of 370 patients were included in the analysis: 154 without FLD and 216 with FLD. Mean age was 57.9 \u00b1 9.2 years and 71.2% (263/370) of the cohort were men. Of the 154 patients without FLD, 40.3% (62/154) were patients with MD (non-FLD with MD) and 59.7% (92/154) were patients without MD (non-FLD nor MD). Of the FLD cohort: 13.0% (28/216) were patients with hepatic steatosis but without metabolic dysfunction (non-MD FLD), 69.9% (151/216) were patients with MAFLD due to the presence of overweight/obesity (MAFLD-OW), 2.8% (6/216) were patients with MAFLD due to the presence of two metabolic abnormalities (MAFLD-MD) and 14.4% (31/216) were patients with MAFLD due to the presence of T2D (MAFLD-T2D). Of the 151 patients with MAFLD-OW, 58.3% (88/151) were patients with MAFLD-MHO and 41.7% (63/151) were patients with MAFLD-MUHO.p < 0.05). Additionally, a higher prevalence in males with metabolic syndrome disorders  was detected in patients with FLD (p < 0.05). Indices of adiposity  were higher in participants with FLD compared with patients without FLD (p < 0.05). Cardiac function was assessed on 69 of the patients , VAT/SCAT ratio , EATi , and with the presence of CAC .In patients with FLD, BAAT fibrosis score significantly correlated with VAT . Interestingly, adiposity may influence the effect of genetic variants on NAFLD. Lin et al. . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CP, GB, GF, JA, and JE designed the study. CP, FM, and AE performed data collection. CP wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Japanese Black cattle (Japanese Wagyu) beef is attracting attention for its aroma and marbling, and its handling is increasing worldwide. Here, we focused on the origin discrimination of Wagyu beef and analyzed the nutritional components of Japanese Wagyu (produced in multiple prefectures of Japan), Hybrid Wagyu , and Australian Wagyu beef using mass spectrometry (MS). Triple-quadrupole liquid chromatography\u2013MS was used to clarify the molecular species of lipids in Wagyu beef. Fourteen classes of lipids were separated, and 128 different triacylglycerides (TGs) were detected. A simple comparative analysis of these TGs using high-performance liquid chromatography revealed significantly higher levels of triolein  and C18:1/C18:1/C16:1 (OOPo) in Japanese Wagyu. Wagyu elements beef were comprehensively analyzed using inductively coupled plasma (ICP)\u2013MS and ICP\u2013optical emission spectrometry. We found significant differences in the rubidium, cesium, and lithium levels of Japanese and Australian Wagyu beef. On comparing metabolites using gas chromatography\u2013MS, we identified significant differences in the levels of amino acids and other components of the Japanese and Australian Wagyu beef. These results suggest the possibility of determining the origin of Wagyu cattle breeds using MS and genetic discrimination. The global population is projected to reach 9.8 billion by 2050, and livestock production is expected to increase by 455 million tons until 2050. One solution to this challenge is establishing a system for sustainable food production. However, improving the productivity of agricultural products suitable for local environments is challenging. The efficient crossborder trade of livestock products can reduce global food-production challenges ,2,3. TheConsumer sensory testing has revealed that the fat content responsible for marbling is correlated with favorable beef flavor ,12,13. IInternational law mandates the tracking and certification of food ingredients and the regions in which they are processed and manufactured by food-safety management. The European Union has implemented relevant laws that protect the names of products linked with the production region, including Protected Designation of Origin and Protected Geographical Indication ,20,21. TFood certification is a procedure for verifying product and labeling conformity and compliance with applicable legal and regulatory provisions. However, food fraud occurs in various forms, including brand imitation, reduced manufacturing costs, and extended shelf life. Food fraud represents an economic and potential food-safety issue ,24. TherRecently, genetic analysis of food products has been used to identify the breed of livestock products; however, this method is inadequate to determine the origin of livestock products that have extremely similar DNA sequences . TherefoFoods contain unique elemental components that vary with the producing region\u2019s feed, water, and soil. Foods also have characteristic distributions of metabolites and lipids that depend on the breed. MS-based metabolomics techniques have been developed for foods such as olive oil, honey, milk, coffee, tea, saffron, and fruit juices to assess their geographic origin .In this study, we compared the nutritional composition of Japanese Wagyu beef and other Wagyu beef bred in other countries using various MS techniques. We obtained the lipid profiles of Japanese Wagyu beef using high-performance liquid chromatography (HPLC)\u2013MS/MS and also investigated the differences in fatty acid, triacylglyceride (TG), and trace-element profiles among Wagyu breeds. These differences in nutrients can be used to determine the production area of Wagyu cattle.We performed a genetic analysis of the Wagyu cattle from Japan and other countries using single-nucleotide polymorphism (SNP) arrays. Principal component analysis (PCA) of SNPs indicated the crossbreeding of Japanese Black cattle genes in the analyzed samples produced outside Japan (World Wagyu) a. In addThe heterozygosity values were indicative of genetic polymorphism between Japanese and World Wagyu beefs c. ComparWe focused on the lipid molecules in the meat to distinguish the geographic production area of Wagyu beef using methods other than genetic analysis. In a previous study, the total lipids fractionated via solvent extraction were comprehensively analyzed using LC\u2013MS/MS , and 108 lipid molecules were identified . Thus, wFourteen lipid classes were detected from the total lipid fraction extracted from the longissimus thoracis of Japanese Wagyu cattle a. TGs weUnder these LC\u2013MS/MS conditions, 259 lipid molecular species were identified, which exceeded the number reported previously . Among tFingerprinting with many lipid molecular species is highly accurate in discriminating Wagyu breeds. However, the use of LC\u2013MS/MS is limited by its high cost. Therefore, we performed a comparative analysis of TGs in Wagyu beef using a conventional HPLC method .Japanese, Australian, and Hybrid Wagyu beef were used as samples to distinguish between the geographic production areas of Wagyu beef. Hybrid Wagyu is a crossbreed between Angus and Wagyu breeds in Australia, and the calves of this breed are fattened for 16 months in Japan. The HPLC of TGs in the marbling of Wagyu beef revealed 16 TG fractions.Orthogonal least-square discriminant analysis (OPLS-DA) score plots demonstrated a clear left\u2013right separation of scores for the Japanese and Australian Wagyu a. The loOn comparing the molecular species of TGs, Japanese Wagyu beef featured significantly more triolein (OOO), which comprised three oleic acids , and OOPo, which comprised two oleic acids and palmitoleic acid , than Australian and Hybrid Wagyu beef d. MoreovNext, we compared the TGs in the fat tissue from Japanese, Australian, and Hybrid Wagyu cattle. Fat tissue samples were collected from intermuscular fat around the rib-eye of the longissimus thoracis muscle . The TG Comparing the fatty acids in the marbling , JapanesComparing the fatty acid composition of the marbling and intermuscular fat from various Wagyu breeds, we found that oleic acid, palmitoleic acid, and myristoleic acid (C14:1) were more abundant in the intermuscular fat .Elemental analysis using a mass spectrometer is used to distinguish the geographic production areas of meat ,36. In tp < 0.001), cesium (p < 0.001), cobalt (p < 0.001), and lithium (p < 0.05) were present at significantly higher levels in Australian Wagyu beef than in Japanese Wagyu (p < 0.001), cesium (p < 0.001), and lithium (p < 0.05) were also present at significantly higher levels in Australian Wagyu beef than in Hybrid Wagyu beef.The score plot of OPLS-DA with 20 elements a revealsse Wagyu c. Rubidip < 0.01), and cadmium (p < 0.05) were present at higher levels in Japanese Wagyu beef. No elements were present at significantly higher levels in Hybrid Wagyu than in Japanese and Australian Wagyu beef.However, molybdenum (p < 0.01) and coefficients of variation (<20%) are presented in p < 0.01) and coefficients of variation (<20%) are also presented. The overview of the metabolite sets enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis is presented in Various metabolites, such as organic and amino acids, are used to discriminate Wagyu breeds ,37. TherVarious cattle breeds are produced globally, and their flavor profiles have been studied ,38,39. WJapanese Wagyu is known to have low rates of genetic polymorphism because of its closed breeding system . World WJapanese Wagyu has a characteristic lipid profile with high oleic acid content . In thisUsing conventional HPLC methods, the comparison of TGs in Japanese, Hybrid, and Australian Wagyu beef revealed significant differences in OOO, OOPo, POP, POS, PPP, and SOS content. The expression of lipid-metabolizing enzymes that synthesize unsaturated fatty acids, such as acetyl-CoA acetyltransferase and the elongation of very long-chain fatty acids protein, is significantly upregulated in Japanese Black cattle compared with that in other breeds . The difWe assume that the PCA data of marbling were less tightly clustered than those of intermuscular fat because of the variability of fat\u2013muscle intermingling. The comparison of OOO containing three oleic acids among Japanese, Hybrid, and Australian Wagyu revealed more significant differences in intermuscular fat than in marbling tissue. TGs in Wagyu beef feature high levels of oleic acid. Oleic acid is an omega-9 fatty acid with the physiological function of having anti-inflammatory effects . Oleic aICP\u2013MS detects trace elements (detection sensitivity: pg/g to ng/g) by analyzing ionized elements, but major elements present at high levels are detected by ICP\u2013OES, thus reducing blind spots in the nutritional components and improving evaluation accuracy . A compaA comparison of Japanese and Australian Wagyu beef by metabolomics analysis demonstrated that many amino acids derived from lean meat are present in Australian Wagyu beef. Amino acid metabolism and biosynthesis were the top enriched metabolite sets in KEGG analysis . This reFurther, elaidic acid, sedoheptulose 7-phosphate, ribose 5-phosphate, and other amino acids were present at higher levels in Japanese Wagyu beef. Elaidic acid is a transisomer of oleic acid. In a previous study, we reported that elaidic acid is a metabolic marker of intermuscular fat . SedohepConversely, metabolite changes during aging after slaughter have been identified in beef via metabolomic analysis . TherefoThe longissimus thoracic or adductor muscles of Japanese Black cattle (production area: multiple prefectures in Japan) or Hybrid Wagyu  were commercially purchased from livestock farmers in Japan. The longissimus thoracic or adductor muscle of World Wagyu  was commercially purchased through a trading company. No animal experiments were included in this study.http://pngu.mgh.harvard.edu/purcell/plink/ accessed on 13 January 2020) [Genomic purification from tissues was performed using the phenol\u2013chloroform method. Purity was checked using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific). SNP genotyping was conducted using Illumina Bovine SNP50K BeadChip ver. 3.0 . Of the SNPs in the chip, SNPs located on autosomal chromosomes were used in the analysis, excluding the SNPs on unknown or sex chromosomes. The observed heterozygosity, expected heterozygosity, and minor allele frequencies were calculated using 50,868 SNPs, excluding 861 SNPs for which genotyping was unsuccessful in the sample. After quality control, PCA was performed using 44,546 SNPs with a SNP call rate of \u226495%, minor allele frequency of \u22645%, and a Hardy\u2013Weinberg equilibrium of \u22640.001. The SNP data were analyzed using PLINK v.1.9  5d for TG, PC (18:17d/15:0) for LPC and PC, phosphatidylethanolamine  for PE and LPE, DG (17:0/17:0) 5d for DG, phosphatidylserine  for PS, phosphatidylinositol  for lysophosphatidylinositol and PI, monoglyceride  for MG, phosphatidylglycerol  for PG, cardiolipin  for CL, sphingomyelin  for SM, and ceramide  for Cer. These standards were purchased from Avanti Polar Lipids, Inc. . The TG molecular species were identified using Lipid Search v.4.2.23  lipid-identification-analysis software.The total lipid fraction was extracted from 1 mg of the Bligh and Dyer variant sample. The extract was dried with nitrogen gas, redissolved in methanol, and subjected to LC\u2013MS/MS. LC\u2013MS/MS was performed using DIONEX UtiMate 3000 (Thermo Fisher Scientific) with an L-column3 C18 metal-free column  and Q Exactive Plus (Thermo Fisher Scientific). The HPLC system comprised solvents A  and B . The HPLC conditions included a column temperature of 40 \u00b0C, an injection volume of 10 \u03bcL, and a flow rate of 0.1 mL/min. MS was performed using the Full MS/dd-MS2 mode (TopN:20) from 200 t-butyl methyl ether/methanol (2:1) following a previously described method [Total lipids were extracted from ground beef (10 g) with d method . The fatd method .For the TG analysis, samples dissolved in isopropyl alcohol were subjected to HPLC using an Agilent Technologies 1260 Infinity equipped with a refractive index detector and Poroshell 120 EC-C18 LC column  following a previously described method . The quaA ceramic knife  was used to grind 50 g samples. The ground samples were then lyophilized in a freeze-dryer . Each dried sample was crushed finely (to <3 mm) with a plastic hammer.Defatted samples were prepared by removing the lipid using organic solvent (hexane and 2-propanol 3:2) and washed with hexane.A 20-fold volume of 61% nitric acid  was added to the defatted samples, which were heated at 120 \u00b0C on an electric hot plate . Next, after nitric acid digestion, 70% perchloric acid  was added to the solution. The samples were then heated at 200 \u00b0C until the organic compounds in the solution were completely digested. The solvent was evaporated by heating, and 1% nitric acid was added to the dried sample to completely dissolve the residue. An internal standard (5 \u00b5g/L) was added to the sample. The sample solution was used for the elemental concentration measurement after constant volume addition with 1% nitric acid. All samples were analyzed in duplicate.We used different analytical instruments depending on the element characteristics. The elements were divided into two groups according to their properties. The first group comprised Li, Co, Cu, Rb, Y, Mo, Ag, Cd, Cs, and Ti, which were analyzed by ICP\u2013MS . The second group comprised Na, Mg, P, K, Ca, Mn, Fe, Zn, Sr, and Ba, which were analyzed by ICP\u2013OES . A summary of the analyzer conditions and elemental references is presented in g for 10 min at 4 \u00b0C to collect the supernatant as the water-soluble fraction of the chloroform/methanol extraction. The water-soluble extract was completely dried via centrifugal drying  for 40 min and freeze-drying for 16 h. The dried samples were dissolved in pyridine containing 20 mg/mL methoxyamine hydrochloride for 90 min at 37 \u00b0C with shaking at 1200 rpm at 37 \u00b0C. The mixture was then derivatized by shaking with N-methyl-N-trimethylsilyl trifluoroacetamide (GL Sciences) for 30 min at 1200 rpm at 37 \u00b0C. After centrifugation, the supernatant was used for GC\u2013MS and processed on a GCMS-TQ8030 (Shimadzu Co.) with a BPX-5 capillary column . The column temperature was maintained at 60 \u00b0C for 2 min, increased by 15 \u00b0C/min to 330 \u00b0C, and then maintained at 330 \u00b0C for 3 min. The front inlet temperature was maintained at 280 \u00b0C. The helium gas flow rate was 39.0 cm/s. The interface and ion-source temperatures were 280 and 200 \u00b0C, respectively. The analysis mode was set to selected reaction monitoring with a split ratio of 1/30 and ionization voltage of 70 V. The mass spectrum was analyzed using GC/MS Metabolite Database v.2 (Shimadzu).Here, 1 mL of methanol containing an internal standard  was added to 100 mg of tissue crushed with zirconia beads . After centrifugation, 100 \u03bcL each of ultrapure water and chloroform were added to 250 \u03bcL of supernatant and shaken at 1200 rpm for 5 min at 37 \u00b0C using a thermomixer comfort shaker . Subsequently, 250 \u03bcL of ultrapure water were added to this solution and centrifuged at 16,000\u00d7 A mixture of all samples was used for quality control, and only the molecular species with high confidence in the measured data\u2019s peak shape, intensity, and elution time were quantified. Analyzed samples were measured by randomizing their order. The height of the detected peak in each sample was corrected by an internal standard and sample weight. The solvent blank value was subtracted, and those with negative values were treated as nondetects.t-test with Excel 2019  or Tukey\u2019s method with Bellcurve . Statistical processing, such as log transformation and scaling, and multivariate analysis (OPLS-DA and PCA) were performed using SIMCA 14.1 software  [Statistical significance was determined using Student\u2019s , Japan) ,34.To distinguish between the geographic origins of Wagyu beef via omics analysis, we compared the differences in the nutritional composition of Wagyu beef from different countries using MS. Quadrupole-Orbitrap LC\u2013MS/MS systems were used to detect 259 lipid molecules, including TG, PC, LPC, DG, and LPE. Because the significant TGs dominate the lipid profile of Wagyu beef, we compared the TG content of Japanese, Hybrid, and Australian Wagyu beef using conventional HPLC. OOO and OOPo were significantly more abundant in Japanese Wagyu beef, whereas POP, POS, PPP, and SOS were significantly more abundant in Australian Wagyu beef. Next, elemental comparisons based on ionomics analysis using ICP\u2013MS and ICP\u2013OES demonstrated that Japanese Wagyu beef contained higher levels of the Mo and Cd trace elements, whereas Australian Wagyu beef contained significantly more Rb, Cs, and Li. Comparing metabolites via metabolomics analysis using GC\u2013MS illustrated that Australian Wagyu beef contained significantly more amino acids and other components derived from lean muscle than Japanese Wagyu beef. These results of omics analyses revealed that the nutritional composition of Wagyu cattle differed with the country of production. This study suggests that combining multiple omics analyses enables the discrimination of the origin of genetically similar Wagyu cattle."}
