{"text": "Adjuvant psychological therapy (APT) is a newly developed cognitive behavioural treatment which has been designed specifically to improve the quality of life of cancer patients by alleviating emotional distress and inducing a fighting spirit. We report a phase I/II study which evaluates APT in routine clinical practice. A consecutive series of 44 outpatients with various cancers referred for psychiatric consultation and receiving APT at the Royal Marsden Hospital was studied. Standardised self-report questionnaires were used to measure anxiety, depression and four principal categories of mental adjustment to cancer, namely, fighting spirit, helplessness, anxious preoccupation and fatalism. Statistical comparisons between pre-therapy scores and scores after an average of five APT sessions revealed significant improvement in anxiety, depression, fighting spirit, anxious preoccupation and helplessness. Fatalism scores showed the same trend, but the changes were smaller. Patients with advanced disease showed as much improvement as those with local or locoregional disease. Present results indicate improvement in both psychiatric symptoms and mental adjustment to cancer associated with APT. Whether this association is causal remains to be determined by randomised controlled trials. Such a trial is in progress."}
{"text": "To pursue these findings, we sought to elucidate the mechanisms underlying target gene selectivity for HIF-1\u03b1 and HIF-2\u03b1. Using chromatin immunoprecipitation to probe binding to hypoxia response elements in vivo, and expression of chimaeric molecules bearing reciprocal domain exchanges between HIF-1\u03b1 and HIF-2\u03b1 molecules, we show that selective activation of HIF-\u03b1 target gene expression is not dependent on selective DNA-binding at the target locus, but depends on non-equivalent C-terminal portions of these molecules. Our data indicate that post-DNA binding mechanisms that are dissimilar for HIF-1\u03b1 and HIF-2\u03b1 determine target gene selectivity in RCC cells.Inactivation of the von Hippel\u2013Lindau tumour suppressor in renal cell carcinoma (RCC) leads to failure of proteolytic regulation of the \u03b1 subunits of hypoxia-inducible factor (HIF), constitutive upregulation of the HIF complex, and overexpression of HIF target genes. However, recent studies have indicated that in this setting, upregulation of the closely related HIF- However, there is increasing evidence for the functional non-equivalence of HIF-1\u03b1 and HIF-2\u03b1, and in cancer, several recent studies have indicated that, at least in certain settings, they have contrasting effects on tumour growth TTGATTGGTAACTTCG 3\u2032- and 5\u2032-ACG aga tct(BglII) ATA GTT CTA CCA GGC 3\u2032. Primers for the latter were 5\u2032-ACT aga tct(BglII) TCT TCA TTG TGC TCA TCC CC 3\u2032 and 5\u2032 CGc cat gg(NcoI)T CGC CCG CAG AAT CGA GGT CC-3\u2032. PHD3.HRE-SV40-luc was created by cloning the PHD3 first intron fragment into the multiple cloning site of pGL3promoter . A mutant HRE version of this reporter was created using site-directed mutagenesis with: 5\u2032-CCACCCTCACGCAGCGacatTAGCCCTGTCACTCAGCC-3\u2032 and a complementary oligonucleotide 5\u2032 GGC TGA GTG ACA GGG CTA atg tCG CTG CGT GAG GGT GG 3\u2032 (mutated nucleotides in lower case). Replacement of the SV40 promoter in pGL3promoter with the cloned PHD3 promoter gave rise to PHD3.HRE-PHD3pro-luc. PHD3pro-luc-PHD3.HRE was created by placing the PHD3 first intron fragment into a BamHI site downstream of the poly(A) signal in pGL3promoter using the following primers: 5\u2032-Ggg atc c(BamHI)TC GAG TTG ATT GGT AAC TTC G-3\u2032 and 5\u2032-Ggg atc c(BamHI)AT AGT TCT ACC AGG C-3\u2032. pCMV-\u03b2-galactosidase  and a 1\u2009kb fragment of the PHD3 promoter upstream to the translation start site  were amplified by PCR from human genomic DNA using primers containing the appropriate restriction sites . Primers for the former were 5\u2032-ATggtacc or hypoxia (1% oxygen and 5% carbon dioxide for 16\u2009h) in an Invivo2 hypoxic workstation .The VHL competent cell lines used were Caki-1 . Hep 3B  and MCF-7 . The VHL defective cell lines 786-O , A498  and RCC4 (a gift from CHMC Buys) were all derived from renal clear cell tumours. All cell lines were maintained in Dulbecco's modified Eagles Medium supplemented with 10% fetal calf serum, 2\u2009m\u03b1 and HIF-2\u03b1 were used as described previously  using Oligofectamine reagent  on day 1, cotransfected with siRNA duplexes (20\u2013100\u2009nM), 800\u2009ng luciferase reporter plasmid and 200\u2009ng pCMV-\u03b2-galactosidase plasmid using Lipofectamine 2000 Reagent (Invitrogen) on day 2, and harvested on day 3. In overexpression experiments, cells were cotransfected with 500\u2009ng HIF-\u03b1 expression plasmid (pcDNA.Hs.HIF-1\u03b1 or pcDNA.Hs.HIF-2\u03b1), 300\u2009ng HRE-linked luciferase reporter plasmid  and 200\u2009ng pCMV-\u03b2-galactosidase plasmid using Lipofectamine 2000 reagent on day 1, and harvested on day 2.Two sets of siRNA duplexes targeting HIF-1\u03bcl 1 \u00d7 passive lysis buffer (Promega) and assayed for luciferase activity according to the manufacturer's instructions. \u03b2-Galactosidase assay was performed to normalise for transfection efficiency.Cells were collected in 100\u2009\u03b1 , HIF-2\u03b1 , CA9 (M75) . Horseradish peroxidase-conjugated goat anti-mouse secondary antibody  and ECL Plus system  were used for detection.Whole cell extracts from six-well plates were prepared in urea-sodium dodecyl sulphate (SDS) buffer, normalised for total protein content, resolved and transferred as previously described  and anti-HIF-2\u03b1 (PM9) antibodies were used in the IP. PM14 and PM9 were raised by immunising a rabbit with a fusion protein consisting of glutathione- S-transferase fused to amino acids 445\u2013553 of mouse HIF-1\u03b1 or amino acids 357\u2013439 of mouse HIF-2\u03b1, respectively. Both antisera cross react with the relevant human HIF isoforms. Dilution buffer and rabbit immunoglobulin were used as negative controls. The resulting DNA was analysed by PCR with primers flanking the HREs of PHD3 intron 1 and CA9 promoter  using standard ChIP protocols . Hep3B cpromoter .\u03b1 and HIF-2\u03b1.For ChIP\u2013Western analysis, urea\u2013SDS lysis buffer was added directly onto the beads used for IP of DNA\u2013protein complexes after the washing steps and boiled at 95\u00b0C for 5\u2009min. After centrifugation at 4000\u2009r.p.m. for 5\u2009min, the supernatant was separated by polyacrylamide gel electrophoresis, transferred and blotted with mouse monoclonal antibodies against HIF-1\u03b1 , HIF-1\u03b1 protein with enhanced electrophoretic mobility (A498) or no immunoreactive HIF-1\u03b1 protein (786-0). The concordance of results produced with independent sets of siRNA duplexes indicates that they are due to specific effects on the targeted isoform. Thus, PHD3 expression conforms to the pattern that has previously been described for HIF target genes such as VEGF and GLUT1  or HIF-2\u03b1 (PHD3). Further analysis of immunoprecipitates from 786-O cells revealed enrichment of both HREs by the antibody to HIF-2\u03b1, but as expected neither HRE was captured in immunoprecipitates using the antibody against HIF-1\u03b1  implicated in DNA binding into the HIF-2\u03b1 molecule did not enable activation of the CA9 gene. In contrast, replacement of the entire C-terminal HIF-2\u03b1 sequence (amino acids 95\u2013870) with the equivalent HIF-1\u03b1 sequence (amino acids 96\u2013826) was sufficient to convey full activity on the CA9 target gene. Comparison of the effects of the chimaera containing HIF-1\u03b1 amino acids 96\u2013826 with those containing less extensive C-terminal sequences  implicated in DNA binding into the HIF-1\u03b1 molecule did not enable activation of the PHD3 gene, whereas replacement of the entire C-terminal HIF-1\u03b1 sequence with the equivalent HIF-2\u03b1 sequence (amino acids 95\u2013870) was sufficient to convey full activity on the PHD3 target gene. So far the relationship between HIF-1\u03b1 and CA9 and that between HIF-2\u03b1 and PHD3 appeared to be reciprocal. However, further analysis revealed that whereas extension of the N-terminal HIF-2\u03b1 sequence to amino acids 1\u2013542 did not restore PHD3 activation to the chimaera, the HIF-2\u03b1 C-terminal sequences 543\u2013870 conveyed full activity, indicating that these sequences were both necessary and sufficient for HIF-2\u03b1 activation of PHD3  promoter appears exclusively responsive to HIF-1\u03b1.To analyse further sequences conveying HIF-1\u03b1 on the endogenous PHD3 gene and effects on the juxtaposed enhancer/promoter sequence in PHD3.HRE-PHD3pro-luc. We considered whether altering spacing between the promoter and enhancer might affect HIF-\u03b1 selectivity. The PHD3 enhancer was therefore moved to a more distant position 3\u2032 to the luciferase reporter gene in PHD3pro-luc-PHD3.HRE. Analysis of activity in transiently transfected RCC4 cells again showed that activity was dominantly mediated by HIF-1\u03b1 rather than HIF-2\u03b1 , siRNA suppression of the inactive isoform did not clearly enhance transcription, suggesting that competition for binding is not important at endogenous levels of the proteins, at least under the circumstances examined.Our findings indicate that this target gene selectivity is achieved by post-DNA-binding mechanisms. Chromatin immunoprecipitation experiments demonstrated non-selective binding for HIF-1\u03b1 target CA9 and the HIF-2\u03b1 target PHD3. First, the results of the HIF-1\u03b1/HIF-2\u03b1 domain exchange experiments were non-reciprocal, and implicated non-equivalent sequences in activation of CA9 and PHD3 expression. Second, experiments directed towards defining the cis-acting DNA sequences at the CA9 and PHD3 loci that might mediate HIF-\u03b1 isoform-selective responses yielded quite different results. Thus, for CA9, these experiments suggested that sequences at the minimal promoter could convey selective responses to HIF-1\u03b1. siRNA-mediated suppression of HIF-\u03b1 isoforms indicated that the minimal CA9 promoter, like the endogenous gene, was specifically responsive to endogenous HIF-1\u03b1 and not HIF-2\u03b1. Although some breakdown in selectivity was observed under conditions of overexpression by transient transfection, in comparison with other HREs, the CA9 promoter again appeared specifically responsive to HIF-1\u03b1, suggesting that specificity determining sequences in HIF-1\u03b1 operate through direct or indirect interactions with factors binding the CA9 promoter.Further analysis suggested that the post-DNA binding mechanisms activating target gene expression are different for the HIF-1\u03b1. Rather siRNA suppression experiments revealed that the sequence was dominantly responsive to HIF-1\u03b1. This was observed irrespective of whether the PHD3 HRE was linked to the PHD3 promoter or a heterologous promoter, for different promoter/HRE spacings, and in both transiently and stably transfected RCC4 cells. Interestingly, in contrast with the lack of effect of siRNA-mediated suppression of HIF-2\u03b1, overexpressed HIF-2\u03b1 powerfully activated the PHD3 HRE. This unexpected finding has recently been noted by others who have proposed that HIF-2\u03b1 transcriptional activity is downregulated by a titratable repressor that is overcome by overexpression of the molecule , NF-\u03baB essential modulator (NEMO) has been identified as a HIF-2\u03b1 binding partner that promotes transcriptional activity (\u03b1 with its endogenous target genes in RCC will require further investigation. Our studies provide a new focus for such investigations and indicate that they should be directed at post-DNA-binding mechanisms regulating HIF-2\u03b1 target gene expression.A number of other recent studies have addressed possible mechanisms underlying differential activity of HIF-1"}