+{"text": "The prevalence of diabetes is increasing in Bangladesh from \u223c5% in 2001 to \u223c13% in 2017/18 (\u223c8.4 million cases). The prevalence of undiagnosed diabetes was also found to be higher at 6% in 2017/18. However, very little is known about the management of diabetes assessed by diabetes awareness, treatment, and control. We aimed to estimate the age-standardised prevalence of awareness, treatment, and control of diabetes and its associated factors.  Cross-sectional data from 1,174 Bangladeshi adults aged 18 years and older available from the most recent nationally representative Bangladesh Demographic and Health Survey (BDHS) 2017\u201318 were analysed. Outcomes were age-standardised prevalence of awareness, treatment, and control of diabetes, estimated using the direct standardisation. Multilevel mixed-effects Poisson regression models were used to identify factors associated with awareness, treatment, and control of diabetes.  Of the respondents we analysed, 30.9%  were aware that they had the condition, and 28.2%  were receiving treatment. Among those treated for diabetes, 26.5%  had controlled diabetes. The prevalence of diabetes awareness, treatment, and control was lower in men than women. Factors positively associated with awareness and treatment were increasing age and hypertension, while factors negatively associated with awareness and treatment were being men and lower education. Factors associated with poor control were secondary education and residing in Rajshahi and Rangpur divisions.  This study provides evidence of poor management of diabetes in Bangladesh, especially in men. Less than one-third of the people with diabetes were aware of their condition. Just over one-fourth of the people with diabetes were on treatment, and among those who were treated only one-fourth had controlled diabetes. Interventions targeting younger people, in particular men and those with lower education, are urgently needed. Government policies that address structural factors including the cost of diabetes care and that strengthen diabetes management programmes within primary healthcare in Bangladesh are urgently needed. Globally, the prevalence of diabetes mellitus is steadily increasing . CurrentDiabetes mellitus was responsible for 6.7 million deaths in 2021 . MoreoveBangladesh has one of the highest burdens of diabetes among countries in the Southeast Asian region (\u223c25 million cases), and there is evidence that this disease burden is increasing . For insSeveral studies have reported a low level of diabetes management in Bangladesh , 18\u201320. The data used were extracted from the latest BDHS 2017/18 . This suHave you ever been told by a doctor or other health worker that you have high blood sugar or diabetes?\u201d). Treatment of diabetes refers to participants using medication at the time of the survey to control their diabetes. To collect these data, participants were asked \u201cAre you currently receiving treatment advice by a doctor or other health worker for your high blood glucose or diabetes?\u201d Control of diabetes refers to participants using medication to control blood glucose at the time of the survey with the FBG value of less than 7.0\u2009mmol/L [Awareness, treatment, and control of diabetes were our outcome variables. These outcome variables were generated for the respondents who had diabetes, which was defined as having elevated FBG (\u22657\u2009mmol/L) and/or on blood glucose-lowering medication at the time of the survey . Blood g0\u2009mmol/L .A comprehensive literature review of factors associated with awareness, treatment, and control of diabetes in Bangladesh and its neighbouring countries allowed the identification of the risk factors examined in this study , 22, 23.We used descriptive statistics to describe the individual, household, and community-level characteristics of the respondents. Age-standardised prevalence estimates of awareness, treatment, and control of diabetes were calculated and presented with 95% CI. The age-standardised estimates were calculated by direct standardisation based on the Bangladesh Population and Housing Census 2011.p-value <0\u00b705 was considered statistically significant. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines informed the design and reporting of the study [We used multilevel mixed-effects Poisson regression with a robust variance to identify factors associated with awareness, treatment, and control of diabetes mellitus. The results were presented as adjusted prevalence ratio (aPR) with 95% CI. Poisson regression was used to avoid overestimation of the odds ratios that occur using logistic regression in cross-sectional studies when the outcome of interest is common . Furtherhe study . All anaThe individual, household, and community-level characteristics of 1,174 participants who provided complete data are summarised in p < 0.05). The overall prevalence of treatment was 28.2% . Treatment was higher among women  than men  (p < 0.05). The prevalence of treatment increased with increasing age, in men and women. The prevalence of control of diabetes was 26.5% , increasing with increasing respondent age.The age-standardised prevalence of awareness, treatment, and control of diabetes is presented in Factors associated with awareness, treatment, and control of diabetes are presented in We found the likelihood of treatment of diabetes increased with the increase in respondents' age. The highest likelihood of treatment was in respondents aged 60\u201364 years  compared to the respondents aged 18\u201334 years. Women were more likely to be treated for diabetes than men . Respondents with primary, preprimary, or no education were less likely to be treated than the respondents with higher education . Respondents with hypertension were more likely to be treated for diabetes than the respondents without hypertension .The likelihood of control of diabetes was found to be 51%  lower among secondary-educated respondents than the higher-educated respondents. We found a lower likelihood of control of diabetes among respondents in Rajshahi  and Rangpur  division as compared to residents in the Barishal division.In this nationally representative study of Bangladesh using data from the most recent BDHS 2017\u201318, we assessed diabetes management by estimating the age-standardised prevalence of awareness, treatment, and control of diabetes and identified factors associated with these conditions. Our findings show that diabetes is poorly managed in Bangladesh, especially in men. Among those with diabetes, only 31% were aware of their condition, and 28% were receiving treatment. Only 26% had controlled diabetes mellitus among those who received treatment. Factors independently associated with awareness and treatment were age, sex, hypertension status, and level of education. Factors associated with poor control were secondary education and residing in Rajshahi and Rangpur divisions.We found that the prevalence of awareness, treatment, and control of diabetes was low. Only 31% of the total respondents with diabetes knew their diabetes status. Of these, only 28% used medication to control diabetes. Of those using medication to control diabetes, only 26% had controlled diabetes. These indicate poorer management of diabetes in Bangladesh as compared to other Southeast Asian countries. For instance, awareness of diabetes in Bangladesh was lower than the average of Southeast Asian countries (50%) , and ChiThis study found awareness and treatment increased with increasing age, which is comparable with other studies conducted in LMICs, including Bangladesh, India, Nepal, and China , 28, 29.We found that people with a low level of education were less likely to be aware, be treated, and have controlled diabetes than those with a higher level of education. Possible mechanisms may be through health literacy and employment. In the social structure of Bangladesh, education is the most important marker of a person's employment. People with lower levels of education are most likely to have manual employment involving physical work. This means that there was worse management of diabetes by a measure of low socioeconomic status, low education, and, by association, manual employment. However, employment appeared to be protective, as there were (nonsignificant) trends to higher awareness, treatment, and control in those with \u201ccurrent employment.\u201d Further investigations of the type of employment  in relation to diabetes management in Bangladesh are needed.Though the place of residence was not a significant factor for awareness and treatment of diabetes, we found poorer control of diabetes in respondents of Rajshahi and Rangpur divisions than in Barishal division. Many factors might contribute to such differences in the control of diabetes in these divisions. For instance, Rajshahi and Rangpur divisions are mostly rural and people are mostly engaged in agricultural activity . ConsistSex differences in diabetes management were found in this study in which women were more likely to be aware of diabetes and more likely to be treated for diabetes than men. Although not statistically significant, control was also higher in women than men. This is similar to previous findings in Bangladesh  and ChinOur study shows that people being underweight were less aware of their diabetes condition compared with people having normal weight. One plausible explanation could be the strong association of diabetes with obesity in health promotion literature, reducing the awareness of underweight people about their risk.The National Guideline for Diabetes Management in Bangladesh focused on raising diabetes awareness and associated factors . These iThe implications of our findings are that there needs to be substantial investment in health promotion to raise awareness and changes in healthcare delivery that address treatment and control of diabetes regardless of sociodemographic status. However, the management of diabetes within the healthcare sector in Bangladesh is challenging for several reasons. The major challenge comes from the facility level, in which maternal and child health and other diseases are prioritised . So far A limitation of this study was that outcome data are self-reported. Diet, metabolic, lifestyle, and behavioural factors are important determinants for diabetes management. However, these data were not available in the dataset and so could not be considered in our analyses. Moreover, the design of this survey was cross-sectional, which limits our capacity to draw casual associations. The major strength is that this is the first study in Bangladesh using a large nationally representative dataset that included adults 18 years and older, suggesting the findings have external validity. Our study generates findings with increased precision because of the use of multilevel mixed-effects Poisson regression that corrects the overestimation of effect size produced by conventional logistic regression employed in cross-sectional studies.This study provides evidence of poor management of diabetes in Bangladesh, especially in men. Less than one-third of the people with diabetes were aware of their condition. Just over one-fourth of the people with diabetes were on treatment, and among those who were treated only one-fourth had controlled diabetes. Interventions targeting younger people, in particular men and those with lower education, are urgently needed. Government policies that address structural factors including the costs of diabetes care and that strengthen diabetes management programmes within primary healthcare in Bangladesh are urgently needed."}
+{"text": "A total of 104 disease nodes, 496 target gene nodes, 35 ingredient nodes, and one drug node were extracted. According to the TCMSP database, 6-hydroxykaempferol, which reportedly promotes the proliferation of microvascular endothelial cells, is a molecule found in SHT. We found that it promoted the proliferation and migration of tendon fibroblasts and elevated tendon repair-related gene expression. Purified 6-hydroxykaempferol promoted the proliferation and migration of tendon fibroblasts and increased their mRNA expression in tendon proliferation.Tendon impairment is a common injury associated with impairment of range of motion and pain. Currently, evidence has confirmed that natural herbs contribute to orthopedics and have shown excellent results in the clinical management of tendon impairment. Shujin Huoxue tablet (SHT) and its complex prescriptions are regularly used in tendon rupture therapy with positive results. This study aimed to discover the potential molecules that promote tendon healing. The Chinese traditional medicine system pharmacological database analysis platform (TCMSP) is the primary resource. The Traditional Chinese Medicine Integrated Database and Encyclopedia of Traditional Chinese Medicine database were used as secondary databases. The GeneCards database was used to search for reported tendinopathy-related genes by keywords. Functions of the targeted genes were analyzed using Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes. Protein\u2013protein interaction information was extracted from the STRING database. Docking study, MTT assay, quantitative real-time PCR, and migration assays were performed to obtain a better understanding of the herbs according to cell function to test the basic pharmacological action  Tendon injuries, such as pain and lack of mobility, are common in both athletes and nonathletes . In modeCurrently, evidence confirms that natural herbs contribute to orthopedics with excellent results in clinical management . Shujin The Chinese traditional medicine system pharmacological database analysis platform (TCMSP) was used as the primary database in this study. Moreover, the Traditional Chinese Medicine Integrated Database (TCMID) and Encyclopedia of Traditional Chinese Medicine (ETCM) database were used as secondary databases. These databases were used in combination to bridge any gaps in key information, such as the name or number of ingredients. Even with the same database and screening strategy, the components included in the analysis vary . TherefoTCMID collects TCM-related information from different sources through text mining. Furthermore, it links common drug and disease databases, including DrugBank, OMIM, and PubChem. Chemical changes may occur during the torment of prescriptions, resulting in the generation of new ingredients. Data containing prescription ingredients were collected from the website, and 778 herbal mass spectra (MS) related to 170 herbs were added. Spectral data were analyzed to show the variation in the quality of herbal medicines from different sources and to differentiate between genuine medicinal materials and common medicinal materials. A massive increase in website analysis data will facilitate the study of combination therapy and the understanding of the underlying mechanisms of TCM at the molecular level .ETCM data on 402 herbs, 3,959 TCM compounds, 7,284 TCM chemical constituents, 2,266 drug targets, and 4,323 related diseases were collected. Data collected on herbal medicines included details of their origin, medicinal taste , medicinal properties , return meridians (lung meridian and liver meridian), indications, ingredients, and quality control standards. Furthermore, other information such as compound name, dosage form, composition, indications, and ingredients, including the molecular formula, molecular weight, various physical and chemical indicators, ADME parameters, and drug-like properties at the compound level, was collected. Quantitative standards were set according to the Pharmacopoeia of the People\u2019s Republic of China (2015 version) which are the official TCM quality evaluation standards in China .The absorption, distribution, metabolism, and excretion (ADME) processes of the active compounds are examined. In this process, the oral bioavailability (OB), drug similarity (DL), and half-life (HL) were the crucial parameters of ADME. The active compounds are indicated with OB \u2265 30% and DL \u2265 0.18 .RRID:SCR_002773) database was used to search for reported tendinopathy-related genes by entering keywords of tendon injuries, tendon rupture, tendon healing, and tendon adhesion for target-based drug applications. The potential tendon-healing targets of the active components of SHT matched the potential gene targets of the active ingredients of SHT. According to the UniProt database (https://www.uniprot.org/), the full names of the target proteins were transformed into gene symbols based on the UniProt ID  v6.8. The examination of the function target genes based on DAVID was carried out through the Gene Ontology (RRID:SCR_002811) biological process (GOBP) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. A p-value of 0.05 or less represents importance in the analysis. The capabilities provided by DAVID accelerate the analysis of genome-scale datasets by facilitating the transition from data collection to biological meaning, and the analysis results and graphical displays remain dynamically linked to raw data and external data repositories, providing in-depth and extensive data cover.Various biological processes and molecular functions were provided through the Database for Annotation, Visualization, and Integrated Discovery  database. The molecular structure documents of active ingredients and key target genes were imported into AutoDock (RRID:SCR_012746) Tools 1.5.6 software for molecular docking, and the documents in the PDBQT format were imported into Open Babel software and converted them into the PDB format. PyMOL 2.5.0 software was used for visual analysis of the target protein and the compound with a high docking score and relatively stable conformation.The molecular structure of active ingredients in the mol2 format was obtained from the TCMSP database. The PDB format of the 3D molecular structure of the corresponding target (protein) gene was obtained from the PDB . According to the manufacturer\u2019s instructions, cells were digested with 0.25% trypsin and seeded in six-well plates, with 2\u00a0ml culture medium made of Dulbecco\u2019s modified Eagle\u2019s medium with 10% fetal bovine serum (FBS), 100\u00a0U/ml penicillin, and 100\u00a0mg/ml streptomycin in each well and incubated at 37\u00b0C in a humidified atmosphere of 5% CO3) were seeded in each well of a 96-well culture plate with culture medium containing DMEM (0.1\u00a0ml of DMEM), 10% FBS, 100\u00a0U/ml penicillin, and 100\u00a0mg/ml streptomycin in each well. Then, 6-hydroxykaempferol  was added to each well at concentrations of 50, 100, 200, and 500\u00a0\u03bcg/ml. After incubation at 37\u00b0C for 24\u00a0h, cells were washed once with 1\u00d7 phosphate-buffered saline (PBS), followed by adding 0.1\u00a0ml DMEM with 0.1\u00a0ml 3--2,5-diphenyl-2-H-tetrazolium bromide . After incubation at 37\u00b0C for 30\u00a0min, the media were removed, and formazan crystals in the cells were solubilized in 0.2\u00a0ml DMSO and processed for OD reading at 570\u00a0nm using a spectrophotometer. Five wells were used for each concentration for each experiment, and the entire experiment was repeated three times.Tendon fibroblasts (3 \u00d7 10Col1a1 and Tenascin C (TNC) was achieved by quantitative real-time PCR using an ABI ViiA7 system and PowerUp SYBR Green Master Mix . The data were normalized to the GAPDH expression. The PCR program consisted of 95\u00b0C for 5\u00a0min, 40 cycles of 95\u00b0C for 30\u00a0s, 56\u201358\u00b0C for 30\u00a0s, and 72\u00b0C for 30\u00a0s, followed by a final extension step at 72\u00b0C for 10\u00a0min. All reactions were conducted in triplicate. The data were analyzed using the threshold cycle (Ct) method. The primer sequences for the target genes were as follows: Col1a1 forward primer, 5\u2032-CCC\u200bAGC\u200bGGT\u200bGGT\u200bTAT\u200bGAC\u200bTT-3'; Col1a1 reverse primer, 5\u2032- TCG\u200bATC\u200bCAG\u200bTAC\u200bTCT\u200bCCG\u200bCT-3'; TNC forward primer, 5\u2032- CAG\u200bAGT\u200bTGC\u200bCAC\u200bCTA\u200bCTT\u200bGCC-3'; TNC reverse primer, 5\u2032-TCT\u200bCTC\u200bCCT\u200bCAT\u200bCTT\u200bCTT\u200bTGT\u200bTCA-3'; GAPDH forward primer, 5\u2032-CTG\u200bGAG\u200bAAA\u200bCCT\u200bGCC\u200bAAG\u200bTAT\u200bG -3\u2032; and GAPDH reverse primer, 5\u2032-GGT\u200bGGA\u200bAGA\u200bATG\u200bGGA\u200bGTT\u200bGCT-3'.Total cellular RNA was extracted using a miRNeasy Mini Kit  according to the manufacturer\u2019s instructions. Total RNA . Quantification of the RNA levels of 6) were seeded in each well of a six-well culture plate with culture medium containing 2\u00a0ml of DMEM with 10% FBS, 100\u00a0U/ml penicillin, and 100\u00a0mg/ml streptomycin in each well and cultured overnight at 37\u00b0C. After cell confluency reached over 95%, the medium was removed, and the cells were washed once with 1\u00d7 PBS. Then, a linear scratch within the cell monolayer was generated using a sterile 1,000-\u03bcl plastic pipette tip. The cellular debris was washed with 1\u00d7 PBS. Next, 2\u00a0ml starvation medium composed of DMEM with 1% FBS, 100\u00a0U/ml penicillin, and 100\u00a0mg/ml streptomycin was added to each well, followed by treatment with 400\u00a0\u03bcg of 6-hydroxykaempferol in three wells, and the cells were incubated at 37\u00b0C to allow migration. At 0 and 3\u00a0h, the images were photographed (10\u00d7 magnification). The percentage of the open wound area (3\u00a0h relative to 0\u00a0h) was calculated using ImageJ software.A scratch wound assay was performed to test the migration of tendon fibroblasts. Tendon fibroblasts (1 \u00d7 10RRID:SCR_002798), San Diego, CA, United States). p < 0.05 was considered significant and presented with * in the figure.Data are the mean \u00b1 SD. The significance of the results was determined based on one-way analysis of variance using Prism 8.0.1  were used to produce a PPI network. It shows the protein\u2013protein interaction between the disease, including tendon injury, tendon adhesion and tendon healing, and the target gene of the active compound. The simple tabular text is extracted from the graphic to run the R program (version 3.6.2). From RRID:SCR_012773) enrichment analysis. As shown in p = 2.88E-22), \u201cfluid shear stress and atherosclerosis\u201d (p = 2.94E-20), \u201cIL-17 signaling pathway\u201d (p = 5.69E-20), and \u201cTNF signaling pathway\u201d (p = 1.02E-19) in the healing process (p = 2.63E-12), \u201ctranscription factor activity, direct ligand-regulated sequence-specific DNA binding\u201d (p = 2.63E-12), \u201ccytokine receptor binding\u201d (p = 3.31E-10), \u201ctetrapyrrole binding\u201d (p = 5.01E-09), \u201ccytokine activity\u201d (p = 1.97E-09), \u201cheme binding\u201d (p = 2.50E-09), \u201csteroid hormone receptor activity\u201d (p = 1.78E-08), \u201creceptor ligand activity\u201d (p = 2.43E-07), \u201cDNA-binding transcription activator activity, RNA polymerase II-specific\u201d (p = 5.48E-07), \u201cgrowth factor receptor binding\u201d (p = 5.64E-07), and \u201cscaffold protein binding\u201d (p = 6.86E-07) were significantly enriched in the healing process . Our results showed that treatment with 6-hydroxykaempferol was able to promote tendon fibroblast proliferation, highly related gene expression, and migration , and migration in tendon proliferation. Further studies should be conducted to determine the potential characteristics of 6-hydroxykaempferol.Clinically, SHT has been used to treat tendon injury. However, a molecular validation of its effects has not previously been conducted. Based on"}
+{"text": "Chlamydomonas reinhardtii. The results suggest that both the MPs and SDZ alone and in combination inhibited the growth of microalgae with an increasing concentration of MPs and SDZ (5\u2013200 mg l\u20131); however, the inhibition rate was reduced by combination. Upon exposure for 7 days, both the MPs and SDZ inhibited algal growth, reduced chlorophyll content, and enhanced superoxide dismutase (SOD) activities, whereas glutathione peroxidase (GSH-Px) activity was elevated only with the exposure of 1 \u03bcm MPs. Fluorescence microscopy and scanning electron microscopy also indicated that particle size contributed to the combined toxicity by aggregating MPs with periphery pollutants. Further, the amount of extracellular secretory protein increased in the presence of MPs and SDZ removal ratio decreased when MPs and SDZ coexisted, suggesting that MPs affected SDZ metabolism by microalgae. The particle size of microplastics affected the toxicity of MPs on microalgae and the combined effect of MPs and SDZ could be mitigated by MPs adsorption. These findings provide insight into microalgae responses to the combination of MPs and antibiotics in water ecosystems.Despite the fact that microplastics (MPs) facilitate the adsorption of environmental organic pollutants and influence their toxicity for organisms, more study is needed on the combination of MPs and antibiotics pollutant effects. In this study, polystyrene MPs (1 and 5 \u03bcm) and sulfadiazine (SDZ) were examined separately and in combination on freshwater microalga,  Furthermore, oxidative stress increases the activity of SOD and glutathione reductase, while decreases the activity of catalase. This made the antioxidant response inadequate to cope with the rising ROS and prevent oxidative damage  and some of them are relatively stable and can persist in surface water and even drinking water, raising concerns about their potential dangers . Among te damage . Studiese damage ,b.Chlorella vulgaris; the levofloxacin  removal rates for the microplastics group (35 items\u22c5L\u20131) and the control group were 23.34 and 46.71%, respectively, on the third day, but the combined toxicity on microalgae was not extensively studied  has a great prospective to easy cultivation, considered as highly susceptible to environmental pollution, used as potential candidate for aquatic contamination assessments, and demonstrated high biosorption and removal efficiency of PPCPs , labeled with green fluorescence (excitation wavelength: 470 nm and emission wavelength: 526 nm), were purchased from BaseLine ChromTech Research Centre  and IR absorption spectra confirmed the chemical composition of the microspheres. The diameters of PS-MPs particle were detected using the scanning electron microscope (SCM). Sulfadiazine (SDZ) sodium salt was purchased from Sigma-Aldrich (Cat. No. S6387-25G and purity 99.9%).Chlamydomonas reinhardtii CC124 strain was obtained from the Chlamydomonas Genetic Center of Duke University  and cultured in a tris-acetate-phosphate (TAP) medium. Algal cells were cultivated in a constant temperature light incubator at 22 \u00b1 2\u00b0C and 20 \u03bcmol photon m\u20132s\u20131 illumination. Algae were grown in 250 ml Erlenmeyer flasks and were shaken daily and randomly arranged to reduce any minor differences in photon irradiance  and six concentrations of PS-MPs  were selected in this study and then the combination of fixed 50 mg l\u20131 PS-MPs and six concentrations of SDZ  to analysis their conjoint effects based on the results of individual toxicity experiments. C. reinhardtii cells were cultured in a 12-well plate  and seeded in the concentration of 105 cells ml\u20131; the cell concentration was counted using a hemocytometer for inhibition rate estimation.To assess the acute effects, a 24\u201396 h period is the ideal time . In ordeotolysis , six conC. reinhardtii generally reached stationary stage after 5\u20136 days of incubation. According to the results of 96 h inhibition tests, the microalgae were subjected to a 7-day toxicity test with 50 mg l\u20131 SDZ, 50 mg l\u20131 PS-MPs, and combined 50 mg l\u20131 SDZ with 50 mg l\u20131 PS-MPs. The microalgae were cultured in 250 ml flasks and kept the other culture conditions consistent with the previous conditions. After exposure, cell concentration and biomass were calculated. A 50-ml culture was collected in order to determine the concentration of extracellular secretory protein and the rest of the culture was centrifugated at 4,500 g and 20\u00b0C for 10 min to collect algal cells. The total protein was extracted to measure biochemical activities.It is known that The contents of chlorophyll (Chl) were determined according to a modified method of the previous study . The culThe parameters of photosynthetic activity of algae were measured using a pulse amplitude modulated (PAM) fluorometer water-PAM . The concentration of protein was determined using a protein quantification kit .The activities of total antioxidant enzyme SOD and glutathione peroxidase (GSH-Px) were measured by using and following the recommended instructions of the colorimetric commercial kits . The activity of SOD was assayed with the reaction based on its inhibition on the scale of superoxide anion generated by xanthine and xanthine oxidase reaction system. A SOD unit was measured as the amount of enzyme that led to a half inhibition of the nitroblue tetrazolium reduction rate using the plate reader at 550 nm. GSH-Px was measured according to the manufacturer\u2019s protocol. Based on the reaction ability of dithodinitrobenzoic acid with sulfhydryl compounds at 405 nm absorption peak in producing a relatively stable yellow color, GSH activity was measured. GSH-Px is preferably represented by catalyzed GSH reaction rate by measuring absorbance at 412 nm for 5 min. In this study, the activities of SOD and GSH-Px were expressed as units per milligram of protein  and analyzed by image analysis system with HCX PL APO 40X 0.85 dry objective lens. Fluorescence images were recorded for MPs particles with excitation and emission wavelengths of 554 and 586 nm, respectively, and 649/670 nm for the determination of fluorescence for microalgal cells, respectively. MPs particles are shown in green color and microalgal cells are shown in red color. After 96 h of exposure under toxicity assay, the microalgal cells were collected by centrifugation . Samples were initially fixed overnight at 4\u00b0C with 2.50% glutaraldehyde and then washed three times with phosphate-buffered saline . Afterward, the samples were dehydrated for 15 min through a series of 30, 50, 70, 80, 90, 95, and 100% alcohol solutions. The dehydrated samples were placed in an oven at 40\u00b0C and dried for 24\u201348 h. After gold spraying, the samples were observed by scanning electron microscopy .g centrifugation at 20\u00b0C for 15 min, supernatant solution was pipetted and filtered through a 0.45-\u03bcm glass fiber (GF) membrane . Colorimetric analysis of EPS protein content was carried out using the protein quantification kit . The absorbance of the samples was read at 595 nm using the Synergy Neo2 Plate Reader  and protein concentration was calculated by comparison with the standard curve , was quantified and analyzed as follows: by 10,000 rd curve .g and 20\u00b0C for 15 min; the supernatant was collected and SDZ concentration was measured at 254 nm using the plate reader and calculated by comparison with the standard curve.The quantum yield is a critical factor that employs to quantify the efficiency of sulfonamides photodegradation reaction and an absorption peak at 254 nm indicates the photolysis products from SDZ degradation . Therefop > 0.05) to confirm normal distribution and homoscedasticity, respectively. The differences among the treatments were analyzed by single-factor ANOVA and taking a level of p < 0.05 as significant to Duncan\u2019s multiple range test. The difference and interactive effects between antibiotics and MPs were examined using the two-way ANOVA followed by the Student\u2013Newman\u2013Keuls (SNK) tests for multiple comparisons (with significant level of p < 0.05).Each experiment was performed in triplicate and graphs were plotted by the Prism 8 (GraphPad Software Incorporation). For statistical analysis, data were subjected to IBM SPSS version 25.0 (IBM SPSS) with the Shapiro\u2013Wilk\u2019s and Levene\u2019s tests , also showed that MPs with smaller sizes of particles were more toxic than those with larger sizes of particles  of PS-MPs combined with different concentrations of antibiotics was subsequently analyzed  at SDZ concentration of 100 mg l\u20131 compared to SDZ alone, while this value decreased by 11.0% (p < 0.05) at SDZ concentration of 200 mg l\u20131. In contrast, there was no significant difference between SDZ alone and in combination with SDZ with 5 \u03bcm PS-MPs at SDZ concentrations of 100 and 200 mg l\u20131. This evidence is suggesting that the presence of MPs may reduce the inhibition of algal growth by excessive antibiotics. This effect is notable for MPs with smaller sizes of particle. The two-way ANOVA results indicated significant effects of MPs and SDZ for growth inhibition of C. reinhardtii and the interaction between SDZ and 5 \u03bcm PS-MPs  and copper (0.5 mg l\u20131) caused severe cellular damage and increased the concentrations of intracellular SOD and malondialdehyde , which is subsequently removed by catalase and GSH-Px. These enzymes catalyze the reduction of H2O2 to harmless products and the intracellular antioxidative system scavenges excess oxygen radicals, leading to avoiding or reducing oxidative damage  did not affect the photosynthesis of various microalgae, including diatoms, Dunaliella salina, and freshwater Chlorella  for 6 days and EPS decreased slightly compared to the control group, but remained much higher than the level at the start of this study , while the removal rate decreased by 11 and 18% in the medium supplemented with 1 \u03bcm PS-MPs and 5 \u03bcm PS-MPs, respectively (C. vulgaris was significantly inhibited when PS-MPs co-exposed (The most antibiotics are difficult to biodegrade due to their pharmacological stability  or resis0 mg l\u20131 . The stu0 mg l\u20131 . There a0 mg l\u20131 , while c0 mg l\u20131 . In addi0 mg l\u20131 . A studywith EPS . To studectively . The inc-exposed ; even thC. reinhardtii. SDZ exposure and the attachment of MPs to microalgae increased antioxidant enzyme activity, resulting in an increase in extracellular secretory proteins and a decrease in chlorophyll content. This was accompanied by a reduction in the growth rate of microalgae and noticeable aggregation of MPs. Exposure toxicity increased with the concentration and interaction of PS-MPs and SDZ, but different particle sizes and their interactions with SDZ had differential effects and when co-exposed with 5 \u03bcm PS-MPs, SDZ removal rates by algae were compromised. Therefore, considering the increasing trend of global antibiotics and microplastics production in response to the demand of the human population growing rapidly and the fundamental role of microalgae as primary producers in aquatic ecosystems, more studies on the combined effects of microplastics and emerging contaminants are required.In this study, we examined