{"text": "Patients with Parkinson's disease (PD) sometimes develop impulsive compulsive behaviours (ICBs) due to their dopaminergic medication. We compared 26 impulsive and 27 non-impulsive patients with PD, both on and off medication, on a task that examined emotion bias in decision making. No group differences were detected, but patients on medication were less biased by emotions than patients off medication and the strongest effects were seen in patients with ICBs. PD patients with ICBs on medication also showed more learning from negative feedback and less from positive feedback, whereas off medication they showed the opposite effect. The basal ganglia, the prefrontal cortex, the amygdala and the fusiform gyrus are all believed to be involved in face processing In this study we tested PD patients on and off their usual dopaminergic medication to examine the effects of impulsivity and dopamine in decision making in a task relying on emotional cues. Participants were asked to learn by trial and error, which of two faces (a happy and an angry face) was associated with a small monetary reward and then to pick that face as often as possible. In theory the facial expression should not influence decision making, however studies in healthy volunteers showed that subjects would select the happy face more often than the angry one, especially when learning was difficult A functional imaging study has shown that emotion bias correlates with activation in the anterior cingulate, which is known to have the greatest dopamine innervation in the cortex Impulsive compulsive behaviours (ICBs) such as pathological gambling and compulsive sexual behaviour are triggered by dopaminergic medication. Although some risk factors for ICBs and behavioural changes on metric tasks have been described Thus we speculated that PD patients with ICBs (PD+ICB) would be less biased by emotions in their \u201con\u201d versus \u201coff\u201d state and hypothesised that controls would learn better on this task than PD+ICB patients.To assess the effects of dopaminergic medication on sensitivity to social bias in PD patients with and without ICBs.Twenty five PD patients without ICBs (PD\u2212ICB) and 26 PD+ICB patients were recruited from the National Hospital for Neurology and Neurosurgery, Queen Square, London. All patients fulfilled the Queen Square Brain Bank criteria for the diagnosis of PD PD patients were tested before and after their usual anti-Parkinson medication in a counterbalanced sequence to account for order effects. All patients who were tested in their \u201coff medication state\u201d did not take their usual anti-Parkinson medication, including both L-dopa and any dopamine agonists, for at least 12 hours and performed the task between 8.00am and 9.00am. They were then retested in their \u201con medication\u201d state the following day, usually mid mornings. Those patients who were tested \u201con medication\u201d first performed this task usually in mid-morning when their motor symptoms were well controlled. They were re-visited on the following day prior to their medication for the second test. Results were compared with 16 controls who were matched for age to the PD+ICB group and who were tested over two days but did not take any anti-Parkinson medication. At the end of the study all participants received a modest honorarium depending on their final score .We used a probabilistic face decision task, which has been described previously All participants provided written informed consent according to the declaration of Helsinki and the study was approved by the UCLH Trust ethics committee.All data analysis was carried out in Matlab. Details about the statistical analysis have been reported elsewhere Analysis of variance was carried out on emotion bias, overall learning, and the effects of positive vs. negative feedback. In all cases, these parameters were computed for each subject, and then an ANOVA was run with the corresponding behavioural measure as a dependent variable. Emotion bias and overall learning were characterized by compiling a 2\u00d72 contingency table for each subject. Each cell in the contingency table contained counts of choice patterns of the model and the observer. Based on the counts in the table, emotion bias was computed as:\u200a=\u200a5.3, p\u200a=\u200a0.008). Post hoc analysis showed that the PD\u2212ICB group was older than the PD+ICB group (p\u200a=\u200a0.009). There was no difference between controls and PD or PD+ICB patients (p>0.08). PD+ICB patients had a significantly younger disease onset relative to PD\u2212ICB patients . There was no difference in LEU dose, disease duration or years of education between both patient groups. Further there was no group difference in UPDRS part 3 motor scores prior to (\u2018off-state\u2019) and similarly no group difference after medication (\u2018on-state\u2019). Significantly more PD\u2212ICB than PD+ICB patients were treated with a dopamine agonist, which is in line with accepted clinical guidelines of managing an ICB in PD , thus all patients chose the angry picture more often in their \u2018on\u2019 compared to their \u2018off\u2019 state. There was no difference between groups in emotion bias  and no interaction between medication and group . Further, there was no effect of fraction correct performance (reward learning), when included as a covariate, on emotion bias . Thus, participants who had a larger emotion bias did not necessarily learn less. When the PD groups were analysed individually to examine medication effects, there was a significant decrease in emotion bias in the PD group on vs. off medication  but not in the ICB group . In contrast to the emotion bias, there were no effects of medication  or group  on overall learning , off medication  and ICB group off medication  and controls . The PD+ICB on medication group just missed significance . For overall learning performance, the PD\u2212ICB on  and control  groups were above chance, but all other groups were at chance (p>0.156).Thus, all patients, except the PD+ICB on group, chose the happy face significantly more often than the angry face, given equivalent reward feedback.We next compared the PD\u2212ICB and PD+ICB groups, off and on medication, to controls, pair-wise. However, there were no significant group differences in emotion bias (p>0.071). Following this, we carried out planned comparisons on emotion bias separately in each group, to see if each group was significantly affected by the facial expression . A signi1, 24\u200a=\u200a0.23, p\u200a=\u200a0.629) or medication by feedback type interactions . However, in the PD+ICB group we found that there was a significant medication by feedback type interaction  but no main effect of medication . Thus, consistent with our previous study In the final analysis we split overall learning into parameters which separately measured learning from positive and negative feedback in the PD groups off and on medication . Results\u2018anticipated\u2019 the correct choice. This anticipation may be mediated via the ventral striatum and anticipation of a conflict is processed via the anterior cingulate All patients chose the happy face more than the angry face, when expressions were matched for reward history, in their \u2018off\u2019 versus \u2018on\u2019 state. Furthermore, when examined pair-wise there were no differences in emotion bias or learning between the patient groups and the control group. This might be because participants generally were not able to learn and integrate the reward information effectively, since their behaviour did not correlate well with the ideal observer, which is a purely statistical model of how one should learn. When each group was analysed individually, we found that all groups, other than the PD+ICB group \u2018on medication\u2019, had a significant emotion bias and preferred the happy to the angry face. Our finding is consistent with previous studies using the same task Thus, PD+ICB patients may learn poorly in some conditions because they might use irrational estimates of correct responses, rather than trying to learn from previous trials.We found that both patient groups selected angry faces less than controls, although none of the groups differed significantly from controls. Patients with schizophrenia also select angry faces less often than controls, given equivalent reward evidence The nonapeptide oxytocin can decrease emotion bias in this task, specifically by increasing choices of angry faces when the evidence supports angry faces In an fMRI study of this task, larger modulation of blood-oxygen-level dependent (BOLD) responses by reward prediction error in the anterior cingulate and temporal parietal junction, were correlated with smaller emotion bias effects. Further those subjects that learned best in this task had the smallest modulation of BOLD responses by reward prediction error In summary, dopaminergic medication decreased sensitivity to social bias in both groups of PD patients, in the absence of group effects and none of the groups differed from matched controls. Our results might imply that PD+ICB patients \u2018on medication\u2019 are less affected by emotional feedback, which could be partly responsible for their difficulties integrating into society and following social norms. This is of interest in relation to the finding of increased schizotypy in PD with ICBs"}
{"text": "The correct title and legend of Figure 3 presently appears with Figure 4. The correct, complete Figure 3 legend and title are:Time to negative NS1 antigenaemia.Kaplan \u2013 Meier survival analysis of time to resolution of NS1 antigenaemia by treatment group (CQ or placebo) and population; A) Intention to treat population and B) Per Protocol population. Two patients NS1 negative at enrollment were later positive (both in the CQ arm) and for the purposes of analysis were considered positive at the time of enrolment.doi:10.1371/journal.pntd.0000785.g004"}
{"text": "The correct title and legend of Figure 4 presently appears with Figure 2. The correct, complete Figure 4 legend and title are:Time to fever clearance.Kaplan \u2013 Meier survival analysis of time to fever clearance by treatment group (CQ or placebo) and population; A) Intention to treat population and B) Per Protocol population. Three patients afebrile at enrollment developed fever later (1 in the CQ arm and 2 in the Placebo arm) and for the purposes of analysis were considered positive at the time of enrolment.doi:10.1371/journal.pntd.0000785.g002"}
{"text": "The correct title and legend of Figure 2 presently appears with Figure 3. The correct, complete Figure 2 legend and title are:Time to resolution of viraemia.Kaplan \u2013 Meier survival analysis of time to resolution of plasma viraemia by treatment group (CQ or placebo) and population; A) Intention to treat population and B) Per Protocol population.doi:10.1371/journal.pntd.0000785.g003"}