the single and combined effects of PS-MPs and SDZ on freshwater alga The original contributions presented in the study are included in the article/ZL, YZ, and ZH conceived and designed the experiments. ZL and LL performed the experiments. ZL, SD, and FH analyzed the data. ZL and YZ contributed reagents, materials, analysis tools, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Penicillium sp. YT2019-3321, an endophytic fungus derived from traditional Chinese medicine Lonicera japonica, was chemically studied.Endophytic fungi associated with medicinal plants have proven to possess a high potential to produce structurally diverse metabolites, some of which are valuable for medicinal applications. In this study, 1 by chiral HPLC yielded individual enantiomers (+)-1 and (\u2013)-1, and their stereochemistry were solved by X-ray diffraction crystallography, respectively.The chemical structures of the isolated compounds were established by a correlative interpretation of HRESIMS and NMR spectroscopic data. The optical resolution of (\u00b1)-1) and penicidone E (2), were isolated and identified from Penicillium sp. YT2019-3321. Compound 2 possessed the \u03b3-pyridone nucleus, which is rarely found in natural products. Cytotoxic assay revealed that the new compound 2 demonstrated a dose-dependent cytotoxicity against the human pancreatic tumor cells PATU8988T with the IC50 value of 11.4 \u03bcM. Further studies indicated that 2 significantly induced apoptosis of PATU8988T cell lines, characterized by the morphologies abnormity, the reduction of cell number, the upregulation of proportion of apoptotic cells, and the ratio of Bcl-2 to Bax. Our study demonstrates that fungal secondary metabolites may have important significance in the discovery of drug leads.Eight structurally diversified secondary metabolites, including two previously unreported polyketides, named (\u00b1)-chrysoalide B ( Filamentous fungi from both marine and terrestrial sources are inherently regarded as a treasure house of structurally diversified secondary metabolites with potent pharmacological activity . Fungi pAlternaria sp. YUD20002, an endophytic fungus derived from the tubers of Solanum tuberosum, yielded five previously undescribed epoxy octa-hydronaphthalene polyketides altereporenes A-E -chrysoalide B (1) and penicidone E (2) -1 and (\u2013)-1, and their stereochemistry were solved by X-ray diffraction crystallography. The new compound 2 possessed the \u03b3-pyridone nucleus, which is rarely found in natural products. In addition to the structural elucidation, the cytotoxic activity of the isolated compounds is also described herein.Endophytic fungi are recognized as microorganisms that spend the whole or part of their lifetime colonizing inter-and/or intra-cellularly plant tissues without causing any apparent disease symptoms . Endophyenes A-E . Acrocalm acutum . Acrocal19.64 \u03bcM . It shoune E (2) . The opt1H and 13C, respectively) and 2D  NMR spectra were performed by an Agilent DD2 500 MHz spectrometer . X-ray diffraction data were collected on an Agilent Xcalibur Gemini E diffractometer equipped with Eos charge-coupled device (CCD) detector with graphite monochromated Cu K\u03b1 radiation (\u03bb = 1.54178 \u00c5). Column chromatography was undertaken by using various packing materials including silica gel , octadecylsilyl (ODS) reversed-phase gel , and Sephadex LH-20 .Optical rotations were measured with a JASCO P-1020 digital polarimeter . UV spectra were obtained on a Lambda 35 UV/Vis spectrophotometer . HRESIMS data were acquired with a scientific LTQ Orbitrap XL spectrometer . 1D  of the rRNA locus. The ITS sequence showed 99% identical to that of P. oxalicum (GenBank accession no. KY400080.1). A voucher specimen of this fungal strain was stored at \u201380\u00b0C at the Third Affiliated Hospital of Wenzhou Medical University. This fungus was cultured on potato dextrose agar medium  at 28\u00b0C for 5 days. Then all of agar plugs were cut into small pieces (0.5 \u00d7 0.5 cm2). Each piece was inoculated in a 1 L Erlenmeyer flask containing 250 mL of potato dextrose broth (PDB) medium (Solarbio). A total of 100 flasks were statically fermented at room temperature for 30 days.The fungal strain 2O . This afforded a total of eight subfractions (Fr. 4.1-Fr. 4.8). Fr. 4.6 was further purified over an open silica gel column chromatography by using the solvent system CH2Cl2 and MeOH with the ratio 20:1 to afford 16 mg of compound 2. Fr. 5 (2.5 g), eluting with EtOAc/petroleum ether 2:1, was applied to ODS silica gel with gradient elution of MeOH/H2O  to yield eight subfractions (Fr. 5.1-Fr. 5.8). Compound 1 (10.2 mg) was isolated from a two-step purification process, first from Fr. 5.3 over an open silica gel column chromatography using the solvent system CH2Cl2 and MeOH with the ratio 20:1, followed by preparative TLC . Compound 1 was further resolved into the pure enantiomers (+)-1  and (\u2013)-1  by chiral HPLC using a . Whelk-O1 chiral column . Compound 3  was isolated from Fr. 5.4 by semipreparative HPLC . Compound 7 (5.6 mg) was isolated from Fr. 5.5 by preparative TLC . Compound 6 (11.3 mg) was isolated from Fr. 5.6 by preparative TLC . Fr. 6 (4.0 g), eluting with EtOAc/petroleum ether 1:1, was fractionated by Sephadex LH-20 column chromatography in MeOH to give subfractions Fr. 6.1-Fr. 6.3. Fr. 6.1 was subjected to semipreparative HPLC (65% MeOH/H2O) to give compounds 4  and 5 , respectively. Finally, compound 8 (4.9 mg) was obtained by preparative TLC  from Fr. 6.3.The fermentation materials were adequately extracted with EtOAc (3 \u00d7 25 L), and the organic solvent was evaporated in vacuum to yield ca. 20 g of crude extracts. The crude extracts were subjected to a silica gel vacuum liquid chromatography column, which was eluted with an increasing gradient of EtOAc/petroleum ether (from 30:1 to 1:1) to afford six fractions (Fr. 1-Fr. 6). Fr. 4 (3.2 g), eluting with EtOAc/petroleum ether 5:1, was further fractionated over an ODS reversed-phase silica gel with a mixed solvent system of MeOH/H1): white amorphous powder; [\u03b1]20D + 9.6  for (+)-1 and [\u03b1]20D \u201310.2  for (\u2013)-1; UV (MeOH) \u03bbmax (log \u03b5) 213 (2.16), 239 (1.60), 331 (1.49) nm; 1H and 13C NMR data (measured in DMSO-d6) .(\u00b1)-Chrysoalide B (-d6) see ; HRESIMS2): colorless oil; [\u03b1]20D + 13.5 ; UV (MeOH) \u03bbmax (log \u03b5) 220 (3.88), 254 (3.26), 309 (2.98); 1H and 13C NMR data (measured in DMSO-d6) (see m/z 390.1547 [M + H]+ (C20H24NO7) and 412.1369 [M + Na]+ (C20H23NO7Na).Penicidone E (-d6) see ; HRESIMS1 and (\u2013)-1 were obtained by slowly evaporating the solvent mixture of MeOH and H2O. Single-crystal X-ray diffraction data were obtained on an Agilent Xcalibur Gemini E diffractometer equipped with Eos CCD detector with graphite monochromated Cu K\u03b1 radiation (\u03bb = 1.54178 \u00c5). Structures were solved by direct methods using the SHELXTL software package - package . All non package .1: C22H26O11 (2 C11H12O5 + H2O), F.W. = 466.43, monoclinic space group P21, unit cell dimensions a = 7.3590 (9) \u00c5, b = 14.4044 (16) \u00c5, c = 10.4189 (12) \u00c5, \u03b1 = \u03b2 = \u03b3 = 90\u00b0, V = 1103.9 (2) \u00c53, Z = 2, dcalcd = 1.403 mg/m3. Crystal size: 0.08 \u00d7 0.05 \u00d7 0.04 mm3, \u03bc = 0.967 mm\u20131, F (000) = 492.0. Reflections collected/unique: 19,174/4,377 [R (int) = 0.0438]. Final indices resulted in R1 = 0.0424 and wR2 = 0.1067 [I > 2\u03c3(I)] Flack parameter = 0.13 (6).Crystal data for (+)-1: C22H26O11 (2 C11H12O5 + H2O), F.W. = 466.43, monoclinic space group P21, unit cell dimensions a = 7.3638 (2) \u00c5, b = 14.3632 (4) \u00c5, c = 10.4176 (3) \u00c5, \u03b1 = \u03b2 = \u03b3 = 90\u00b0, V = 1101.11 (5) \u00c53, Z = 2, dcalcd = 1.407 mg/m3. Crystal size: 0.12 \u00d7 0.07 \u00d7 0.04 mm3, \u03bc = 0.970 mm\u20131, F (000) = 492.0. Reflections collected/unique: 25,073/4,450 [R (int) = 0.0590]. Final indices resulted in R1 = 0.0358 and wR2 = 0.0827 [I > 2\u03c3(I)] Flack parameter = 0.05 (10).Crystal data for (\u2013)-2 at 37\u00b0C. Cells were treated with the positive control doxorubicin (dox) at the dose of 10 \u03bcM and the test compounds at the dose of 20 \u03bcM, respectively, for 48 h when they reached \u223c80% confluence.The human pancreatic cancer cell line PATU8988T was acquired from Shanghai Fuheng Biotechnology Co., Ltd., RPMI 1640 medium containing 10% fetal bovine serum  was used. The cells were cultured in 5% CO2 at 37\u00b0C. Cell viability was detected at absorbance of 450 nm.CCK-8 (Solarbio) was applied to detect the cell viability according to the manufacturer\u2019s instruction as previously described . In brieCell apoptosis was examined by flow cytometry using Annexin V-FITC Apoptosis Detection Kit  according to the manufacturer\u2019s instruction. Cells were incubated with or without test compounds at 20 \u03bcM for 48 h, followed by being treated with 200 mL binding buffer and stained with Annexin V-FITC and PI for 40 min in the dark. After that, the cells were assessed by flow cytometry .RIPA buffer  containing protease inhibitors  was applied to extract protein lysates of the cells. The concentration of protein was examined by the Bradford assay. The samples were diluted in loading buffer and denatured at 95\u00b0C for 5 min. Then they were separated in SDS PAGE gel followed by being transferred into nitrocellulose membranes for next steps. After being treated with blocking solution for 1 h at room temperature, the membranes were incubated with the following primary antibodies: Bax and Bcl-2 purchased from ABclone. After that, membranes were washed with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20 for 15 min for three times followed by being treated with secondary antibodies for 1 h at room temperature. Bands were visualized with ECL substrate (Bio-Rad Laboratories).S-2 and DFT calculations were performed with the Gaussian 16 program (The conformer rotamer ensemble sampling tool (crest)  was used program . The con1) was isolated as white amorphous powder. Its molecular formula, C11H12O5, was established by HRESIMS at m/z 223.0644 [M-H]\u2013 . Observation of the 1H NMR data of 1  and 3.80  as well as two coupled aromatic methines at \u03b4H 7.13  and 7.05 . The 13C spectroscopic data of 1 (C 165.1 (C-1), five quaternary carbons including one oxygenated sp3 at \u03b4C 106.8 (C-3), two aromatic methines at \u03b4C 123.5 (C-5) and 115.2 (C-6), and three methyl groups including two methoxy groups at \u03b4C 50.7 (C-9) and 55.9 (C-10). Considering the functional groups observed for compound 1 as well as the characteristic UV absorption peaks at 213, 239, and 331 nm -Chrysoalide B -1 and (\u2013)-1 with a ratio of 1:1. Both (+)-1 and (\u2013)-1 were cultured into suitable single crystals in MeOH/H2O mixed solution. Single-crystal X-ray diffraction of (+)-1 and (\u2013)-1 (1 and (\u2013)-1. It should be pointed out that R based on ECD calculations. However, in our study, the absolute configuration of (+)-chrysoalide B (1) was revised as 3S by single-crystal X-ray diffraction.Compound D-201810 . Surprisnd (\u2013)-1  not only2), isolated as colorless oil, was found to possess the molecular formula of C20H23NO7 on the basis of HRESIMS (m/z 390.1547 [M + H] + for C20H24NO7 and 412.1369 [M + Na] + for C20H23NO7Na). Overall inspection of the 1H and 13C NMR spectra of 2 (C 192.4 (C-8) and 175.8 (C-10), one ester carbonyl at \u03b4C 166.3 (C-1), ten sp2-hybridized carbons which resonated between \u03b4C 103.2 and 160.0, one methylene at \u03b4C 39.2 (C-15), one oxygenated sp3 methine at \u03b4C 75.3 (C-16), and five methyls including four methoxy groups at \u03b4C 52.5 (1-OMe), 56.0 (4-OMe), 56.5 (6-OMe), and 56.0 (16-OMe). The 1H and 13C NMR spectra of 2 were partially similar to that of penicidone C, a cytotoxic alkaloidal metabolite isolated from an endophytic Penicillium sp. (C 125.3 and 134.2 ascribable to the two sp2 methine groups in penicidone C were replaced by a methylene (C-15) and an oxygenated sp3 methine (C-16). Moreover, an extra methoxyl signal at \u03b4H 3.23 and \u03b4C 56.0 (16-OMe) appeared in the 1H and 13C NMR spectra of 2. The observation could be explained by assuming that 2 was an oxidative derivative of penicidone C at the location of C-15 and C-16. This assumption was reinforced by the HMBC correlations from H3-17 to C-15 and C-16, from H2-15 to C-11 and C-12, and from 16-OMe to C-16  level in methanol (\u03bb = 589 nm). The calculated optical rotation was \u201324.4, which was of the opposite sign to the experimental value ([\u03b1]20D + 13.5). Therefore, the absolute configuration of 2 was established as 16R.Penicidone E  (4) (5) (6 (7) (8) (In addition to the new compounds lide (3) , oxister (3) (4) , penicil (4) (5) , a diphe) (5)  (6 (7) , and 3-[ (7) (8) .1-8 were detected for their cytotoxicity against human pancreatic cancer cell line PATU8988T by using the CCK-8 method  and 2-treated groups while the cells in the control group and DMSO group performed normally  and penicidone E (2), were isolated from Penicillium sp. YT2019-3321, an endophytic fungus derived from traditional Chinese medicine Lonicera Japonica. The optical resolution of (\u00b1)-1 by chiral HPLC yielded individual enantiomers (+)-1 and (\u2013)-1, and their stereochemistry were solved by X-ray diffraction crystallography, respectively. The \u03b3-pyridone nucleus found in 2 is rare in natural products, with only four analogs having similar structures. Tumor cell proliferation might be inhibited by cell apoptosis. Our study demonstrated that the new compound 2 significantly induced apoptosis in human pancreatic tumor cells (PATU8988T), characterized by the morphologies abnormity, the reduction of cell number, the upregulation of proportion of apoptotic cells, and decrease in the ratio of Bcl-2 to Bax.Eight structurally diversified secondary metabolites, including two previously unreported polyketides, named (\u00b1)-chrysoalide B (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/CZ and XL: conception or design. SJ, WW, CS, XP, and LX: acquisition, analysis, and interpretation of data. WW: drafting the work and revising. CZ, XL, and WW: final approval of the manuscript. All authors contributed to the article and approved the submitted version."}
+{"text": "Escherichia coli, Staphylococcus aureus, and Candida albicans, utilizing specifically designed primers. The method for preparing membrane-damaged bacteria was optimized to improve the ability of the PMA dye to distinguish between live and dead indicator bacteria. Finally, this method could simultaneously detect viable numbers of the indicator bacteria after the disinfectants were used. The R2 values of the PMA-qPCR standard curves were 0.9986, 0.9980, and 0.9962 for E. coli, S. aureus, and C. albicans, respectively, and the detection range was 103\u2009~\u2009106\u2009CFU/ml, showing no significant difference in accuracy compared to that of the plate counting method (p > 0.05). The method established here is the first application of PMA-qPCR to detect the antibacterial and bacteriostatic activity of disinfectants. This technique markedly simplifies the detection steps of antibacterial and bacteriostatic activity, reduces the detection time (3\u2009h compared to 48\u2009~\u200972\u2009h for the plate counting method), improves the quality supervision efficiency of disinfectants, and guarantees healthy and safe lives.Rapid detection of antibacterial and bacteriostatic properties is an important part of the quality and safety supervision of disinfectants. In this study, propidium monoazide (PMA) was used in combination with real-time PCR (PMA-qPCR) to detect the antibacterial and bacteriostatic activity of disinfectants against three commonly used indicator bacteria,  The novel coronavirus has caused a worldwide infection since its outbreak in 2019, and the situation remains dire. The market share of disinfectants has proliferated to maximize people\u2019s health status; however, their quality control is facing an unprecedented challenge. Disinfectants products include disinfectant solution, disinfection devices , hygiene products, and single-use medical supplies . DisinfeEscherichia coli 8099 stands for enteric bacteria, Staphylococcus aureus ATCC 6538 stands for septic cocci in bacterial colonies, and Candida albicans ATCC 10231 stands for pathogenic fungi. The specific method of this assay is to perform a plate count of the indicator bacteria from the treated samples and calculate the bactericidal inhibition rate from the difference in the number of viable bacteria compared to that of the control samples. The plate count method is simple, but the number of steps and workload is large, the turnaround time for test results is approximately 48\u2009h (bacteria) to 72\u2009h (yeast), and many sublethal indicator bacteria cannot form colonies because of the limitations of the culture environment , with the following main indicators: ironment . TherefoIn recent years, real-time PCR (qPCR) has been widely used for the rapid quantitative detection of microorganisms . The priE. coli  and propidium monoazide (PMA) are a class of photoreactive dyes with a high affinity for DNA . They arE. coli , Salmonelmonella , Lactobabacillus , Vibrio  , which is consistent with our previous reports . The DNAThe qPCR reaction system was (20\u2009\u03bcl):2\u00d7 SYBR Premix ExTaq 10\u2009\u03bcl, 0.4\u2009\u03bcl each of upstream and downstream primers and probe primer (10\u2009\u03bcmol\u00b7L-1), 2\u2009\u03bcl of template DNA (10\u2009ng\u00b7\u03bcL-1), and dd H2O was added to 20\u2009\u03bcl. A negative control reaction was set without DNA.The amplification conditions were: pre-denaturation at 95\u00b0C for 30\u2009s, denaturation at 95\u00b0C for 5\u2009s, annealing at 58\u00b0C for 30\u2009s, and extension at 72\u00b0C for 30\u2009s for 40\u2009cycles. The fluorescence signal was collected at the time of warming to establish a melting curve.Primer specificity was verified by extracting genomic DNA from the three experimental strains and eight reference strains mentioned above, amplifying the samples by qPCR using the three primers mentioned above, and determining the specificity of the primers based on the results of the amplified Ct values .1 to 106 dilutions) were prepared to obtain samples with different initial DNA concentrations. The samples were amplified by qPCR using the above primers, and the linear equation of the Ct value versus initial DNA concentration was plotted to calculate the qPCR amplification efficiency E value (E\u2009=\u200910-1/slope). Three replicates were performed for each qPCR sample.Primer amplification efficiency was verified by taking 1\u2009ml of the prepared experimental broth with OD600\u2009\u2248\u20091 , and the number of viable bacteria in the experimental broth was determined by plate counting . At the One milliliter of the experimental bacterial solution with OD600\u2009\u2248\u20091 prepared as above was placed in a 1.5\u2009ml centrifuge tube, washed twice in phosphate buffer solution (PBS), and resuspended in 1\u2009ml of PBS buffer. The bacteria were treated in a water bath at 100\u00b0C for 15\u2009min for heat lethality, and the untreated suspension was used as the control group. Three replicates were performed for each sample.One milliliter of the experimental bacterial solution with OD600\u2009\u2248\u20091 prepared as described above was placed in a 1.5\u2009ml centrifuge tube, washed twice in PBS buffer, and resuspended in 1\u2009ml of PBS buffer. The homogenization program was set to a homogenization speed of 6.0\u2009m/s, working time of 30\u2009s, and interval of 30\u2009s. A total of 15, 20, 25, 30, and 35\u2009cycles were performed, with the untreated bacterial suspension as the control group. Three replicates were performed for each sample.The samples obtained using the above method were directly coated with 200\u2009\u03bcl of the corresponding solid medium plates and incubated at 37\u00b0C for 24\u2009~\u200948\u2009h. These results were used to determine the effectiveness of the preparation of membrane-damaged bacteria. Three replicates were performed for each sample.The working concentration of PMA dye-treated samples  in this study was 40\u2009\u03bcg/ml . The PMAg for 1\u2009min, washed twice with PBS buffer solution, and resuspended in an equal volume of PBS buffer solution. The suspensions were divided equally into two groups: one group without any treatment, that is, the live group, and one group with membrane-damaged bacteria prepared first according to the optimized method, followed by PMA treatment. DNA was extracted from both groups, and the DNA of the live group was diluted in a gradient (100\u2009~\u2009106 times) with the DNA of the membrane-damaged group. The Ct values were obtained by qPCR amplification using the designed primers. Ct values were obtained using an ABI 7500 FAST fluorescent quantitative PCR instrument. Three replicates were performed for each sample. A standard curve of Ct values versus the initial DNA concentration of live bacteria after PMA treatment was plotted.One milliliter of the experimental bacterial solution with OD600\u2009\u2248\u20091 prepared by the above method was collected by centrifugation at 12,000\u2009\u00d7\u2009The disinfectant products tested were: Refreshing Hand sanitizer, produced by Shanghai Jahwa United for germicidal experiments; Willows Foam Antibacterial Hand sanitizer, produced by Willis (Guangzhou) Household Products for germicidal experiments; Jierou sanitary wipes, produced by Zhongshun Jierou (Sichuan) Paper for sterilization experiments; Vida sanitary wipes, produced by Vida Paper (Beijing) for germicidal experiments; 84 disinfectants, produced by Jiangsu Atef 84 for sterilization experiments; and Hand disinfectant, produced by Nanjing Zhuhai Biotechnology for sterilization experiments.A 10\u2009g sample of hand sanitizer was weighed, added to an equal mass of PBS , homogenized, and prepared to obtain the sample solution to be tested.5\u2009~\u20094.5\u2009\u00d7\u2009106\u2009CFU/ml suspension; the three indicator bacterial suspensions were mixed in equal volumes; 300\u2009\u03bcl of the mixed suspension was added to 5\u2009ml of the sample solution, and 300\u2009\u03bcl of the control sample was added to 5\u2009ml of PBS for the control group. After 2\u2009min, the experimental and control samples (0.5\u2009ml) were placed in a test tube containing 5\u2009ml of PBS and mixed well to terminate the inhibition experiment. The experimental and control samples were treated with PMA, and DNA was extracted and subjected to qPCR assays. The Ct values were recorded, and the number of viable bacteria in the sample solution was calculated using the PMA-qPCR standard curve.Plate counting detection refers to China Standard GB15979-2002: Hygienic standard for disposable sanitary products. The PMA-qPCR method was slightly modified on the basis of the plate counting method, as follows: the 24\u2009h slant culture of a single indicator bacterium was washed with PBS to make a bacterial concentration of approximately 5\u2009\u00d7\u2009105\u2009~\u20094.5\u2009\u00d7\u2009106\u2009CFU/ml. Next, 300\u2009\u03bcl of the indicator bacterial suspension was added dropwise to the control sample. After the reaction was terminated by the neutralizer, the samples of the experimental group and the control group were treated with PMA, and DNA was extracted and subjected to qPCR. Ct values were recorded, and the number of viable bacteria in the samples was calculated using the PMA-qPCR standard curve.The neutralizing agent was identified according to China Standard GB15979-2002: Hygienic standard for disposable sanitary products, and the neutralizing agent of the sanitary wipes used in this study was determined to be \u201cTryptic Soybean Peptone Liquid Medium (TSB) containing 1% sodium thiosulfate and 1% Tween 80.\u201d The PMA-qPCR method was modified slightly by mixing three indicator bacterial suspensions in equal volumes, each with a concentration of 5\u2009\u00d7\u200910The neutralizer identification test was performed according to China Technical Standard For Disinfection 2002, and the neutralizer used in this study for the 84 disinfectant samples was PBS containing 0.5% sodium thiosulfate, 0.2% lecithin, and 2% Tween 80\u2033. The disinfectant sample to be tested was prepared using sterile hard water at a concentration of 1.25 times the concentration to be tested.8\u2009CFU/ml to 1.5\u2009\u00d7\u2009109\u2009CFU/ml. The samples were treated with PMA, and DNA was extracted and subjected to qPCR. Ct values were recorded, and the number of viable bacteria in the samples was calculated using the PMA-qPCR standard curve.The PMA-qPCR method was slightly modified in the experimental method, and the indicator bacterial suspension was mixed with three types of suspensions in equal volumes, and the concentration of each indicator suspension was approximately 3\u2009\u00d7\u200910t-test, and the significance level was set at 0.05.The experiments were repeated three times with all indicators in three parallel groups. The results are expressed as x\u2009\u00b1\u2009s. The SPSS software (version 2.0) was used for the statistical analysis of the experimental data. The two groups were analyzed using independent samples The specificity of qPCR primers was verified according to a previously described method. The qPCR results obtained for the Ct values are shown in E. coli, S. aureus, and C. albicans, were 1.1\u2009\u00d7\u2009109, 5.6\u2009\u00d7\u2009108, and 4.5\u2009\u00d7\u2009108\u2009CFU/ml, respectively. The genomic DNA of the three indicator bacteria was then extracted separately, diluted in a gradient to obtain 102\u2009~\u2009109\u2009CFU/ml bacterial DNA concentrations, and used sequentially as templates for qPCR amplification. The Ct values of the single indicator bacteria were plotted against a standard curve of log10 CFU/mL, and the experimental results are shown in The experimental bacterial solutions prepared at OD600\u2009\u2248\u20091 for the indicator bacteria were subjected to plate colony counting. The results showed that the experimental concentrations of the three different indicator bacteria, 3\u2009~\u2009106\u2009CFU/ml, and the amplification efficiency was in the range of 90\u2009~\u2009105%.The results showed that DNA from the three indicator bacteria was successfully amplified using the corresponding qPCR in the range of 10Based on these results, we concluded that the qPCR primers had good specificity and high amplification efficiency. To further improve the detection efficiency, three indicator bacteria primers were added to the qPCR system simultaneously in the actual disinfectant sample testing to achieve a one-step detection of different indicator bacteria.The three indicator bacteria were treated separately in a 100\u00b0C water bath for 15\u2009min, and the samples before and after treatment were subjected to plate counting and qPCR. In addition, the heated samples were subjected to PMA treatment and subsequent qPCR analysis; the results are shown in The heating method was effective for fragmenting all three indicator bacteria, and no colonies grew on the corresponding plates after heating . HoweverTo determine the appropriate homogenization intensity that could simultaneously target the three indicator bacteria, a one-way experiment on the number of homogenization cycles was conducted, and the results are shown in E. coli has a sparse cell wall, is easily fragmented, and was completely fragmented after 15 homogenization cycles, whereas the gram-positive bacterium S. aureus and C. albicans required increased mechanical fragmentation intensity owing to their dense cell walls. According to the experimental results, the final condition of the homogenization crushing treatment was determined as 6.0\u2009m/s (work 30\u2009s and stop 30\u2009s) per cycle for 35\u2009cycles.The homogenization method did not have consistent fragmentation effects for the three different indicator bacteria . The graThe three indicator bacteria were homogenized and separately fragmented. The samples were subjected to qPCR before and after treatment, and homogenized samples were subjected to PMA and subsequent qPCR analyses. E. coli, S. aureus, and C. albicans, respectively, indicating that PMA could distinguish between live and membrane-damaged bacteria. These results fully illustrated the feasibility of PMA-qPCR for the quantification of viable bacteria among the three indicator bacteria.The Ct values of qPCR before and after homogenization of the three indicator bacteria were not significantly different, which revealed that this treatment did not affect the amplification of genomic DNA of the strains involved in subsequent PCR reactions . In addiE. coli, S. aureus, and C. albicans were 103\u2009~\u2009106\u2009CFU/ml, respectively, which met the requirements for antibacterial and bacteriostatic effects of disinfectants (China Standard GB15979-2002: Hygienic standard for disposable sanitary products).Standard curves of PMA-qPCR for the three indicator bacteria were established according to the methods described above . The resThe antibacterial and bacteriostatic activity of the three disinfectants were tested using the PMA-qPCR method and were compared with those of the corresponding national standards adopted for plate counting.In the present study, the established PMA-qPCR method was applied to test six disinfectants in three categories, hand sanitizers, sanitary wipes, and disinfection solutions, as shown in According to China Technical Standard For Disinfection 2002, the plate counting method is the national standard detection method for the numerical detection of viable microorganisms in disinfectants. This method is simple; however, test results depend on the growth of microorganisms, which takes approximately 48\u2009h (bacteria) to 72\u2009h (yeast). In addition, owing to the limitations of the culture medium and conditions, the plate counting method can only detect bacteria suitable for growth under the corresponding culture conditions. Many indicator bacteria in a sublethal state (VBNC) cannot form colonies in conventional culture media . TherefoS. aureus, E. coli, and C. albicans (At the same time, antibacterial and bacteriostatic activity tests of disinfectants usually require two to three indicator bacteria: albicans . During S. aureus and C. albicans, where the \u0394Ct values were 6.24 and 4.87 before and after heat treatment, respectively. These results revealed that the treatment caused different degrees of DNA damage in the three indicator bacteria and affected the amplification of the PCR reaction. Therefore, the heating method was not suitable for the preparation of membrane-damaged bacteria in this study. In addition, three indicator bacteria with membrane damage were prepared using the optimized homogenization method and analyzed using PMA treatment and qPCR. The results showed that the \u0394Ct values of qPCR before and after PMA treatment of the three indicator bacteria were 12.83, 12.73, and 11.10 for E. coli, S. aureus, and C. albicans, respectively (Currently, PMA combined with qPCR is primarily used for microbial detection in food, medicine, and the environment. However, there are no reports on the antibacterial and bacteriostatic activity detection of disinfectants. The standard curve of PMA-qPCR was drawn by preparing the membrane-damaged bacteria of the target microorganism using the DNA from the membrane-damaged bacteria to conduct a gradient dilution of its living bacterial DNA, and then drawing it according to the Ct value of the qPCR reaction. Therefore, the preparation method of membrane-damaged bacteria is the key to distinguishing between live and dead bacteria of the target microorganism by PMA dye, and it also determines the accuracy of PMA-qPCR for the detection of living target microorganisms. Heating is the most commonly reported method for preparing membrane-damaged bacteria . Howeverectively . PMA canFinally, the PMA-qPCR quantitative detection method established herein was used to rapidly detect the antibacterial and bacteriostatic activity of the six disinfectants in three categories. The quantitative detection results of living bacteria before and after contact with the indicator bacteria of each disinfectant was not significantly different from those of the plate counting method, and the detection time was shortened to 3\u2009h compared to 48\u2009~\u200972\u2009h for the plate counting method . The preThe current study is the first application of PMA-qPCR for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants. There was no significant difference between the method established here and the plate counting method in the rapid detection of the antibacterial and bacteriostatic activity of the six disinfectants in the three categories, indicating the high accuracy of this method. In addition, this method inspects disinfectants in a reaction system concurrently to carry out quarantine on three commonly used indicator bacteria/antimicrobial resistance testing. Furthermore, it significantly simplifies the testing steps, reduces the testing time, improves the quality of disinfectant regulatory efficiency, and safeguards people\u2019s health and safety.