{"text": "Escherichia coli and evaluated for its biological activity.Interleukin 7 (IL-7) is a \u03d2c family cytokine involved in the homeostatic proliferation and maintenance of immune cells. In the present study, we report the expression of bovine IL-7 (bIL-7) in E. coli (BL 21 DE3). The expressed protein was purified by nickel-nitrilotriacetatechromatography, and the reactivity of the protein was confirmed by western blotting using monoclonal antibodies raised against human IL-7. The biological activity of expressed bIL-7 was evaluated by analyzing its effect on the expression of anuclear factor for activated T-cells c1 (NFATc1), B-cell lymphoma 2 (Bcl2), suppressor of cytokine signaling 3 (SOCS3) molecules in bovine peripheral blood mononuclear cells (PBMCs) by quantitative polymerase chain reaction. Ability of the expressed protein was also analyzed by its effect on phosphorylating signal transducer and activator 3 (STAT3) molecule by immunostaining in human embryonic kidney cells 293 (HEK293) cells.The sequence coding for bIL-7 (mature protein) was amplified from primary bovine kidney cell culture and cloned into pET28-a vector and expressed in The bIL-7 was able to induce the expression of Bcl2 and NFATc1expression in bovine PBMCs by 7 and 5-folds, respectively, whereas a 2-fold decrease was observed in the case of SOCS3 expression. Immunostaining studies in HEK293 cells using antihuman phospho-STAT3 showed activation and nuclear translocation of STAT3 molecule on bIL-7 treatment.E. coli expressed bIL-7 protein is biologically active. Considering the role of IL-7 in T-cell homeostasis and memory cell generation, this molecule can be used for enhancing the vaccine response and that has to be proved by further experiments.bIL-7 gene was successfully amplified, cloned, and expressed in a prokaryotic expression system. The biological activity study showed that the  Interleukin 7 (IL-7) is a vital cytokine for the homeostasis and development of both T- and B-lymphocytes. It is produced by intestinal epithelial cells, keratinocytes, hepatic tissues, peripheral blood dendritic cells, endothelial cells, smooth muscles, and fibroblast cells in human, however in mouse, thymic stromal cells, fetal thymus cells, bone marrow, spleen, kidney, keratinocytes, and fetal intestine are identified as the sites of IL-7 production . IL-7 isL, and MCI-1, which are also anti-apoptotic in nature [IL-7 activation promotes survival and proliferation of lymphocytes . The surn nature ,7. Supprn nature ,9. The mn nature . Very ren nature .The crystal structure of IL-7 receptor and their binding properties with IL-7 were well-studied in the case of human IL-7 .in vitro.Since there is no report on the characterization of bovine IL-7 (bIL-7), the present study reports the cloning of full-length sequence of bIL-7 and expression of the bIL-7 mature (bIL-7 mat) protein in prokaryotic expression system and evaluation of its biological activity All the procedures were carried out with prior ethical permission of Institute Bio-Safety Committee (IBSC) and Institute Animal Ethics Committee (IAEC), Indian Veterinary Research Institute, Hebbal, Bangalore. (Ref.No. F.8-56-Vol.II/RCSS/2014-15/dtd 8-06-2015).Bovine kidney cells were collected in normal saline from the local slaughter house, and bovine primary kidney cell culture was prepared. Total RNA was isolated from the primary bovine kidney cell culture using TRIZOL reagent .cDNA was prepared by reverse transcriptase reaction using oligo dT primers. The cDNA was used to amplify the IL-7 full-length sequence using IL-7L (5\u2019-ggcgggtaccatgttccatgtttcttttagg-3\u2019) and IL-7R (5\u2019-ggcggcggccgctcagtgttctttaatgccc-3\u2019) primers which were designed using IL-7 sequence available in the database (Gene Bank Accession No: BC142466.1). The purified polymerase chain reaction (PCR) product was ligated into EcoRV digested pcDNA (+3.1) vector. The cloned insert was subjected for sequencing by an automated sequencer, and the sequence was confirmed by comparing with known related sequences present in the database by NCBI blast tool.E.coli. Overnight grown cultures of four randomly selected recombinant colonies were inoculated to 10 ml Luria-Bertani broth medium and grown to optical density600= 0.6-0.8 at 37\u00b0C. Protein expression was induced with 1 mM isopropyl thiogalactoside (IPTG) for 5 h at 30\u00b0C. The pellet was lysed and analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel for protein expression under denaturing conditions. After confirming the protein expression, the fusion protein was purified by nickel-nitrilotriacetate (Ni-NTA) chromatography  using denaturing conditions for eluting the protein by elution buffer containing 250 mM imidazole. Protein was partially refolded by reducing the molarity of urea in the washing buffer and different fractions of elute were analyzed by SDS-PAGE. The purified samples were ran on 10% SDS-PAGE and transferred to nitocellulose membrane for western blotting using human Il-7 specific monoclonal antibodies .bIL-7 mat sequence was amplified from the full-length IL-7-pcDNA plasmid using IL-7Lmat (5\u2019-gcggatccgattgtgatattagaggtaaagac-3\u2019) and IL-7R (5\u2019-ggcggcggccgctcagtgttctttaatgccc-3\u2019) primers. The PCR product was cloned into pET28a vector at BamHI and NotI sites to generate pET28-bIL-7 mat and transformed into DH5\u03b1 cells. The recombinant plasmid pET28-bIL-7mat confirmed by restriction digestion analysis and expressed in BL21c DE3 strain of 6 cells were dispersed in each well in a six-well plate. About 100 ng of the recombinant protein was used per well for the biological activity assay. PBMCs were incubated with mature IL-7 for 3 h, and total mRNA was collected by TRIzol reagent . cDNA was synthesized using oligodT primers. Real time primers were designed for Bcl2 , NFATc1 , and SOCS3  molecules using Primer blast software of NCBI. ABI prism 7300 (Applied Biosystems) was used to carry out qPCR with conditions consisting of an initial step at 50\u00b0C for 2 min and 95\u00b0C for 2 min followed by 40 cycles at 95\u00b0C for 15 s and at 60\u00b0C for 1 min. The qPCR results were analyzed by relative quantification method [The biological activity of purified mature IL-7 was studied by its effects on expression of Bcl2, nuclear factor for activated T-cells c1 (NFATc1), and suppressor of cytokine signaling 3 (SOCS3) molecules. Peripheral blood mononuclear cells (PBMCs) were isolated from cattle blood using histopaque 1077 . Approximately 2 \u00d7 10n method , where g5 human embryonic kidney cells 293 (HEK293) cell per well were seeded in six-well plate and allowed to grow for 24-36 h. Cells were incubated with 10 \u03bcg/ml of purified E.coli expressed IL-7 mature protein for 30 min. After incubation cells were washed twice with 1 \u00d7 phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde in 1 \u00d7 PBS  for 15 min. The plate was again washed twice with 1 \u00d7 PBS and treated with 0.5% triton x-100 (1 \u00d7 PBS) for 15-20 min to permeabilize the cell and nuclear membrane. Cells were washed twice with 1 \u00d7 tris-buffered saline (TBS)-Tween  and blocked with 3%BSA (in TBS-Tween) for 1h. Washing was repeated thrice with 1 \u00d7 TBS-Tween and incubated with the anti human phospho-STAT3 antibody (1:1000 in TBS-Tween) for 1 h at 37\u00b0C. After washing secondary antibody conjugated with fluorescein isothiocyanate (1:2000 in 1 \u00d7 TBS-Tween) was added to the plates and incubated for 1h at 37\u00b0C. The plates were washed with 1 \u00d7TBS-Tween and observed under fluorescence microscope.Immunostaining analysis of STAT3 molecule activation was carried out in a six-well culture plate. Approximately 1 \u00d7 10E.coli, and protein expression was induced by 1mM IPTG. On SDS-PAGE analysis, an extra band of 20 kDa was observed in induced cultures when compared with uninduced cultures (In this study, the recombinant plasmid was transformed into BL21c DE3 strain of cultures . Since tcultures , a singlcultures .A thick cultures .E.coli expressed bIL-7 was evaluated by assessing itseffect on the expression of Bcl2, NFATc1, and SOCS3 molecules. In this study, the effect of bacterial expressed IL-7 on these molecules was analyzed by qPCR by keeping GAPDH gene as an internal control. The results showed 7.2-fold increase in the expression of Bcl2, 4.8-fold increase in the NFATc expression, and 2.3-fold decrease in the expression of SOCS3 compared to the control in bovine PBMC culture (Biological activity of  culture .E.coli expressed IL-7 on phosphorylating STAT3 molecule is analyzed by immunostaining. HEK293 cells were incubated with 10 \u03bcg/ml of recombinant protein for 30 min. Later cells were processed for immunostaining according to the above-described procedure. Cells incubated with recombinant bIL-7 showed prominent fluorescence in the nucleus (The effect of  nucleus  and c, w nucleus  and f.IL-7 is believed to be the most important cytokine involved in the homeostatic proliferation of T- and B-cells. IL-7 signals through a receptor complex of IL-7R\u03b1 and \u03b3 chains . IL-7 is+T-cells [Homeostatic proliferation and survival of immune cells are mainly attributed to the function of IL-7. The survival effect of IL-7 is mainly elicited by up-regulating anti-apoptotic proteins such asBcl2-family proteins by the JAK3/STAT5 pathway. Expression of Bcl2 decreases in effector cells and increases to the maximum in memory cells, when compared to na\u00efve CD8+T-cells . The exp+T-cells ,16. Rece+T-cells . A paralSOCS molecules are the physiological suppressors of cytokine signaling. They function as internal regulators of cytokine signaling, and many of the SOCS molecules are activated as a response to cytokine signaling. Because of their cytokine regulatory function, SOCS molecules are exploited by many infectious agents to escape from host immune system. It has been shown that IL-7 therapy augments IL-6 production by inhibiting SOCS3 activation by regulating FoxO transcriptional activity. Our resE.coli expressed IL-7 on phosphorylating STAT3 molecule is analyzed by immunostaining. In the present experiment, HEK293 cells incubated with IL-7 showed fluorescence in the nucleus indicating the nuclear translocation of the phosphorylated STAT3 molecules,whereasthe nucleus of the control cells did not show any fluorescence and remained restricted to the cytoplasm. On activation, STAT molecules would be translocated into the nucleus and elicit effects at the transcription level. Phosphorylation of STAT molecule is one of the modes of action of IL-7. STAT5 is documented as the major candidate for the JAK/STAT signaling pathway by IL-7. In the development of B-cells, especially pre-B-cells depend on IL-7-mediated STAT3 signaling. Studies revealed that STAT3 deficient pre-B-cells are highly susceptible for apoptosis [27-thymocytesin adult mice and promote the capacity of \u03b3\u03b427- to produce IL-17, which help in the polarization of T-cells. This selectivity of IL-7 on \u03b3\u03b427-thymocytes to produce IL-7 mainly owes for its capacity to activate STAT3 in such cells [The effect of poptosis . IL-7 inch cells . Like STch cells .Imperative role by IL-7 in T-cell memory prompted the molecule to be tested for its adjuvanted effect in its DNA and protein forms. It showed increased T-cell response against hepatitis C virus infection  and aviaE.coli expressed bIL-7 matare biologically active. Uponstimulation with the recombinant bIL-7, there was an increase in the mRNA levels of NFATc1 and Bcl2 and a decrease in the expression of the SOCS3 molecule in PBMCs. Use of IL-7 protein and IL-7 encoding DNA constructs are reported to be useful against cancer and many viral infections. Biologically active bIL-7 expressed in E.coli will be a good candidate to evaluate further it as a therapeutic agent as well as an adjuvant to the vaccine in bovines.The present study shows that JL- The research work mentioned in this article is a part of the Ph.D research work of the first author and first author carried out all the work mentioned in this article. NV - The immunostaining of HEK cells to analyze the biological activity of the bIL-7 protein. JHD - The real time PCR assay described in the present study. GRR- The entire work mentioned in this article was carried out under the guidance of the fourth author."}
{"text": "Diffraction gratings are an essential optical component of high-power, short-pulse lasers. The maximum output of high-power pulsed lasers is always determined by laser resistance of gratings and this resistance is strongly dependent on the local near electric field intensity in the grating structure. We presented a novel method of reducing electric-field-enhancement in metal-dielectric grating by designing asymmetric grating ridge while maintaining high diffraction performance. Compared with the common isosceles trapezoidal grating, the grating with asymmetric ridge got a 0.04% reduction of diffraction efficiency in TE polarization at 1053\u2009nm incident wavelength but a 21.3% reduction of maximal electric-field-enhancement in grating structure. This method can be applied to any surface-relief gratings to reduce the electric-field-enhancement for improving the laser induced damage threshold (LIDT) of grating and supporting the grating-based chirped pulse amplification (CPA) system to develop into higher peak-power levels. Metal gratings were firstly and commonly used as PCGs in CPA systems because metals can naturally reflect light with broadband high efficiency3, but they were limited to the laser induced damage threshold (LIDT)2. Compared with metal gratings, all-dielectric gratings feature negligible losses and much higher LIDTs5 but cannot fulfill the broader bandwidth specifications6 and the mechanical stress will increases significantly when the number of layers and size become large7. For avoiding these disadvantages mentioned above, mixed metal-dielectric grating with high diffraction efficiency (DE) and broad bandwidth is a viable candidate for the realization of PCG with high LIDT8.The pulse compression grating (PCG) is one of the most critical components of a high-power grating-based chirped pulse amplification (CPA) laser system9 and recently we found that this grating was initially damaged at the grating ridge by femtosecond laser in transverse-electric (TE) polarization. The initial damage was occurred at the location where the enhancement of the electric field intensity  is highest in the grating as shown in Fig.\u00a0et al.5. Steve Hocquet et al. also reported this phenomenon in multilayer dielectric (MLD) grating and they improved the LIDT from 2.9\u2009J/cm2 up to 4.8\u2009J/cm2 in normal beam by illuminating the MLD grating from in TE to transverse-magnetic (TM) polarization with a reduction of electric-field- enhancement from 1.38 to 1.16. But they stressed that MLD grating have been designed so far to be used in TE polarization because the DE is much lower in TM polarization10. So polarization conversion seems not a practical method and direct reducing electric-field-enhancement in grating is required to improve the LIDT of the grating used with femtosecond laser. Bonod and Neauport reported a drop of the maximum field intensity by increasing the groove width11, but Liu et al. reported a similar method but decreasing the groove width12, so the method of adjusting the groove width seems to be uncommon because it strongly relies on the specific grating itself. In any case, achieving high LIDT while maintaining high diffraction performance is a significant and challenging work for supporting the grating-based CPA system to develop into much higher peak-power levels. In this paper, we present a metal-dielectric grating solution of reducing electric-field-enhancement in the structure and achieving high DE at the same time.A broadband metal-dielectric grating with a sandwich-ridge was designed and fabricated in our previous work9. For the sake of simplicity, the grating ridge with single material was set to be rectangle in the beginning. So our grating contains a rectangle grating ridge (height hg), a residual layer (thickness tr), a matching layer (thickness tm), a high reflective Ag layer (thickness tAg) and the substrate. The material of the grating ridge and the residual layer is Ta2O5 because high-refractive-index material can lower the etching depth and increase the DE and also can be compared to the material in sandwich grating structure mentioned in Fig.\u00a02 is set to match the phase for achieving higher DE. We focus on designing the grating with broadband high DE around 1053\u2009nm wavelength. Multi-parameter optimization is carried out in our design because multiple parameters of the grating influence the DE and bandwidth of grating. These parameters included the period of the metal-dielectric grating \u039b, the duty cycle f, the height of grating ridge hg and all the thicknesses . The incident angle was fixed at -1st reflective Littrow-mount constraint by g\u2009=\u20090.439\u2009\u03bcm, tr\u2009=\u20090.163\u2009\u03bcm, tm\u2009=\u20090.025\u2009\u03bcm and tAg\u2009=\u20090.2\u2009\u03bcm. The calculated -1st reflective DE spectrum in TE polarization and the schematic structure of the grating are shown in Fig.\u00a013. So we sliced the rectangle ridge into 10 thin planar slabs and these slabs were randomly shifted with a slight displacement away from the center. The calculated DE spectrum and the schematic structure of the deformed grating are shown in Fig.\u00a0The asymmetric distribution of electric field intensity see Fig.\u00a0 is resul15. So we re-optimized the metal-dielectric grating into isosceles trapezoid shape to go a step further for describing our designing method. Based on our previous experiments15, the base angle of \u03b2 in the grating was mostly measured near 80\u00b0 with a \u00b110\u00b0 range, so the isosceles base angle of \u03b2 was set to 80\u00b0 for convenience. The period of grating \u039b was fixed at 0.667\u2009\u03bcm as the same as the rectangle one for keeping consistent working situation. The trapezoidal grating ridge was sliced into 40 slabs to increase the accuracy of simulation. The re-optimized parameters of the isosceles trapezoidal metal-dielectric grating were: f\u2009=\u20090.72, hg\u2009=\u20090.59\u2009\u03bcm, tr\u2009=\u20090.236\u2009\u03bcm, tm\u2009=\u20090.178\u2009\u03bcm and tAg\u2009=\u20090.2\u2009\u03bcm. The calculated DE of the -1st order reflected spectrum in TE polarization and the schematic structure of this grating are shown in Fig.\u00a0Design of isosceles trapezoidal grating ridge with base angle of \u03b2 rather than the rectangular shape can lower the difficulty of fabrication based on our previous worksL and \u03b2R) were added to describe our grating shape and they were set to be 70\u00b0 and 90\u00b0 respectively when the incident light irradiated from left top. The two angles 70\u00b0 and 90\u00b0 are the results from the optimization process, which are separated \u00b110\u00b0 from 80\u00b0 and this equal difference will cause the ridge distortion in a certain direction. Compared with the isosceles trapezoidal grating ridge with 80\u00b0 base angle, the non-isosceles grating can be considered that the sliced slabs were integrally shifted a small displacement opposite to the incoming wave. The other parameters of the asymmetric grating were optimized with slight changes: f\u2009=\u20090.7, hg\u2009=\u20090.6\u2009\u03bcm, tr\u2009=\u20090.23\u2009\u03bcm. The calculated -1st reflective DE spectrum in TE polarization and the schematic structure of this asymmetry grating are shown in Fig.\u00a0et al., they got a 39.58% increasement of LIDT by a 18.96% diminution of electric-field-enhancement10, so we have confidence to believe that the LIDT of the metal-dielectric grating can be improved by designing asymmetric grating ridge because of these obvious reductions of electric-field-enhancement in grating.We want to design specific asymmetric grating shape to modulate the distribution of electric field intensity of the grating while maintaining high DE. According to the result from Fig.\u00a016 to find minimum of merit function and the merit function was taken as a mean-error expression around the considered wavelength range N is the number of wavelength discrete points and 17 was employed to calculate the DE and the distribution of the enhancement of the electric field intensity. The grating bandwidth can be optimized depended on the wavelength range in the merit function. Using evolutionary algorithms such as GA has been proved to be a straightforward and efficient method to optimized grating based on our previous works18 and GA is also widely used in fiber optics19, three-dimensional printings20 and other optical components designing21.The optimization is obtained by the genetic algorithm (GA)9. In coating process, a chromium film was deposited on the substrate first to enhance the adhesion of metal film and substrate, and all layers were deposited by electron beam evaporation. The coating sample was annealed at 350\u2009\u00b0C in air to release internal stress for avoiding delamination in the cleaning process. In lithography process, the grating ridge pattern of photoresist was generated from the interference of two monochromatic collimated beams of ultraviolet (UV) light. In etching process, the grating structures were transferred from the photoresist to the dielectric layers by reaction ion beam etching technology and cleaned by hydrochloric/peroxide mixture combined with mega-sonic cleaning method. Besides, the asymmetric grating ridge can be fabricated by specific slant-etching technology, which is common in slanted grating fabrication based on the studies by Tapani Levola et al.22 and Kalle Ventola et al.23. In the damage test procedure, each test site was only exposed to a single shot, and different sites were irradiated with the different laser fluence. The fluence was gradually decreased until the damage didn\u2019t happened. The irradiated sites were preliminarily judged to be damaged or not with the aid of charge-coupled device (CCD) and the details of damaged spots were carried out by SEM.The metal-dielectric grating mentioned in Fig.\u00a0All data generated or analysed during this study are included in this published article.26. This design method can be applied to any surface-relief gratings to reduce the electric-field-enhancement for improving the LIDT of grating and supporting the grating-based CPA system to develop into much higher power levels.In this paper, we presented a novel method of reducing electric-field-enhancement in metal-dielectric grating by designing asymmetric grating ridge. We firstly demonstrated that restricted distortion of grating ridge will not influence much on the DE, which means that grating can be designed with unusual shape while maintaining high DE. Associated with the initial damage at the grating ridge where the enhancement of the electric field intensity is highest in the structure, we modified our optimized isosceles trapezoidal metal-dielectric grating into non-isosceles shape with two base angles for modulating the distribution of electric field intensity in the grating. Compared with the isosceles trapezoidal grating, the grating with asymmetric ridge got a 0.04% reduction of DE in TE polarization at 1053\u2009nm central incident wavelength but a 21.3% reduction of electric-field-enhancement in grating structure. This result indicated that it\u2019s valid to design specific asymmetric grating shape to modulate the distribution of electric field intensity of the grating while maintaining high DE. Asymmetric gratings can be not only used to reduce laser damage but also used for other applications such as near-to-eye display technology and waveguide technology"}
{"text": "A strong and anisotropic interfacial coupling between the charge transfer pairs is demonstrated. The van der Waals heterostructures exhibit pressure dependent sensitivity with a high piezoresistance coefficient of \u22124.4\u2009\u00d7\u200910\u22126\u2009Pa\u22121, and conductance and capacitance tunable by external stimuli (ferroelectric field and magnetic field). Density functional theory calculations confirm charge transfer between the n-orbitals of the S atoms in BEDT\u2013TTF of the BEDT\u2013TTF/C60 layer and the \u03c0* orbitals of C atoms in C60 of the P3DDT/C60 layer contribute to the inter-complex CT. The two-dimensional molecular van der Waals heterostructures with tunable optical\u2013electronic\u2013magnetic coupling properties are promising for flexible electronic applications.Two-dimensional van der Waals heterostructures are of considerable interest for the next generation nanoelectronics because of their unique interlayer coupling and optoelectronic properties. Here, we report a modified Langmuir\u2013Blodgett method to organize two-dimensional molecular charge transfer crystals into arbitrarily and vertically stacked heterostructures, consisting of bis(ethylenedithio)tetrathiafulvalene (BEDT\u2013TTF)/C Two-dimensional van der Waals heterostructures are of interest due to their unique interlayer coupling and optoelectronic properties. Here authors develop a Langmuir-Blodgett method to organize charge transfer molecular heterostructures with externally tunable conductance and capacitance and broadband photoresponse. Currently, 2D heterostructures are predominantly based on inorganic complexes, especially chalcogenides with strong vertical chemical bonds1. In this context, the urgent demand of flexible nanoelectronics and optoelectronics calls for a novel generation of organic heterostructures held by vdW forces for both vertical and horizontal orientation7. Among the organic complexes, the superior optoelectronic properties and the lack of interlayer screening effect for the 2D donor and acceptor CT molecular crystals, as well as the charge density wave induced long-range vdW force in the polarized structure, is a promising candidate to enable 2D CT molecular heterostructures with tunable optical\u2013electronic\u2013magnetic coupling behavior at the atomic level17.Two-dimensional (2D) heterostructures based on weak interlayer van der Waals (vdW) interaction with the lack of superficial dangling bonds afford multiple degrees of freedom for the creation of new high-quality 2D heterojunctions and superlattices without the constrains of lattice parameters, enabling customized and tunable optical\u2013electronic\u2013magnetic properties2. Therefore, it