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.YL, SH, JZ, CZ, FH, YX, HQ, and YY participated in the design and discussion of the study. YL and SH carried out the experiments. YL, SH, and YY wrote the manuscript. JZ, CZ, FH, YX, HQ, and YY discussed, revised, and edited the manuscript. All authors have read and approved the final version to be published.This work was supported by the State Administration for Market Regulation Foundation of China [Grant number 2020MK136], and the Key Laboratory of Biotoxin Analysis and Assessment for State Market Regulation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "BackgroundNeonatal thrombocytopenia is one of the most common clinical entities encountered in the neonatal intensive care unit (NICU); if not identified early, it can lead to significant morbidity and mortality. The aim of this study was to find out the etiological profile of neonatal thrombocytopenia in the NICU and to study the association between the etiology and onset of thrombocytopenia.Methods9/L). The demographic data such as name, sex, gestational age, age at the onset of thrombocytopenia, and birth weight was recorded. Data was collected based on laboratory investigations.It was a single-center, cross-sectional, descriptive study of neonates having thrombocytopenia. The study was carried out in the NICU of the department of pediatrics in a tertiary care center over a period of one year. The study population included neonates admitted to the NICU having thrombocytopenia . A statistically significant result was found in disseminated intravascular coagulation (DIC); the p value was 0.02. The majority of neonates had late-onset sepsis (LOS) (57%). In both early-onset sepsis (EOS) and LOS, 36.84%\u00a0each, the majority of neonates had moderate thrombocytopenia. Statistically significant results were found in respiratory distress syndrome (RDS) and necrotizing enterocolitis (NEC); the p value was 0.004 and 0.03, respectively.ConclusionThrombocytopenia is a universal finding in neonates in the NICU, and it is an important prognostic marker of various disease conditions in neonates. Thus, the timely recognition and management of thrombocytopenia is essential to reduce neonatal morbidity and mortality. The incidence of thrombocytopenia in newborns is 1%-5% at birth, while severe thrombocytopenia\u00a0exists in 0.1%-0.5% of neonates. The incidence of thrombocytopenia is inversely proportional to the gestational age and birth weight of a newborn. Most of the neonates in NICU manifest mild (100-150\u00d7109/L) to moderate (50-99\u00d7109/L) thrombocytopenia, while in about 20% of neonates, severe thrombocytopenia (<50\u00d7109/L) is seen. Neonates are more predisposed to develop thrombocytopenia in response to illness, which may be because of the limited ability of the neonatal megakaryopoietic axis to increase the production of platelets in response to platelet consumption [Thrombocytopenia is one of the frequent and universal hematopoietic entities found in neonates in the neonatal intensive care unit (NICU) . NeonataThe pathophysiology of neonatal thrombocytopenia is similar to that of adults, which consists of decreased platelet production, increased platelet consumption, hypersplenism, or a combination of all of these mechanisms. The etiological factors responsible for neonatal thrombocytopenia are prematurity, birth asphyxia, intrauterine growth retardation (IUGR), low birth weight (LBW), hyperbilirubinemia, meconium aspiration syndrome (MAS), respiratory distress syndrome (RDS), and sepsis. Maternal disseminated intravascular coagulation (DIC) and pregnancy-induced hypertension (PIH) have also contributed to the etiology of neonatal thrombocytopenia. Severe thrombocytopenia is largely manifested in preterm babies with a gestational age of less than 36 weeks, extremely low birth weight babies ELBW, <1000 g), or critically ill neonates in the NICU [000 g, orThe causative factors responsible for early-onset thrombocytopenia (first 72 hours of life) are different from those of late-onset thrombocytopenia (after 72 hours of life) . Most ofIt was a single-center, cross-sectional, descriptive, and observational study carried out on 100 neonates in the NICU of the department of pediatrics in a tertiary care center. The study population was neonates admitted to the NICU having thrombocytopenia (platelet count: <150\u00d7109/L), while neonates having chromosomal or genetic disorders and neonates born outside the hospital having thrombocytopenia and who received platelet\u00a0transfusion in other NICU were excluded from the study. The sample size for this study was calculated with reference to statistical data  using thAfter obtaining the institutional ethics committee's (IEC) approval and the parents' or guardians' written informed consent (IEC number: 7/2021), neonates were recruited for the study. The study was conducted from January 2021 to December 2021, and the duration of the study was one year.The demographic data such as name, sex, gestational age, age at the onset of thrombocytopenia, and birth weight was recorded. Information was procured from parents/guardians of the enrolled neonates, and the details of demographic and clinical parameters were noted. A structured interviewer-administered questionnaire was used as the data-collecting tool; it had been pretested and modified before being used in the study. Data was collected based on laboratory investigations. The sample was collected using the convenience sampling technique. The blood specimens were collected from each neonate before the administration of antibiotics. Blood samples were obtained for the sepsis workup, which included total leukocyte count (TLC), absolute neutrophil count (ANC), immature neutrophils to total neutrophil count ratio (I/T ratio), platelet count, blood culture and antibiotic sensitivity, and C-reactive protein (CRP) estimation. One\u00a0milliliter of blood was drawn and then put up in the autoanalyzer  for the analysis of the blood for complete blood count, differential leukocyte count, and platelet count. The machine works on the flow cytometry principle. A scattergram was used to reduce the platelet bias.Neonatal thrombocytopenia is characterized by a neonate with a platelet count of less than 1.5 lakh/cumm, which is further classified into the following [Neonatal thrombocytopenia can also be classified based on the timing of presentation as early and late, which is used in the diagnostic evaluation : a)\u00a0EarlStructured data-collecting forms were used to gather the data. Every observation and discovery were coded, recorded into a\u00a0Microsoft (MS) Excel  master spreadsheet, and then analyzed with the Epi Info software . Analysis was conducted using the Statistical Package for Social Sciences (SPSS)\u00a0version 25 \u00a0and Epi Info version 7.3. The chi-square test and Fischer's exact test were used to see the association between independent variables.Figure Figure Table Table Table Table Table 9/L by the second trimester of gestation, and then, the same level persists throughout life. Though it has been recognized that the presence of severe thrombocytopenia (platelets of <50\u00d7109/L) in neonates requires special clinical recognition, the relationship between the severity of thrombocytopenia and the likelihood of bleeding is relatively lacking\u00a0[Thrombocytopenia is one of the most prevalent and universal hematopoietic conditions that can be seen in newborns in the NICU . In fetaIn our study, we found that thrombocytopenia was predominantly present in preterm babies 75, 75%) as compared to term babies (25%). Similar results were seen by Madhavi et al.\u00a0 and Shar5, 75% asIn our study, males outweighed females in numbers. Thrombocytopenia was more seen in males  as compared to females 32, 32%). Similar results were obtained by Tirupathi et al.\u00a0 and Rath, 32%. SiThe etiological profile of our study showed neonatal sepsis  as the most common\u00a0cause of thrombocytopenia in neonates; subsequently, prematurity , SGA 38, 38%), and RDS  were the major causes. Likewise, Gupta et al. 8, 38%, a, and MadIn this study, early-onset thrombocytopenia was present in 34 (34%) neonates, while late onset was present in 66 (66%). Other than DIC, all other risk factors had late-onset thrombocytopenia, while in neonatal sepsis complicated with DIC, it was early-onset thrombocytopenia, which was statistically significant . Similar results were found by Nandyal et al. ; in theiWe found that the incidence of LOS  was more as compared to EOS , while there was no evidence of sepsis in five (5%) neonates. Similar results were obtained by Lim et al.\u00a0, with LOThough the common causative factors for early-onset thrombocytopenia are chronic fetal hypoxia, NEC\u00a0and sepsis have been found to be the important risk factors for late-onset thrombocytopenia. However, no etiology was identified in a significant proportion of thrombocytopenia in ELBW neonates . MortaliFailure to include the clinical profile and detailed maternal history in the study was the limitation of the study. The referral of neonates from outside of the study setting who already presented with sepsis\u00a0could have led to selection bias.It was a single-center, small-scale study where the sample size was few. A multicentric study with a large sample size needs to be done for a detailed understanding of the etiology of thrombocytopenia considering both maternal and neonatal factors that are responsible for thrombocytopenia, which will ultimately lead to a decrease in morbidity and mortality of neonates.Neonatal thrombocytopenia is a frequent and universal clinical finding encountered in neonates admitted to the NICU, and it serves as a key prognostic indicator of various disease conditions in newborns admitted to the NICU. Thus, the early identification, evaluation, and timely intervention of thrombocytopenia are essential to reduce morbidity and mortality. Sepsis and prematurity were discovered to be separate independent causal factors for poor prognosis in newborns admitted to the NICU. As thrombocytopenia is relatively common in newborns, it is crucial to check the platelet count, pattern of onset, and degree\u00a0and severity of thrombocytopenia in every newborn admitted to the NICU. This will aid the neonatologist in making a diagnosis, planning interventions, and starting treatment early and promptly."}
+{"text": "Understanding actions performed by others requires us to integrate different types of information about people, scenes, objects, and their interactions. What organizing dimensions does the mind use to make sense of this complex action space? To address this question, we collected intuitive similarity judgments across two large-scale sets of naturalistic videos depicting everyday actions. We used cross-validated sparse non-negative matrix factorization to identify the structure underlying action similarity judgments. A low-dimensional representation, consisting of nine to ten dimensions, was sufficient to accurately reconstruct human similarity judgments. The dimensions were robust to stimulus set perturbations and reproducible in a separate odd-one-out experiment. Human labels mapped these dimensions onto semantic axes relating to food, work, and home life; social axes relating to people and emotions; and one visual axis related to scene setting. While highly interpretable, these dimensions did not share a clear one-to-one correspondence with prior hypotheses of action-relevant dimensions. Together, our results reveal a low-dimensional set of robust and interpretable dimensions that organize intuitive action similarity judgments and highlight the importance of data-driven investigations of behavioral representations. Our ability to rapidly recognize and respond to others\u2019 actions is remarkable, given the wide variety of human behaviors that span different contexts, goals, and motor sequences. When we see a person acting in the world, we integrate visual information, social cues and prior knowledge to interpret their action. These daily actions in context are often described as activities, which differ from other more basic-level or kinematic-based definitions of action, and despite their ubiquity, still pose a challenge to even state-of-the-art machine learning algorithms. How does the mind make sense of this complex action space?2, social and affective features5, and visual features6 as essential components in visual action understanding. However, such an approach requires the experimenter to pre-define actions and their potential organizing dimensions, necessarily limiting the hypothesis space. Action categories have commonly been defined based on the verbs they represent7 or everyday action categories as listed, for example, in the American Time Use Survey (ATUS)9. Given the diversity of actions, a low-dimensional, flexible representation may be a more efficient way to organize them in the mind and brain; but generating the hypotheses that could uncover this representation remains difficult, especially for naturalistic stimuli that vary along multiple axes.Previous work on action understanding in the mind and brain has focused on hypothesis-driven efforts to identify critical action features and their neural underpinnings. This work has highlighted semantic content10. Recent work has extended this method to near scenes, known as reachspaces, and identified 30 dimensions capturing their most important characteristics11. Low-dimensional representations have been also proposed that explain how people perceive others and their mental states13 or psychologically meaningful situations15.Data-driven methods provide an alternative to pre-defined representational spaces and have achieved great success in mapping perceptual and psychological representations in other visual domains. In object recognition, a data-driven computational model revealed 49 interpretable dimensions capable of accurately predicting human similarity judgments16, as well as guide predictions about actions17. However, since this taxonomy was generated from text data, most of these dimensions were relatively abstract , and it is unclear whether a similar set of dimensions would emerge from visual action representations. In the visual domain, six broad semantic clusters were shown to explain semantic similarity judgments of controlled action images1, suggesting that actions may be semantically categorized at the superordinate level. However, it remains unclear how this finding would generalize to more natural and diverse stimulus sets.To date there has been only limited data-driven work in the action domain. Using principal component analysis (PCA) of large-scale text data, a low-dimensional taxonomy of actions has been shown to explain neural data and human action judgments18 collected in our prior study5. Behavioral similarity has often been used as a proxy for mental representations21 and has been shown to correlate with neural representations26. Specifically, the perceived similarity of actions has been found to map onto critical action features, such as their goals or their social-affective content, as well as onto the structure of neural patterns elicited by actions9.We analyzed a dataset containing unconstrained behavioral similarity judgments of two sets of natural action videos from the Moments in Time dataset27 (NMF) to recover the dimensions underlying behavioral similarity. This approach has two main advantages. First, it allows dimensions to be sparse, so that they need not be present in every action. For example, a single-agent action would have a value of 0 along a social interaction dimension. Second, the method requires the dimensions to be non-negative. Thus, dimensions can add up without canceling each other out, and no dimension can negate another\u2019s importance. Together, these criteria help recover interpretable dimensions, with values that are interpretable as the degree to which they are present in the data.Here, we employ a data-driven approach, sparse non-negative matrix factorization28.We show that a cross-validated approach to dimensionality reduction produces a low-dimensional representation that is interpretable by humans and generalizes across stimulus categories. Importantly, the dimensions recovered by NMF are more robust than those generated by the more commonly used PCA. The non-negativity constraint is known to yield a parts-based description, supporting dimension interpretabilityUsing human labeling and semantic embeddings, we find that dimensions map to interpretable visual, semantic, and social axes and generalize across two experiments with different experimental structure, stimuli, and participants. Together, our results highlight the semantic structure underlying intuitive action similarity and show that cross-validated NMF is a useful tool for recovering interpretable, low-dimensional cognitive representations.18. In two previously conducted experiments5, participants arranged two sets of 152 and 65 videos from 18 everyday action categories8 according to their unconstrained similarity29. The first dataset also included videos of natural scenes as a control category . In Experiment 1, participants arranged different subsets of 30 videos from the 152-video set. In Experiment 2, participants arranged all 65 videos.During the experiments, participants arranged a maximum of 7\u20138 videos at a time inside a circular arena, and the task continued until sufficient evidence was obtained for each pair of videosIn both experiments, participants were instructed to arrange the videos according to how similar they were, thus allowing participants to use their own criteria to arrange the videos, as well as to use different criteria for different groupings of videos. This method allowed us to recover a multidimensional, intuitive representation of naturalistic actions.31 with a nested cross-validation approach (see Methods) to recover the optimal number of underlying dimensions in the behavioral data . Using only behavioral similarity matrices as its starting point, this method can thus recover interpretable features that may shed light on how actions are organized in the mind.We used sparse non-negative matrix factorizationDespite differences in stimulus set size and sampling, both experiments were characterized by similar numbers of dimensions  than as a function of number of action categories  these dimensions are reproducible and (2) to what degree they are interpretable.34, a 300-dimensional word embedding pretrained on 1 million English words.To test reproducibility, participants in an online experiment selected the odd video out of a group consisting of seven highly weighted videos and one low-weighted video along each dimension. In a separate online experiment to test interpretability, participants were asked to provide up to three labels for each dimension after viewing the eight highest and eight lowest weighted videos. Their labels were quantitatively evaluated using FastTextP\u2009<\u20090.004), though participants performed significantly better on average in Experiment 1 (mean accuracy 0.8 \u00b1 0.13) than in Experiment 2 \u2009=\u20093.69, P\u2009=\u20090.002).All dimensions were reproducible in the odd-one-out experiments .Participants\u2019 labels were consistent for most dimensions Fig.\u00a0B. Agreemnature/outdoors), to action-related , as well as social and affective . Dimensions in Experiment 2 included more social information overall, with four dimensions labeled with social or affective terms , compared to one in Experiment 1 (children). Although many dimensions reflected action categories included in the dataset  or labeled features that explained the most variance\u00a0in our previous experiment (relating to people and affect), the information they provided was richer than the a priori category labels and crossed predefined category boundaries. For example, some videos were highly rated along several different dimensions (e.g. work and learning), thus capturing the complexity of naturalistic stimuli which often depict several actions or lend themselves to different interpretations.The most common labels Fig.\u00a0 capturedeating included both eating and preparing food), while other related actions remained separated (e.g. work was split into office work vs chores/cleaning).Further, not all action categories were reflected in NMF dimensions, suggesting that certain action categories are more important than others in organizing behavior. Certain action categories were absorbed by others . This analysis revealed several dimensions that were present in both datasets: ork Fig.