is critical to investigate the coupling behavior between two pairs of different CT layers, which holds promise to show unique coupling behavior distinct from traditional inorganic counterparts due to the inter-conversion between singlet and triplet CT states under external stimuli combined with the internal hyperfine interaction and spin-orbit coupling of organic semiconductors. Moreover, the combination of the distinct properties from different layers is not only complementary but also will enhance the performance of the whole system18.The atomically regulated interface of 2D heterostructures affords an ideal platform to understand the heterointerfacial coupling behavior. However, the conventional 2D heterostructures are composed of two layers of opposite carrier type with CT interaction1. Moreover, the choice of lattice-matched substrate is needed for the growth of 2D materials, which limits the scalable preparation20. Thus, a universal method for the preparation of organic 2D vdWHs is in urgent demand. On the other hand, the understanding and control of heterointerfacial coupling behavior for the organic vdWHs is of key importance towards the further design and applications of the nano-devices. Among all the approaches to control the interfacial state, extrinsic charge injection by ferroelectric field generated from flexible P(VDF-TrFE) polymer has drawn much attention. The large polarization of the crystallized ferroelectric polymer, P(VDF-TrFE) induces a net polarization of organic CT components with enhanced optoelectronic and even magnetoelectric performance24.To prepare 2D vdW heterostructures (vdWH), conventional chemical vapor deposition approach is widely used. However, the weak interlayer interaction between the monolayer 2D film and the substrate leads to the island growth rather than continuous monolayers60) acceptor layer (DTC60) and bis(ethylenedithio)tetrathiafulvalene (BEDT\u2013TTF) donor with C60 acceptor layer (ETC60). The interfacial coupling can be tuned in over a large range by external fields (ferroelectric and magnetic) with strong enhancement of current and capacitance, due to the coupling across two CT pairs along the vertical and horizontal orientations.Here, we report the large scale assembly of 2D CT heterostructures with controlled orientation and unique physicochemical properties, consisting of vertically stacked poly (P3DDT) donor with fullerene  solvent with large surface tension, good spreading ability, and the low vapor pressure of 1,2-dichlorobenzene (DCB) solvent for ETC60 and DTC60 are all crucial for the assembly and stacking of 2D heterostructures  vibration of C60 in the ETC60-DTC60 heterostructure nanosheets compared to that in single ETC60 and DTC60 nanosheets confirms the existence of coupling between the ETC60 and DTC60 nanosheets , forming the segregated stacking 2D ETC60 nanosheets with the distance between each layer close to 6.46\u2009\u00c528. The P3DDT layers self-organize through the \u03c0-\u03c0 interactions16. The distinct structure of P3DDT and C60 molecules lead to the segregated stacking of P3DDT and C60 in the 2D DTC60 nanosheets, where the closest distance between the backbone of P3DDT and C60 molecules is 8.08\u2009\u00c516. Each ETC60 nanosheet crystalline unit contains six molecules of BEDT-TTF and four molecules of C60, while that of DTC60 contains two molecules of P3DDT and two molecules of C60. The orthorhombic crystalline unit cell of ETC60/DTC60 has lattice parameters of a\u2009=\u200926.71\u2009\u00c5, \u03b1\u2009=\u200990\u00b0, b\u2009=\u200932.51\u2009\u00c5, \u03b2\u2009=\u200990\u00b0, c\u2009=\u200958.90\u2009\u00c5, \u03b3\u2009=\u200990\u00b0. The horizontal stacking of the molecular chains on water surface forms a stable structure, which can also be verified by the following anisotropic properties. The CT interaction mainly exists along the vertical orientation, while \u03c0\u2013\u03c0 stacking and interchain interaction dominate the horizontal orientation.The crystallized 2D nature of such heterostructures is further confirmed by transmission electron microscopy and selected-area electron diffraction patterns Fig.\u00a0. The shi60-DTC60 heterostructure devices. The optical, band alignment and anisotropic photoresponse properties are shown in Fig.\u00a060 can be attributed to \u22125.68 and \u22124.37\u2009eV, respectively. In addition, the HOMO and LUMO levels of the DTC60 nanosheet are \u22126.23 and \u22124.46\u2009eV, respectively. The energy offset between ETC60 and DTC60 nanosheets facilitates the CT interaction for the coupling between ETC60 and DTC60 nanosheets , where h is planck\u2019s constant, c demonstrates the speed of light, R is photoresponsivity, e represents the charge of an electron, \u03bb refers to the wavelength of the irradiated light. The EQE for the horizontal and vertical orientations are 41% and 155%, respectively. According to the photocurrent formula\u03b8 is the fitted slope related to the trapping and recombination process for the photogenerated charge carriers32. The \u03b8 values for the horizontal and vertical orientations are 0.961 and 0.959 under light intensity lower than 7\u2009mW/cm2, which is close to one unit, revealing that monomolecular recombination is the dominant recombination process at low light intensity , we performed first principles density functional theory (DFT) calculations. The electronic density of states (DOS) and the partial density of states (PDOS) of the ETC60-DTC60 are shown in Fig.\u00a060-DTC60 heterostructure results in shifting of the LUMO level closer to the Fermi energy. This decreases the HOMO-LUMO gap, and thus, contributes to the CT in the system. The HOMO is comprised mainly of the 2p states of S atoms in BEDT-TTF molecules of ETC60, while the 2p states of C atoms in C60 molecules of DTC60 dominate the LUMO band  in the heterostructure. There is also a significant contribution of the intra-complex charge transfer within the ETC60, but with a higher HOMO-LUMO gap than the inter-complex CT state  and I(0) is the current with and without magnetic field. The MC along vertical orientation is larger than that along horizontal orientation, and increases with the increase of magnetic field under dark  is much longer than that of singlet exciton (~\u2009ps)38, triplet excitons contribute to the formation of dipoles, and thus the capacitance can be changed by magnetic field, i.e., magneto-capacitance effect  and C(0) are the capacitance under light illumination and dark, respectively , the heterostructure can also demonstrate magneto- and photoexcited capacitance. However, as discussed in the inset of Fig.\u00a060 nanosheets and ETC60 nanosheets is much weaker than that of the opposite stacking. Consequently, C% for magneto-capacitance and photoexcitation is lower than 1% and 4%, respectively ark Fig.\u00a0. It alsoark Fig.\u00a0. Magnetiect Fig.\u00a0. Consequect Fig.\u00a0. The ampely Fig.\u00a0. Photoex60 and DTC60 nanosheets, confirming the tuning ability of the CT states by ferroelectric field. Two different device structures are compared to reveal the mechanism of interface coupling. For the first structure, the stacking sequence from bottom to top is P(VDF-TrFE) layer, ETC60 nanosheet and DTC60 nanosheet, respectively  layer and DTC60 nanosheet, respectively  layer on the bottom of ETC60 nanosheet due to the screening of CT interaction between ETC60 and DTC60 nanosheets by the inserted P(VDF-TrFE) layer  ferroelectric field influences the behavior of dipoles, leading to the change of dielectric behavior. As shown in Fig.\u00a060 and DTC60 nanosheets, it also shows the same enhancement behavior  ferroelectric polymer with high electrostatic field are applied to tune the interfacial coupling. The ferroelectric field from the P(VDF-TrFE) layer enhances the CT density and MC effect Fig.\u00a0. The MC ely Fig.\u00a0. The seqyer Fig.\u00a0, reveali40. Two approaches are proposed to demonstrate the flexible electronic behavior of such 2D CT vdWHs, including tension strain and compress strain along the horizontal orientation. The inset of Fig.\u00a0, \u03b5 is strain. The maximal gauge factor is 8.5. The flexibility can also be expressed by the piezoresistance coefficient41 according toX is the stress, \u22126\u2009Pa\u22121 below a strain of 4.3%, and it decreases to \u22124.1\u2009\u00d7\u200910\u22126\u2009Pa\u22121 under higher strain. The piezoresistance coefficient of such 2D heterostructures is much larger than that of one dimensional nanowires42. The repeatability of the mechanical flexibility is confirmed by the pressure dependent electronic behavior  (P3DDT) donor with a fullerene acceptor layer and bis(ethylenedithio)tetrathiafulvalene donor with a fullerene acceptor layer into scalable free-standing sequential stacking vdWHs. The coupling between two CT layers endows the heterostructure with broadband fast photoresponse of milliseconds. The anisotropic packing structure leads to a much larger coupling extent of charge transfer along the vertical orientation than that of horizontal orientation, leading to anisotropic optical\u2013electronic\u2013magnetic stimulated optoelectronic properties. Moreover, the ferroelectric field effect from P(VDF-TrFE) is applied to tune the coupling and charge transfer in the heterostructures. The DFT calculations confirm the charge transfer between the n-orbitals of the S atoms in BEDT-TTF of ETC60 (Sigma-Aldrich Co.) was added to above solution and stirred at room temperature for 2\u2009h. After centrifuged at 6000\u2009rpm for 20\u2009min, the supernatant solution was isolated and used for the preparation of the film. For P3DDT/C60 solution preparation, 5\u2009mg/mL P3DDT (Ossila Ltd.) was dissolved in 1,2-DCB at 70\u2009\u00b0C for 3\u2009h. Then, 5\u2009mg/mL C60 was added into the solution and stirred at room temperature overnight.5\u2009mg/mL BEDT-TTF  was dissolved in 1,2-dichlorobenzene  solvent, and stirred at 80\u2009\u00b0C for 3\u2009h. Then, 5\u2009mg/mL C60 solution was dropped slightly onto the surface of 20\u2009mL distilled water/DMF mixed solution (volume ratio of 1:1) in a glass vessel and dispersed homogeneously. After several hours of slowly evaporation of 1,2-DCB solvent, thin two-dimensional nanosheet forms. The preparation method for P3DDT/C60 nanosheet is the same, except that only 20\u2009\u03bcL solution is needed. The preparation details for the heterostructure devices, the preparation of P(VDF-TrFE) layer and the flexible devices on PDMS substrates for mechanical characterization are discussed in Supplementary information.In total 50\u2009\u03bcL BEDT-TTF/CTopography images were taken by Bruker dimension icon atomic force microscopy (AFM) under tapping mode with Si probe at a scanning rate of 1\u03bcm/s. The radius and resonant frequency are 25\u2009nm and 13\u2009kHz, respectively.CHI 422 Series Electrochemical Workstation was used to get current and voltage signals. Driel\u2019s 50\u2013150\u2009W Research Arc Lamp Sources was used to output simulated solar light. In addition 365\u2009nm, 650\u2009nm, and 850\u2009nm light comes from UVL-21 compact UV lamp, THORLABS red light and THORLABS LIU 850 A near-infrared light, respectively. The signal was obtained at a sample interval of 0.001\u2009s.60 and DTC60 solution were self-assembled onto the working electrode and dried at 40\u2009\u00b0C in glove box to form uniformly coated nanosheets. The measurement step was 25\u2009mV/s. The HOMO level was obtained from the equation: EHOMO\u2009=\u2009[\u2212exp(Eonset (vs.Ag/AgCl)\u2212Eonset (Fc/Fc\u2009+\u2009vs.Ag/AgCl))]\u22124.8\u2009eV. The LUMO level was calculated from the obtained bandgap from the optical spectra.Cyclic voltammetry experiment was carried out in a three-electrode cell with glassy carbon electrode as the working electrode, Pt electrode as counter electrode, and Ag/AgCl electrode in saturated KCl water solution as the reference electrode. All the electrodes were cleaned and blown dry by nitrogen gas before use. The 0.1\u2009M tetrabutylammonium tetrafluoroborate (Sigma Aldrich) was used as the electrolyte and dissolved in dehydrated acetonitrile (ACN). The stability of Ag/AgCl electrode was checked by ferrocene (Sigma Aldrich) as internal standard substance. The ETCCapacitance signal from 40\u2009Hz to 10\u2009M\u2009Hz was captured by Agilent 4294a Precision impedance equipment at 0.2\u2009V and room temperature. Samples were collected by 16047E fixture. P(VDF-TrFE) ferroelectric layer was polarized by Keithley 2400 source meter at 15\u2009V for 10\u2009s.\u22121. Pressure was loaded by finger print. The pressure through finger was weighed and calibrated by a balance.Tension stress was loaded by Instron 5944 Single Column Tabletop Low-Force Universal Testing System at a speed of 0.5\u2009\u03bcm\u2009s43. We use a generalized gradient approximation (GGA) with the Perdew\u2013Burke\u2013Ernzerhof (PBE)44 parameterization of exchange correlation energy functional with ultrasoft pseudopotentials45. An energy cutoff of 30\u2009Ry was used for truncating the plane wave basis set to represent wave functions. Our recent work on ETC60 crystals gives excellent result with these parameters46. The structures were relaxed until the magnitude of the Hellman\u2013Feynman force on each ion became smaller than 0.03\u2009eV/\u00c5. Brillouin zone integrations were carried out with a uniform small mesh of k-points. The Grimme parameterization47 was used to include the vdW interactions. For the purpose of this study, the heterostructure comprising of BEDT-TTF/C60 (ETC60) and P3DDT/C60 (DTC60) complexes (monolayer structure) was built using the Avogadro48 and Gauss View5 packages49. For the ETC60 section of the heterostructure, the crystal coordinates were used from the Cambridge Crystallographic Data Centre50. The monolayer structures of the ETC60 and DTC60 complexes were formed by removing the structures of DTC60 and ETC60, respectively from those of the heterostructure. The heterostructure contains total of 690 atoms out of which 396 belongs to ETC60 and remaining 294 associated with DTC60, respectively.Our calculations are based on first-principles density functional theory (DFT) as implemented in the Quantum ESPRESSO codehttps://doi.org/10.6084/m9.figshare.5005331.v1 Remaining data that support the findings are available from the corresponding author upon reasonable request.The data that support the findings of this study are available from the online data repository Figshare with identifier Peer Review FileSupplementary Information"}
{"text": "We report rabies virus transmission among solid organ transplantation recipients in Changsha, China, in 2016. Two recipients were confirmed to have rabies and died. Our findings suggest that more attention should be paid to the possibility of rabies virus transmission through organ transplantation for clinical and public health reasons. In 2016, Zhou et al. reported a case of rabies virus transmission in China that was probably a result of organ transplantation , where he exhibited anemophobia, convulsions, limb rigidity, and hypersalivation. The patient was moved to hospital B (days 7\u201314) in Changsha. At admission, some examination findings indicated a possibility of viral encephalitis  Table 1.On day 14, the patient was transferred to hospital C, where he became comatose and was declared brain dead. Permission was granted for organ donation, because no specific pathogen had been detected and China\u2019s organ transplant policy allows for organ donations in cases of viral encephalitides without laboratory-confirmed pathogens. Rabies virus\u2013specific binding antibodies were also not detected in the serum samples. The donor had possibly been exposed to rabies but had not been vaccinated against rabies . The patient\u2019s kidneys and liver were removed for transplantation in hospital D in Changsha on the following day.Kidney and liver transplantations were performed on December 10, 2015. Two female recipients in hospital D received the kidneys, and an 8-month-old girl in hospital E in Shanghai, China, received the liver. No other organs were collected or transplanted from this donor. The surgeries were all uneventful, and the recipients were discharged after transplantation. However, all 3 recipients were eventually readmitted to the hospital with complex symptoms . On the 40th day after transplantation, the left kidney recipient (29 years of age) revisited hospital D, reporting worsening right lower quadrant abdominal pain and vomiting. In the next 3 days, she successively had anemophobia, shortness of breath, chest tightness, anxiety, insomnia, and hypersalivation. During hospitalization, her blood pressure was as high as 248/148 mm Hg. On her 7th day in the hospital, she became comatose and then died. On the 43rd day after transplantation, the right kidney recipient (age 47 years) developed aches in her lower limbs, insomnia, and a decreased appetite. She was readmitted to hospital D the next day. On the 3rd day after admission, she exhibited hydrophobia, anemophobia, photesthesia, and fatigue. She showed agitation, dyspnea, and hypersalivation on the 4th day; she became comatose and died 1 day later. The liver recipient was readmitted with pneumonia on the 34th day after transplantation and died of asphyxia and multiple organ failure within 5 days. This patient did not show any signs or symptoms of rabies or encephalitis. None of the recipients had been exposed to potentially rabid animals or had been vaccinated previously for rabies online Technical Appendix). Both kidney recipients tested positive for rabies virus (. Both kiIn the past 10 years, rabies transmission by solid organ transplantation has been described occasionally worldwide (Epidemiologic information about donor and the 2 kidney transplant recipients in a rabies outbreak associated with solid organ transplantation, Changsha, China, 2015\u20132016."}
{"text": "Kidney transplantation is now a viable alternative to dialysis in HIV-positive patients who achieve good immunovirological control with the currently available antiretroviral therapy regimens. This systematic review and meta-analysis investigate the published evidence of outcome and risk of kidney transplantation in HIV-positive patients following the PRISMA guidelines.Searches of PubMed, the Cochrane Library and EMBASE identified 27 cohort studies and 1670 case series evaluating the survival of HIV-positive kidney transplant patients published between July 2003 and May 2018. The regimens for induction, maintenance therapy and highly active antiretroviral therapy, acute rejection, patient and graft survival, CD4 count and infectious complications were recorded. We evaluated the patient survival and graft survival at 1 and 3\u00a0years respectively, acute rejection rate and also other infectious complications by using a random-effects analysis.At 1\u00a0year, patient survival was 0.97 , graft survival was 0.91 , acute rejection was 0.33 , and infectious complications was 0.41 , and at 3\u00a0years, patient survival was 0.94  and graft survival was 0.81 .With careful selection and evaluation, kidney transplantation can be performed with good outcomes in HIV-positive patients. Traditionally, human immunodeficiency virus (HIV)-positive patients (HIV+) has not been considered to be good candidates for solid-organ transplantation for the poor prognosis of HIV patients. However, with the introduction of antiretroviral combination therapy (cART), the survival of HIV+ patients have been great improved. While the frequency of Acquired Immune Deficiency Syndrome (AIDS)-related events has consequently decreased, mortality due to organ failure has become a significant concern.The initial attempts at kidney transplantation (KT) in HIV+ patients led to poor outcomes, but better results occurred with the availability of highly active antiretroviral therapy (HAART) , 2.In this scenario, KT started to be proposed as a treatment even as \u201cstandard-of-care\u201d for end-stage renal disease (ESRD) in selected HIV+ patients .A multicentre study in the USA found that the survival rates for HIV+ recipients fall between those reported for older KT recipients and for all recipients in the American national database .Despite these encouraging results, many issues still need to be addressed. Among the more relevant are the elevated incidence of acute rejection (AR), lower patient survival (PS) and graft survival (GS), and the hurdles caused by the interaction of immunosuppressive and antiretroviral (ARV) drugs. We conducted a systemic review and meta-analysis to determine the effectiveness of KT in the presence of HIV. Specifically, we examined PS and GS, AR and infectious complications in HIV+ patients who have undergone KT.The study design of a systematic review and meta-analysis was chosen to define the published evidence of the effectiveness of KT in HIV+ patients. The study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement standards . Our revWe searched the Medline (1966 to June 2018), EMBASE (1974 to June 2018), and Cochrane Controlled Trials Register databases to identify studies that referred to KT in HIV+ patients; we also searched the reference lists of the retrieved studies. The following search terms were used: KT, HIV+, AIDS. A combination of subject headings and keywords for KT, HAART, HIV+ recipient, allograft survival, antiretroviral therapy, donor selection, ESRD and immunosuppression was used for the literature search.Cohort studies and case\u2013control studies were all eligible for inclusion if they reported outcomes of KT in HIV+ patients. Studies reporting outcomes shorter than 12\u00a0months post transplantation and transplantation occurring before HAART were introduced were excluded. Articles were independently assessed by 2 reviewers (X Z and WR X) according to the predetermined eligibility criteria. Any disagreement between reviewers was resolved by discussion with a third reviewer (XP H).All data were extracted independently by 2 reviewers (X Z and WR X) onto a Microsoft Excel spreadsheet , and any discrepancies were resolved by consensus. The following information was collected for each study: the study country, sample size, inclusion criteria, exclusion criteria, induction and maintenance immunosuppression, HAART regimen, mean CD4 T-cell counts (CD4 counts) pre-transplant and post-transplant, infectious complications, post-transplant neoplasia, PS and GS at 1 and 3\u00a0years, and AR rate. In order to analyse data of Infectious complications (IC), all infections requiring hospitalization were registered.The quality of each study used for the meta-analysis was assessed based on the Newcastle\u2013Ottawa-Scale (NOS) for cohort studies . The evaWe undertook the descriptive analyses to identify the number of studies with relevant data, the countries where the studies were conducted, and other population attributes. The transplant outcome data were pooled using different transformations according to their different normal distribution conditions.The data for PS, GS, AR, IC at 1\u00a0year and PS at 3\u00a0years were analysed using log transformation.The data for GS at 3\u00a0years were analysed using arcsine transformation.The transformed data were combined to estimate the pooled percentages with 95% confidence intervals using a random-effects model.2 [http://www.R-project.org; version 2.9.0), using the software libraries \u2018meta\u2019 and \u2018metafor\u2019, for the meta-analysis and meta-regression models, respectively.The transformed data were combined to estimate the pooled percentages with 95% confidence intervals using a random effects model  and preson by I2 . StatistThe search strategy identified 86 citations Fig.\u00a0, and amoDetailed characteristics of all included studies are provided in Table\u00a0Regarding immunosuppression and rejection, most of the patients received antibody induction therapy with different regimens containing basiliximab, daclizumab, antithymocyte globulin (ATG) or methylprednisolone. The maintenance regimens were mainly composed of cyclosporin A (CSA), mycophenolate mofetil (MMF), tacrolimus (TAC) and steroids, which were the same as the maintenance regimens for HIV-negative patients.Mean CD4 counts were steady in most of the patients. As we observed, in all the cohorts with available data (22/27 cohorts), mean CD4 counts pre-TX and post-TX were greater than 200 cells/\u03bcL, and even elevated post transplantation.Pneumocystis jirovecii for at least 6\u00a0months.Prophylaxis against opportunistic infection was common in most studies as follows: patients received ganciclovir or valganciclovir for cytomegalovirus and trimethoprim/sulfamethoxazole, dapsone or atovaquone for Kaposi\u2019s sarcoma and skin cancer were the most observed post-transplant neoplasia. As we showed in Table\u00a0Each of the 27 studies included in the meta-analysis was assessed by the NOS to investigate the risk of bias within the studies. Table\u00a02\u2009=\u200936%) of patients survived, while 94%  of patients survived at 3\u00a0years.Twenty-seven studies reporting PS at 1-year post KT included PS estimate to post KT for 1429 patients; however, only nine studies including 509 patients reported PS at 3\u00a0years. The results of the analysis are shown in Figs.