\u00a0. FurtherHere, we used sparse non-negative matrix factorization to recover a low-dimensional representation of intuitive action similarity judgments across two naturalistic video datasets. This resulted in robust and interpretable dimensions that generalized across experiments. Our results highlight the visual, semantic and social axes that organize intuitive visual action understanding.10. Here, we showed that a different approach with the same constraints can recover robust, generalizable and interpretable dimensions of human actions. As opposed to those recovered for objects, the action dimensions were only moderately sparse, potentially due to the naturalistic nature of our stimuli. However, optimizing sparsity enabled us to strike the right balance between categorical and continuous descriptions of our data, thus capturing a rich underlying feature space33.In the visual domain, it is reasonable to assume that features can be either absent or present to variable degrees, and that they can be additively combined to characterize a stimulus. Previous work has demonstrated that sparsity and positivity constraints enable the detection of interpretable dimensions underlying object similarity judgmentsOur approach recovered a similar number of dimensions across the two experiments (ten and nine), despite their different stimulus set sizes (152 vs. 65 videos). While the dimensions all had an interpretable, semantic description, none mapped directly onto previously used visual, semantic, or social features, suggesting that a data-driven approach can uncover additional information beyond hypothesis-driven analyses. Furthermore, the dimensions generalized across important stimulus categories like action category and scene setting , there was higher variance in the number and content of dimensions obtained after manipulating stimulus set composition , environment (nature/outdoors), and social information . A previous data-driven analysis of semantic action similarity judgments found six clusters of actions related to locomotion, cleaning, food, leisure, and socializing1. Here, we found that some semantic categories emerged even in the absence of an explicit semantic task, while other dimensions reflected visual or social-affective features, highlighting the rich and varied information extracted from naturalistic actions.These analyses revealed several interpretable and reproducible dimensions, including those related to common everyday actions (work or eating), while others tended to be grouped together based on other critical features, like scene setting or social structure.Importantly, the NMF procedure did not simply return the action categories used to curate the dataset, and in fact none of the dimensions provided a one-to-one correspondence with semantic action category Figs.\u00a0 and 5. Inature/outdoors dimension. In Experiment 1, this dimension included control videos depicting natural scenes, while in Experiment 2, this dimension emerged in the absence of such control videos, suggesting that the natural environment is a salient organizing feature in itself , highlighting the social structure of the similarity data revealed by our previous hypothesis-driven work5. In Experiment 2, videos depicting different actions were grouped together based on social or affective features like communication  or negative affect . These results are in line with previous work suggesting that social features, including others\u2019 intentions and emotions, are important in action perception9, and provide further insight into the specific social information that is prioritized.Several dimensions were given labels pertaining to people . Despite these differences, the majority of dimensions correlated across experiments, suggesting that the NMF reconstructions form a shared semantic space, emerging in spite of stimulus set and sampling differences across experiments.The dimension labels revealed differences as well as similarities between the two experiments. Notably, dimensions in Experiments 2 included more social-affective information Fig.\u00a0, despitetalking and playing games) versus object-directed  actions, are consistent with prior neural findings38. Sociality has also been identified as a key feature in neural action representations5, as has information about the spatial layout of the environment3.Though the behavioral representations measured here likely reflect a late stage in action processing, they can reveal insights into the underlying neural representations. Key distinctions between our dimensions, such as the separation of person-directed  emerged in both the text data and our two video datasets. This opens exciting avenues for research into visual and language-based action understanding and whether they share a conceptual taxonomy.Naturalistic actions involve interactions between people, objects, and places, and it is thus no surprise that the dimensions we uncover reflect the richness of this information. This renders actions, as defined here, the ideal stepping stone towards higher-level event understanding. Another action taxonomy derived from data-driven text analysis proposed six broad action distinctionsRelatedly, stimulus selection is the biggest factor in determining the structure of similarity judgments. Here, both stimulus sets represented 18 everyday action categories based on the American Time Use Survey, curated so as to minimize visual confounds. These action categories may be described as activities or visual events, comprising sets of related actions that occur in daily life. While the number of stimuli does not impact the dimensionality of the final NMF reconstruction, the number of action categories does . 58 participants recruited through the Department of Psychological and Brain Sciences Research Portal at Johns Hopkins University took part in Experiment 2 .We analyzed data from two previously conducted multiple arrangement experimentsTwo experiments were conducted to validate the dimensions resulting from Experiments 1 and 2. 54 participants validated the dimensions from Experiment 1  and a different set of 54 participants validated the dimensions from Experiment 2 . All subjects were recruited through the Department of Psychological and Brain Sciences Research Portal at Johns Hopkins University.All procedures for online data collection were approved by the Johns Hopkins University Institutional Review Board, and informed consent was obtained from all participants. All research was performed in accordance with the Declaration of Helsinki.www.meadows-research.com). Participants arranged the videos inside a circular arena according to their similarity. In order to capture intuitive, natural behavior, we did not define or constrain similarity. An adaptive algorithm ensured that different pairs of videos were presented in different trials, until a sufficient signal-to-noise ratio was achieved for each distance estimate. Behavioral representational dissimilarity matrices (RDM) were then constructed using inverse multi-dimensional scaling30. See Dima et al. 20225 for more details on the experimental procedure.To measure the intuitive similarity between videos depicting everyday action events, we implemented a multiple arrangement task using the Meadows platform .We used a data-driven approach, sparse NMFIn aiming to represent each action video through a combination of underlying features, some of these may be assumed to be categorical. Such features would be present in some of the videos, but not in others, such that participants would arrange videos from the same category close together, and those outside the category farther apart. Sparse NMF applies sparsity constraints, allowing us to detect such categorical features that may group specific actions together.33.However, the degree to which a feature is present may also distinguish certain actions from others, especially for features that capture non-categorical information. By enforcing positivity, NMF recovers continuous features with interpretable numerical values, reflecting the degree to which each feature is present in each stimulus. These two constraints thus allow both categorical and continuous structure to emerge, an approach well-suited to capture how real-world stimuli are represented in the mind40.Given a data matrix 41. As this matrix was symmetric, the output matrices were highly correlated (Pearson\u2019s r\u2009>\u20090.93), leading in practice to a similar solution to that given by symmetric NMF, where We first converted the behavioral RDM to a similarity matrix as used in symmetric applications of NMFWe used a nested cross-validation scheme for NMF Fig.\u00a0B. In ExpS using the following formula: Sa,b\u2009=\u2009max, min)42.Each training and test matrix used in cross-validation was created by averaging across similarity ratings (Experiment 1) or participants (Experiment 2). Due to the random sampling in Experiment 1, there were different numbers of ratings per video pair. Any missing datapoints after averaging (Experiment 1) were imputed (no more than 0.2% of any given similarity matrix). This was done by replacing each missing similarity value To evaluate the final performance of the NMF procedure,\u2009~\u200910% of the data was held out. In Experiment 1, this consisted of one randomly selected similarity rating for each pair of videos. The final test set was thus a complete similarity matrix with a single rating per pair (amounting to 9.52% of the data). In Experiment 2, the final test set consisted of five randomly selected participants\u2019 data (amounting to 9.43% of the data).For parameter selection, the training data  was divided into three sets Fig.\u00a0B.k (number of dimensions), up to 150 in Experiment 1 and 65 in Experiment 2 (just below the maximum number of videos in each experiment). The two sparsity parameters for W and H were selected using two-fold cross-validation on two thirds of the training data. In a hold-out procedure, the best combination of sparsity parameters for each k was tested on the remaining third of the training data. To speed up computation, we only tested combinations of sparsity parameters (s) ranging between 0 (no sparsity) and 0.8 (80% sparsity) in steps of 0.1. We selected the combination with maximal accuracy across the average of both folds, defined as the Kendall\u2019s We searched for the best sparsity parameters for each k). To avoid overfitting, we identified the elbow point in this performance curve, defined as the point maximally distant from a line linking the two ends of the curve.To increase robustness, this cross-validation procedure for sparsity parameter selection was repeated five times with different training set splits. The average performance curve on the held-out training set was used to select the best number of dimensions  with the selected combination of parameters. The held-out 10% of the data was used to evaluate performance by calculating the Kendall\u2019s We performed a post-hoc control analysis to assess the robustness of NMF dimensions to perturbations in the stimulus set. The NMF procedure was repeated after leaving out key stimulus categories that correlated with identified NMF dimensions . To ensure these stimulus categories did not drive results, the dimensions obtained from each control analysis were correlated to the original dimensions. The correlations were then tested against chance using one-tailed randomization testing with 1000 iterations of component matrix shuffling.5 , we assessed the correlation between each NMF dimension and 12 visual, action-related, and social featuresk) was selected using two-fold cross-validation on the training data (~\u200990% of the data).To asssess whether NMF provides an advantage over the more commonly used PCA, we conducted a similar cross-validated analysis using PCA, and assessed the resulting reconstruction accuracy and robustness to stimulus set perturbations in both experiments. The cross-validation procedure was exactly the same, except that no search for sparsity parameters was conducted. Instead, only the number of dimensions (We used two tasks in two separate online experiments (corresponding to Experiments 1 and 2) to assess the interpretability of NMF dimensions in separate participant cohorts. We presented the eight highest weighted and eight lowest weighted videos along each dimension obtained from NMF as stimuli to the subjects. The experiment was implemented in JavaScript.First, participants were asked to select the odd video out of a group consisting of seven highly weighted videos and one low-weighted video (odd-one-out) for a given dimension. This was done 20 times for each dimension with random resampling (from the top and bottom eight) of the videos shown. Participants were excluded if they did not achieve above-chance performance (over 12.5%) on catch trials involving a natural scene video as the odd-one-out among videos containing people. Dimensions were considered reproducible if participants achieved above-chance accuracy in selecting the odd-one-out .After completing this task, participants were asked to provide up to three labels (words or short phrases) for each dimension based on a visual inspection of the eight highest and eight lowest weighted videos.nature vs home); in these cases, we only kept the first label. Next, we visualized the labels by creating word clouds of the most common labels using the MATLAB wordcloud function.We visually inspected the labels provided by participants to correct spelling errors and identify cases where pairs of antonyms were used to label a dimension . To generate a chance level for participant agreement, we calculated the proportion of related labels across different dimensions.To quantify participant agreement on labels, we used FastTextFinally, we assessed whether the NMF dimension labels replicated across the two experiments. To generate a dissimilarity matrix, embeddings were averaged across labels within each dimension before calculating Euclidean distances between dimensions. This allowed us to visualize which dimensions were most semantically related across experiments.Supplementary Information 1.Supplementary Information 2."}
+{"text": "The reversible and dynamic ubiquitination-deubiquitination process plays an essential role in maintaining protein homeostasis, which is critical to almost all the biological processes. Therefore, the metabolic dysregulation of deubiquitinases often lead to serious consequences, including the growth and metastasis of tumors. Accordingly, deubiquitinases can be served as key drug targets for the treatment of tumors. The small molecule inhibitors targeting deubiquitinases has become one of the hot spots of anti-tumor drug research areas. This review concentrated on the function and mechanism of deubiquitinase system in the proliferation, apoptosis, metastasis and autophagy of tumor cells. The research status of small molecule inhibitors of specific deubiquitinases in tumor treatment is introduced, aiming to provide reference for the development of clinical targeted drugs.Ubiquitin is a small protein that can be added onto target protein for inducing target degradation, thereby modulating the activity and stability of protein. Relatively, deubiquitinases (DUBs), a class catalase that can remove ubiquitin from substrate protein, provide a positive regulation of the protein amount at transcription level, post-translational modification, protein interaction,  Ubiquitprotease . The ubiprotease .Ubiquitination is a reversible dynamic process because deubiquitinases (DUBs) can hydrolyze the peptide bond of glycine at position 76 of ubiquitin, thereby removing ubiquitin from the substrate protein . DeubiquUp to now, over 4 90 DUBs from seven subfamilies  have beein vitro  can suppress tumor cell proliferation through suppressing ER\u03b1 signaling of ER-positive BC, thereby promoting ER-negative BC cell growth . USP14 c pathway , which a pathway . USP22 c pathway . In addi in vivo .via AR, while USP16 inhibits cell proliferation and colony formation , prostate cancer and colon cancer . RelativMany ubiquitin-specific protease family members are also connected with tumor metastasis. USP4 is a vital cell pathway regulator, which is involved in p53 regulation, TGF-\u03b2 response and NF-\u03baB signal transduction, and makes a critical effect on cancer genesis and progression . In CRC In eukaryotes, autophagy is a highly conserved lysosomal degradation pathway that recognizes and degrades dysfunctional organelles, macromolecular complexes, as well as intracellular bacteria and other foreign bodies. Moreover, it generates energy recycling, and makes a critical effect on maintaining homeostasis of cells, tissues, and organisms . AutophaUbiquitination, as an important post-translational modification, participates in several autophagy stages . AccordiMammalian target of rapamycin complex 1 (mTORC1) and ULK1/2 kinase complex are key complexes that induce autophagy. DEPTOR is a mTORC1 inhibitor that can regulate autophagy . OTUB1 cin vitro (Due to the extensive involvement of DUBs in tumour proliferation, apoptosis and metastasis, numerous small molecule inhibitors of DUBs have been identified as tumor drugs. At present, most inhibitors of DUBs are still in the research stage. VLX1570, a USP14/UCHL5 dual inhibitor, was the first DUB inhibitor investigated clinically in 2015. However, its investigation was terminated in the clinical trial stage due to its dose-limiting toxic effect . Most ofin vitro . In addiin vitro .Focusing on autophagy that makes a critical effect on tumor development, researchers have successively identified and screened various specific small-molecule inhibitors of autophagy-related DUBs, including USP1-UAF1 complex inhibitors GW7647 and Pimozide, which can successfully reverse NSCLC cell resistance to chemotherapeutic drugs . Small-mOver the past decades, the structure, function, role, associated mechanism, and the relationship with diseases of DUBs have been investigated. It has been known that DUBs are closely associated with cancer genesis and progression, which have become the new hotspots in tumor treatment. DUBs are extensively involved in various processes of tumor development. They can regulate the levels of proteins, including the \u201cundrugable\u201d targets that are not sensitive to traditional targeted therapies. Therefore, DUBs have a broad prospect as the tumor therapeutic targets. However, most of the DUBs have poor specificity. One DUB can regulate multiple substrates, while one substrate also can be regulated by multiple DUBs. In addition, there are dynamic changes between ubiquitin ligases and DUBs. Therefore, in various types of tumors, DUBs show a dual role of promotion and suppression. The original methods and techniques for screening inhibitors are also associated with some shortcomings such as poor biocompatibility and high off-target rate , causing"}