\u00a02\u2009=\u200969%) of grafts had survived, and GS subsequently declined to 0.81  at 3\u00a0years.Twenty-six studies including 1391 patients reported GS at 1-year post KT, and nine studies including 509 patients reported GS at 3\u00a0years. The results of the analysis are shown in Figs.\u00a02\u2009=\u200960%) of patients had AR.Twenty-five studies including 1051 patients reported AR post KT at 1\u00a0year, and the results of the analysis are shown in Fig.\u00a02\u2009=\u200959%) of patients had IC.Nineteen studies including 584 patients reported IC post KT at 1\u00a0year; the results of the analysis are shown in Fig.\u00a0To our knowledge, this is the first systematic review and meta-analysis of such a large scale to report the outcomes of KT in HIV+ patients. We review and meta-analysis the outcomes in HIV+ KT patients, and looks at the 1- and 3-year GS/PS and AR rate.The availability of cART has made KT a feasible treatment for selected HIV+ patients with ESRD, with outcomes somewhat inferior to those observed among the overall population of KT recipients , 9\u201311.KT is now a viable treatment for select patients with HIV and ESRD. Moreover, the high incidence of morbidity and mortality resulting from cardiovascular issues in HIV+ patients , 13, as However, data regarding long-term outcomes and comparisons with appropriately matched HIV\u2212 patients are still lacking.Locke et al. analysed 510 adult KT recipients with HIV matched 1:10 with HIV\u2212 controls. They found that HIV\u2212 and HIV mono-infected KT recipients had similar GS and PS, whereas HIV/HCV co-infected recipients had worse outcomes .Izzo et al. found that the survival rate of patients was 82.1% and functioning grafts was 71.4% , and a rStock et al.  reportedIn our analyses, at 1\u00a0year, PS was 0.97 , GS was 0.91 , and at 3\u00a0years, PS was 0.94 , GS was 0.81 .One of the most challenging goals in solid-organ transplantation is to tailor the immunosuppressive regimen for each individual patient to minimize immunosuppression while still preventing AR. Opportunistic infections and malignancies often attributed to immunosuppression itself remain a significant cause of death after transplantation. In the field of HIV+ organ transplantation, finding a balanced approach to immunosuppression is even more critical.Currently, the vast majority of KT patients receive induction immunosuppression, which has been shown to greatly reduce the risk of rejection and improve PS and GS . As showGuidelines from the Kidney Disease: Improving Global Outcomes transplant working group recommended anti-IL2R as the first-line treatment for patients at low risk for rejection and ATG for those at high risk .Despite being the standard of care for most HIV patients, the use of induction immunosuppression for HIV+ patients, particularly ATG, remains controversial.On the one hand, HIV+ patients have high rates of rejection and thus stand to benefit significantly from induction. On the other hand, the risks posed from prolonged lymphocyte depletion are of major concern given that HIV+ patients are perceived to already threaten T cell populations and reduced immunity, both states that are associated with an increased risk of opportunistic infections.A recent study showed that ATG induction was associated with long-term impairment of T cell function and related infections, even after the patients had normalized CD4 counts . This fiThe incidence and severity of IC following transplantation are largely dictated by the recipient\u2019s capacity for immune reconstitution. A study by Suarez et al. indicate that ATG-induced CD4 lymphopenia can be prolonged, and even at 1\u00a0year post transplant, a substantial proportion of patients has CD4 counts\u2009<\u2009200/\u03bcL . The basA study by Bossini et al. showed that in HIV+ KT recipients treated with basiliximab and maintained on a calcineurin inhibitor (CNI)-mycophenolic acid (MPA)-based regimen, early corticosteroid withdrawal was associated with a very high incidence of AR and that kidney function was worse in patients with rejection .However, in contrast to the studies above, in a large national cohort of 830 HIV+ KT recipients, Kucirka found wide variation in the use of induction immunosuppression, with >\u200930% of HIV+ patients receiving no induction compared with only 20% of their HIV counterparts. Therefore, the study indicated that the use of induction, including the lymphocyte-depleting agent ATG, was not associated with an increased risk of infections. Despite the fact that induction recipients were at higher risk for AR, the researchers observed lower rates of delayed graft function (DGF), AR, graft loss and days spent hospitalized in the first year after KT as well as a trend towards lower mortality. They suggested that the benefits of induction immunosuppression to prevent graft rejection in HIV+ KT recipients far outweigh the perceived risk of increased infections. Because the study had the largest sample size to date and the cohort was nationally representative rather than a select study population, this study claims to be more credible. Furthermore, the authors accounted for confounding and treatment selection bias, which previous studies did not do, and they did this using inverse probability of treatment weighting (IPTW), a method that allowed them to adjust for many clinical and demographic factors even when modelling relatively rare outcomes such as graft loss and death .A high risk of AR is a well-known concern in HIV-infected kidney graft recipients. With regard to rejection, most studies observed a higher number of events in HIV+ patients than in HIV\u2212 patients. AR may occur as a result of immune dysregulation and the continuous inflammatory state of HIV+ recipients, in whom immunogenicity is increased following allograft implantation , 29.Vicari et al.  evaluateIn the study, the incidence of treated AR was higher in the HIV+ group, and the incidence of biopsy-confirmed AR was numerically higher in this group. Additionally, even though an identical incidence of antibody-mediated AR occurred, the incidence of steroid-resistant rejection was numerically higher in the HIV group. Many reports have revealed an elevated incidence of AR in HIV+ recipients, varying between 31% and 55% , 9, 10, However, Malat et al.  describeGathogo et al.  reportedIn addition, the elevated incidence of acute cellular rejection has been recently hypothesized to partially occur a result of an infiltration of inflammatory cells that occurs in response to tubular cell infection by HIV .A study by Malat showed a relatively higher incidence of mixed rejection in HIV+ recipients compared with that reported for non-HIV transplant recipients. A donor terminal serum creatinine greater than 2.5\u00a0mg/dL predicted mixed rejection and was associated with poor outcomes. Donor selection and optimization of immunosuppression may be critical in these patients . Even if2\u2009=\u200959%), and the rate of incidence of IC observed in this study in HIV+ KT patients is in line with the frequencies reported in a study by Stock et al. [During the first decades of the renal transplantation era, a serious IC developed in up to 70% of patients following transplantation, resulting in fatal outcomes in as many as 11% to 40% of cases . In a reHowever, the long-term patient and graft outcome of the whole cohort were not influenced by HIV status but were adversely influenced by infections, as survival was diminished in patients having at least one infection.Furthermore, one-third of HIV+ KT recipients in a study by Ailioaie et al. did not have any episodes of infection, and repeated infections were not frequent. More importantly, the rate of incident infections was not different between the HIV+ and HIV\u2212 matched groups.As experience with transplantation in HIV+ patients grow, significant drug\u2013drug interactions between ART and maintenance immunosuppression have been identified as a major clinical challenge.Post-transplant management of HIV infection with protease inhibitor (PI) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART is complicated by reciprocal drug interactions with immunosuppressive therapy, especially CNI, because of inhibition or induction of P450 cytochrome enzymes. Co-administration of PIs with CNIs requires significantly decreased CNI doses and prolonged dosing intervals to avoid supratherapeutic trough levels.Despite appropriate CNI dose adjustments, variations of drug serum levels are difficult to control and have been linked to increased graft rejection in HIV+ KT recipients , 39.In a study of 150 HIV+ KT patients, the largest to date, higher-than-expected rates of rejection were reported  [In a study by Rosa et al., patients receiving PI-containing regimens had lower PS at 1 and 3\u00a0years than patients receiving PI-sparing regimens\u201485% vs. 100% (p\u2009=\u20090.06) and 82% vs. 100% (p\u2009=\u20090.03), respectively .The increased risk of AR in HIV+ individuals has been largely attributed to reduced exposure to immunosuppressive agents due to drug\u2013drug interactions with ART , 41, 42.This observation might be due to the effects of PI on tacrolimus levels, considering that the overwhelming majority of these patients were on a PI-containing regimen and that more than half had tacrolimus levels above target at the time of infection. PI could also influence the net state of immunosuppression by increasing the level or effect of other immunosuppressants, such as prednisone and mycophenolate.The most important finding in the present study is the association between PI use and adverse outcomes, namely, reduced 3-year PS and GS, and increased risk of serious non-opportunistic infections. These observations remained true in analyses restricted to patients receiving nucleoside reverse transcriptase inhibitor (NRTI) \u201cbackbone\u201d; thus, even after excluding the potential influence of other agents included in the ART regimen, PI continued to be associated with poor outcomes.However, the use of NNRTI or tenofovir disoproxil fumarate (TDF) did not influence GS. Tenofovir alafenamide (TAF) is a new formulation of tenofovir associated with less kidney (and bone) toxicity . WhetherIn a large single-centre study of HIV+ KT recipients conducted by Boyle et al. , treatmeGiven that specific recipient characteristics, such as hepatitis B co-infection and certain HIV mutations, continue to make TDF-based regimens the most likely to provide adequate viral suppression post-transplant, despite observational data for nephrotoxicity in the non-transplant population.However, given the limitations of this study, TDF should be reserved for patients who have limited ART options and should be used very cautiously in the KT population, with appropriate dose adjustment and surveillance of kidney function, including kidney biopsy when indicated. Substituting TAF for TDF in KT patients is reasonable, but it should be noted that no data are yet available on long-term kidney outcomes with TAF in KT and non-KT recipients in the setting of both preserved and reduced glomerular filtration rate (GFR).Since their introduction in 2007, integrase strand transfer inhibitors (INSTIs) have been proposed as preferred post-transplant ART because of a favourable pharmacologic profile with decreased potential for drug interactions , 42, 46.Recently, Alfano et al., reported that preferred drug included raltegravir and dolutegravir for INSTI class, maraviroc for CCR5 receptor antagonist, lamivudine for NRTI, and rilpivirine for NNRTI, which offered advantage of having no drug interactions .In summary, we believe that INSTI or CCR5-based therapy should be the preferred ART in patients with HIV who undergo KT, primarily because of decreased drug\u2013drug interactions with immunosuppressive medications such as CNIs, enabling easier monitoring of immunosuppressive medications and superior graft outcomes. However, larger and more controlled trials are needed to better assess the long-term outcomes of INSTI-based therapy to elucidate factors related to GS other than direct reciprocal drug interactions.In conclusion, this systematic review and meta-analysis demonstrated that with careful selection of patients and multidisciplinary evaluation, KT can be performed with good outcome in HIV+ patients. Moreover, with the advent of INSTI-based cART regimens, drug\u2013drug interactions between cART and immunosuppressants have been dramatically reduced. Nevertheless, further studies are needed to optimize immunosuppressive therapy regimens for HIV+ patients, with the aim of reducing the high rate of AR after transplantation. Furthermore, this review still has its limitations, such as lack of sufficient studies, possibility of some overlapping patient cohorts, short of comparator. And we are also looking forward to other novel papers as more and more studies regarding KT of HIV+ patients."}