+{"text": "To determine the factors associated with the duration of breastfeeding in mothers of babies cared for in a kangaroo family program.  Quantitative, observational study with a secondary source of a retrospective cohort of 707 babies with monitoring at admission, at 40 weeks, at three and at six months of corrected age in the kangaroo family program of a public hospital in the municipality of Rionegro  from 2016 to 2019.  49.6% of babies were born with low weight for gestational age and 51.5% were female. 58.3% of the mothers were unemployed and 86.2% of them lived with their partner. When entering the kangaroo family program, 94.2% of the babies received breastfeeding and at six months they were 44.7%. The variables that were associated with the duration of breastfeeding up to six months according to the explanatory model were: the mother's cohabitation with her partner  and receiving breastfeeding when entering the kangaroo family program (APR: 2.30).  The factors related to the duration of breastfeeding in mothers of babies cared for in the kangaroo family program were that the mother lived with her partner and that the mother was breastfeeding when she entered the program, therefore they received education and support from the interdisciplinary team, which could favor confidence and willingness towards breastfeeding. The inclusion criterion was attendance at the six-month CA (Corrected Age) control. Patients with hypoxic ischemic encephalopathy and grade III and IV intraventricular hemorrhage were excluded. None of the patients had a contraindication for breastfeeding or palatal defects. The corrected age is defined as the chronological age in days or weeks that the baby was missing to complete 40 weeks of gestation. The sourThe study had the approval of the ethics committee of the health institution and the Catholic University of the East, classifying it as risk-free research in accordance with the Colombian resolution that establishes the scientific, technical, and administrative standards for health research. The idenThe descriptive analysis of the variables was done according to their nature. For the qualitative ones, relative frequencies were calculated; and for the quantitative ones, the average was calculated with its measure of dispersion after evaluating the normality in its distribution with the Kolmogorov Smirnov test. Bivariate analysis was performed to establish the association of the dependent variable (duration of breastfeeding) with each of the selected independent variables using the Chi2 test with a significance level of 5% (p<0.05) and crude RPs were calculated. In the multivariate analysis, all the selected variables were included and through logistic regression with the Enter method, the adjusted ORs were generated. The level of significance was established at 5% (p<0.05). Since the multivariate model generates the OR association measure, the Stromberg (15) formula was used to convert it to RP. The analyzes were performed with the R statistical package.The gender distribution was similar and about half of the cohort were classified as term newborns with low weight for gestational age . The GA average was 35.8 weeks (SD: 2.2), birth weight was 2228.7 grams (SD 382.6), and height was 45.7 centimeters (SD 3.1). The mothers\u2019 average age was 27 years old (SD: 6.4) and the fathers\u2019 average age was 31 years old (SD: 7.6). At the six-month CA control, about half of the children were still being breastfed. Characterization data are presented in The variables that were associated with breastfeeding in the unadjusted analyzes were: number of control appointments attended in the kangaroo family program up to six months of corrected age, height at birth, health system, type of housing, cohabitation of the mother with the couple, the mother's occupation, family nutrition, having worked during the pregnancy, type of birth and breastfeeding upon admission to the program. These variables presented a significant association (p<0.05) with the dependent variable. Once the explanatory model was made, adjusting for all the variables that were associated in the bivariate analysis, only the cohabitation of the mother with the couple  and breastfeeding at the time of admission to the program  maintained their significant association with breastfeeding at six months. See et al. were non-exclusively breastfed at six months of corrected age. Montealegre-Pomar et al.  which reet al.(One of the fundamental axes of the kangaroo mother method is the feeding of the premature or low birth weight newborn, ideally with breast milk.,21 thereet al. report set al.et al.,(Timely hospital discharge with the guarantee of constant monitoring is a component of the KMC, from which emotional, physical and educational support is provided to the mother and family to favor the satisfaction of the kangaroo baby needs. The applet al., advice tThe nursing staff plays an important role in the hospital and outpatient kangaroo care team, since they are responsible for adapting the premature baby to the KMC, monitoring their anthropometric parameters, guiding the mother and evaluating the quality of breastfeeding, and support the family in preparing for the baby care at home. Therefore, it is important that the interventions of the KFP interdisciplinary team contribute to the goal of improving breastfeeding rates in this population.The conclusion of this study is that among the sociodemographic, economic, and clinical factors evaluated, the cohabitation of the premature babies\u2019 mothers with their partner and breastfeeding upon admission to the kangaroo family program were factors that were associated with the duration of breastfeeding in the explanatory model. Mothers who live with their partner have the possibility of sharing the responsibility of caring for the baby, and those who offer breastfeeding upon entering the program receive education and support from the interdisciplinary team, all of which can promote their confidence and willingness to breastfeed.It is necessary to identify more adjustable factors that influence the duration of breastfeeding in kangaroo babies that can be the object of interventions, as well as it must be documented which specific interventions that are carried out in the KFP are more useful to contribute to this purpose.One of the limitations of this study is that it was not possible to establish the prevalence of exclusive breastfeeding up to six months of corrected age because at the time of the study this variable was not defined in the database, since retrospective data were used which were collected with clinical and non-investigative purposes. Therefore, the database did not contain information on the rate of loss to monitoring in the program and on a greater number of economic, social, educational, and work variables that can influence the duration of breastfeeding. Another limitation is that most of the explored variables correspond to sociodemographic or birth factors that are not adjustable.Grant. This article is derived from a research project entitled \"Demographic, social, economic, and clinical factors that influence the duration of breastfeeding in high-risk patients who were monitored in the kangaroo family program in the municipality of Rionegro, between 12/16/2016 and 12/31/2019\u201d, which was financed by the Directorate of Research, Development and Innovation of the Catholic University of the East."}
+{"text": "Eryngium carlinae, have been investigated regarding their medicinal properties in in vitro and in vivo assays, showing favorable results for the treatment of various diseases such as diabetes and obesity. The present study examined the antioxidant and anti-inflammatory effects of the phenolic compounds present in an ethyl acetate extract of the inflorescences of Eryngium carlinae on liver homogenates and mitochondria from streptozotocin (STZ)-induced diabetic rats. Phenolic compounds were identified and quantified by UHPLC-MS. In vitro assays were carried out to discover the antioxidant potential of the extract. Male Wistar rats were administered with a single intraperitoneal injection of STZ (45 mg/kg) and were given the ethyl acetate extract at a level of 30 mg/kg for 60 days. Phytochemical assays showed that the major constituents of the extract were flavonoids; in addition, the in vitro antioxidant activity was dose dependent with IC50 = 57.97 mg/mL and IC50 = 30.90 mg/mL in the DPPH and FRAP assays, respectively. Moreover, the oral administration of the ethyl acetate extract improved the effects of NAFLD, decreasing serum and liver triacylglycerides (TG) levels and oxidative stress markers and increasing the activity of the antioxidant enzymes. Likewise, it attenuated liver damage by decreasing the expression of NF-\u03baB and iNOS, which lead to inflammation and liver damage. We hypothesize that solvent polarity and consequently chemical composition of the ethyl acetate extract of E. carlinae, exert the beneficial effects due to phenolic compounds. These results suggest that the phenolic compounds of the ethyl acetate extract of E. carlinae have antioxidant, anti-inflammatory, hypolipidemic, and hepatoprotective activity.Secondary metabolites such as flavonoids are promising in the treatment of non-alcoholic fatty liver disease (NAFLD), which is one of the complications of diabetes due to oxidative stress and inflammation. Some plants, such as  Diabetes mellitus is a metabolic disorder characterized by increased blood glucose or hyperglycemia . HyperglThe pharmacological treatment of NAFLD in patients with diabetes mellitus (DM) is based on reducing lipid accumulation and stopping the progression of inflammation and fibrosis. In spite of being considered promising for showing potential effects during in vitro and in vivo trials, few positive results have been reported in clinical trials confirming its effectiveness because of the multiple pathways implicated in the etiology of NAFLD ,5. HowevEryngium L. have been used in traditional medicine, such as Eryngium carlinae. It is commonly known as \u201cfrog herb\u201d. In traditional medicine in Mexico, infusions of E. carlinae have been used to treat different conditions such as coughing, indigestion, prostate diseases, lipid disorders and diabetes . E. carlinae was carried out as it was previously reported by Ju\u00e1rez-Trujillo et al. (2018)  before recording the basal fluorescence for 1 min at excitation/emission wavelengths of 352/464 nm. Next, 10 mM \u03b2-NADH was added, and changes in fluorescence were monitored for 2 min. The same procedure was performed for rotenone-insensitive Complex I activity by adding 2 mM rotenone. The specific activity was calculated using a standard curve for \u03b2- NADH.To evaluate the activities of the respiratory chain complexes, mitochondria were permeabilized by freeze-thawing. Complex I activity was assessed by a method previously described, with some modifications . For thi\u22121 cm\u22121 as the molar extinction coefficient. Whereas, for the activity of Complex II + III, 0.1 mg/mL of the solubilized mitochondria was resuspended in a 250 mM phosphate buffer, 20 mM EDTA, 10 mg/mL BSA, 0.5 M succinate, 2 mM rotenone, and 0.2 mM KCN. The mixture was incubated for 3 min at room temperature, then 250 \u00b5g oxidized cytochrome c was added to the mixture, and the basal absorbance was recorded for 1.5 min at 550 nm. Finally, 0.5 mM antimycin A was added, and the changes in absorbance were measured for 3 min. The specific activity was calculated based on the absorbance, with 19.1 mM\u22121 cm\u22121 as the molar extinction coefficient.The activities of Complexes II and II + III were determined as described previously . For Com\u22121 cm\u22121 as the molar extinction coefficient.Complex IV activity was evaluated by the protocol described by Pe\u00f1a-Montes et al. (2020) . For thiw/v) nonfat milk in TBS-T  and 0.05% TWEEN 20) for 1 h at 20 \u00b0C. Next, the membranes were incubated overnight at 4 \u00b0C on a shaker with primary anti- NF-\u03ba\u0392 , anti-NOS2 , or anti-\u03b2-tubulin . The samples were incubated for 2 h or overnight at 4 \u00b0C on a shaker with HRP-conjugated goat anti-rabbit IgG  or m-IgG\u03ba BP . Images were acquired using chemiluminescence and a ChemiDoc XRS+ imaging system , and were analyzed with ImageLab 6.0.1. . The relative protein levels were calculated based on \u03b2-tubulin expression as a loading control.The expression levels of transcription factor NF-\u03ba\u0392 and iNOS protein in the liver homogenate were measured by immunoblotting. Briefly, 50 \u00b5g of protein was resolved on 10% SDS polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes and blocked with 5% , our extract showed lower antioxidant activity [E. carlinae, which was statistically greater than or equal to that of the standard antioxidant used [E. carlinae extract compared with other plants [The antioxidant activity of the ethyl acetate extract of molecule . It was tal ions ,37. Compactivity . Howeverant used . Howeverr plants .E. carlinae [E. carlinae [Justicia spicigera in STZ-induced diabetic rats. This hypolipidemic effect could be related to the high content of rosmarinic acid, promoting fatty acid \u03b2-oxidation via AMPK and by inhibiting fatty acid synthesis, leading to a decrease in hepatic TG content [During diabetes, besides hyperglycemia, a characteristic symptom of the disease is the loss of weight, especially in Type 1 diabetes. This is because lipids and proteins are more prone to be metabolized than carbohydrates, and dyslipidemia, which, due to insulin deficiency or resistance, promotes an increase in TG accumulation in the serum and liver ,41. The carlinae ,42. Nevecarlinae , who demcarlinae . A decrecarlinae ,44 throu content ,46.There is clinical and experimental evidence of the use of different methods for NAFLD/NASH diagnosis. The assessment of liver enzymes such as ALT, AST, and ALP can be taken into account for an initial diagnosis of the disease . In thisOxidative stress derived from lipid accumulation has been shown to dysregulate liver signaling and metabolism, leading to the development of liver disease ,52. TherOne of the effects of lipid accumulation is an increase in ROS production by the mitochondria in an attempt to decrease the lipid load through \u03b2-oxidation . As showDuring lipid overload, oxidation is mediated by the cytochromes and peroxisomes, contributing to the production of ROS, leading to an imbalance between the antioxidant system and ROS . In thisAdditionally, the byproducts of lipid peroxidation cause alterations in the mitochondrial membrane, contributing to dysfunction, an important feature of NAFLD, which leads to cell damage . These aLipid accumulation and the increase in oxidative stress induce an inflammatory response, another main characteristic of NAFLD. This inflammatory response in hepatocytes begins with NF-\u03ba\u0392 (p50:RelA heterodimer) transcription factor activation by release of its inhibitor I\u03ba\u03b2 by phosphorylation and translocation to the nucleus, where it binds to DNA and promotes pro-inflammatory cytokines and inducible enzyme transcription . In thisE. carlinae.Once NF-\u03ba\u0392 has translocated to the nucleus, the transcription of inducible enzymes such as nitric oxide synthase (iNOS) is carried out. Under hyperglycemic conditions, this enzyme promotes inflammation and apoptosis in the liver, as this isoform, unlike the constitutive ones, can produce a large amount of nitric oxide (NO) from L-arginine. In addition, through different stimuli, such as ROS or the cytokines TNF-\u03b1 and IL-1\u03b2, the expression of iNOS is increased not only in Kupffer cells (macrophages) but also in hepatocytes and hepatic stellate cells . This isE. carlinae inflorescences had dose-dependent antioxidant activity in vitro and in vivo at a dose of 30 mg/kg in STZ-induced diabetic rats. The antioxidant activity of the extract was demonstrated through a decrease in oxidative stress markers by inhibiting ROS production, decreasing lipid peroxidation, and restoring mitochondrial complex activity, as well as enhancing the antioxidant system by restoring the activity of the antioxidant enzymes catalase and MnSOD. In addition, the ethyl acetate extract had anti-inflammatory effects due to its ability to decrease NF-\u03ba\u0392 and iNOS expression. It also showed hypolipidemic activity in the serum and the liver of STZ-induced diabetic rats by decreasing TG levels, and it may also have a possible hepatoprotective effect, as observed through the decrease in liver enzymes in the serum. The proposed hypothetical interaction model of the ethyl acetate extract of the inflorescences of E. carlinae is shown in The present results demonstrated that the phenolic compounds such as rosmarinic acid of the ethyl acetate extract of the"}