{"text": "The beneficial effects of silicon and its role for plants are well established; however, the advantages of silicon nanoparticles over its bulk material are an area that is less explored. Silicon nanoparticles have distinctive physiological characteristics that allow them to enter plants and influence plant metabolic activities. The mesoporous nature of silicon nanoparticles also makes them good candidates as suitable nanocarriers for different molecules that may help in agriculture. Several studies have shown the importance of silicon nanoparticles in agriculture, but an overview of the related aspects was missing. Therefore, this review brings together the literature on silicon nanoparticles and discusses the impact of silicon nanoparticles on several aspects of agricultural sciences. The review also discusses the future application of silicon nanoparticles in plant growth, plant development, and improvement of plant productivity. Equisetaceae family cannot survive in nutrient solutions lacking silicon  were successfully employed as an optical sensor for melamine detection. Similarly, Liu et al. (In recent decades, environmental sensing technologies based on metallic nanoparticles have gained enormous attention due to their extraordinary sensing resolution and sensitivity to analytes under optical detection. Developing a nanoparticle-based sensing device has been proposed due to the surface area of nanoparticles and the availability of surface anchored bioactive molecules for analyte detection (Hache et al. u et al.  preparedu et al. . TherefoNanotechnology is a promising area of interdisciplinary research that opens avenues in several fields, such as medicine, pharmaceuticals, electronics, and agriculture. This article presents the potential of Si-NPs in agriculture and brings together the literature relevant to the use of nanoparticles as pesticides, fertilizers, herbicides, genetic and drug transfer agents, soil improving agents, and sensors for soil analysis. Studies show that Si-NPs have the potential to revolutionize the existing technology used in various sectors, such as agriculture and plant biotechnology. Silicon nanoparticle-mediated targeting of biomolecules would be useful for developing new cultivars that are resistant to various biotic and abiotic factors. These nanoparticles can provide green and eco-friendly alternatives to various chemical fertilizers without harming nature. Thus, Si-NPs may have concrete solutions to many agricultural problems regarding weeds, pathogenicity, drought, crop yield, and productivity."}
{"text": "Juvenile dermatomyositis (JDM) is the most common inflammatory myopathy in childhood and a major cause of morbidity among children with pediatric rheumatic diseases. The management of JDM is very heterogeneous. The JDM working group of the Society for Pediatric Rheumatology (GKJR) aims to define consensus- and practice-based strategies in order to harmonize diagnosis, treatment and monitoring of JDM.The JDM working group was established in 2015 consisting of 23 pediatric rheumatologists, pediatric neurologists and dermatologists with expertise in the management of JDM. Current practice patterns of management in JDM had previously been identified via an online survey among pediatric rheumatologists and neurologists. Using a consensus process consisting of online surveys and a face-to-face consensus conference statements were defined regarding the diagnosis, treatment and monitoring of JDM. During the conference consensus was achieved via nominal group technique. Voting took place using an electronic audience response system, and at least 80% consensus was required for individual statements.Overall 10 individual statements were developed, finally reaching a consensus of 92 to 100% regarding (1) establishing a diagnosis, (2) case definitions for the application of the strategies (moderate and severe JDM), (3) initial diagnostic testing, (4) monitoring and documentation, (5) treatment targets within the context of a treat-to-target strategy, (6) supportive therapies, (7) explicit definition of a treat-to-target strategy, (8) various glucocorticoid regimens, including intermittent intravenous methylprednisolone pulse and high-dose oral glucocorticoid therapies with tapering, (9) initial glucocorticoid-sparing therapy and (10) management of refractory disease.Using a consensus process among JDM experts, statements regarding the management of JDM were defined. These statements and the strategies aid in the management of patients with moderate and severe JDM. Juvenile dermatomyositis (JDM) is the most common inflammatory myopathy of childhood and a major cause of morbidity and mortality among patients with pediatric rheumatic diseases . The manThe PRO-KIND  initiative is a sub-committee of the Society for Pediatric Rheumatology (GKJR) in Germany and Austria and aims to define consensus-based plans to harmonize diagnostic and treatment approaches. This initiative was started since it was perceived that children with juvenile rheumatic diseases in Germany and Austria often are either treated too late or not treated with the most up-to-date therapeutic regimens. To meet this challenge, the goal of the PRO-KIND initiative is to foster the use of harmonized diagnostic and treatment plans with defined targets , 26. PreAs a next step, the goal of the PRO-KIND working group on JDM was to harmonize previously identified patterns into plans and statements.www.surveymonkey.com). Nine pediatric rheumatologists participated in an initial face-to-face meeting in January 2016. There was consensus that the expert panel should also include expert pediatric neurologists. Therefore, 2 pediatric neurologists with special expertise in managing JDM (W. M-F. and U.S.) were also invited to join the group, forming an extended interdisciplinary expert panel.The JDM working group was formed in April 2015, initially comprised of 12 pediatric rheumatologists with clinical expertise in the management of patients with JDM. Identification of project goals took place via a formal online survey using the web-based program SurveyMonkey  and a non-voting dermatologist experienced in the care of children with JDM participated in a final face-to-face consensus meeting on January 13, 2017. The extended expert panel overlapped with but was not identical with the members of the initial working group. Prior to the consensus conference, the voting members had received access to various essential data via sciebo, a cloud service hosted by the state of Northrhine-Westphalia. The data provided included detailed results of previous online surveys, previous meeting protocols, essential literature obtained via an extensive literature search , including the 112 papers that were scored by SHARE . The proThe expert status of the panel members is demonstrated by the fact that centers represented in the panel contributed more than half of all patients  with JDM documented within the National Pediatric Rheumatic Disease Database (\u201cKerndokumentation rheumakranker Kinder und Jugendlicher\u201d).Regarding the diagnosis of JDM, there was consensus that the conventional Bohan and Peter criteria should be modified. In particular, there was consensus that typical magnetic resonance imaging (MRI) findings represented an important finding for establishing a diagnosis of JDM. Furthermore, electromyography was removed from the list of findings. The finding of symmetric proximal muscle weakness was modified to also include myalgia as a possible criterion Table\u00a0. ConcernIt was felt to be important to define a minimum dataset that should be routinely collected on all patients with at least moderate JDM Table\u00a0. The groThe group defined a treat-to-target strategy, which means treatment to be adjusted according to the achievement of previously defined targets Table\u00a0. It was There was consensus that all patients with JDM should receive supportive therapy including physical therapy to prevent contractures, muscle weakness, de-conditioning and disability. Early return to sports activities is encouraged as long as it is safely possible. Effective sun and/or ultraviolet light protection using avoidance, textile protection or sunscreen is strongly suggested. Additional supportive therapies may include hydroxychloroquin (HCQ), vitamin D or calcium supplementation Table\u00a0. It was Different glucocorticoid regimens were preferred by experts within the panel. Based on the results of a previous survey , 3 glucoNo consensus was found concerning a specific tapering protocol for glucocorticoids. However, the group defined landmarks for the glucocorticoid taper, i.e. the aim to discontinue glucocorticoids after 12\u00a0months of therapy, and, in case of initial high-dose oral glucocorticoid therapy, PDN doses of 50, 25 and 12.5% 2\u00a0months, 4\u00a0months and 6\u00a0months after initiation of therapy, respectively  therapy, such as AZA, CSA or MMF. Furthermore, there was consensus that high-dose IVIG may be used in conjunction with DMARD therapy, especially in case of severe JDM Table\u00a0. HoweverThe expert group considered refractory disease to be present if the predefined treatment targets were not reached. Furthermore, patients with longstanding (i.e. not new-onset) JDM may be treated according to strategies outlined here. The group considered two options to be viable under these circumstances: (1) Additional therapy with another DMARD, specifically, AZA, calcineurin inhibitos (CSA or tacrolimus), CYC, IVIG, MMF, RTX or TNFi, or (2) changing therapy to one of these medications. However, there was no consensus on which specific sequence of medications would be preferred. In order to reflect this uncertainty, the medications or medication classes are simply listed alphabetically Table\u00a0.The statements developed are condensed in Fig.\u00a0Based on data on the current clinical practice in Germany we defined consensus-based statements and strategies supporting the diagnosis and the management of JDM. The expert panel consisted both of pediatric neurologist and rheumatologists with all of them having practical experience in managing JDM. The group supports the use of \u201cadapted\u201d Bohan and Peter criteria for the diagnosis of JDM, including the use of specific MRI findings as a Bohan and Peter \u201cequivalent\u201d criterion, similar to what others have proposed in the past . There aSince there is a relative lack of data from randomized controlled clinical trials (RCTs), and since it is unlikely that large-scale RCTs for JDM will take place, one possible solution to gather more information on the efficacy or inefficacy of various treatments may be to collect \u201creal life\u201d data on disease outcome, i.e. to pursue comparative effectiveness research . An impoThese harmonized consensus strategies overall fit rather well into the above-mentioned framework set up by the CARRA consensus treatment plans and the SHARE recommendations which had been developed previously or simultaneously, respectively. However, there are important differences in clinical practice in Germany when compared to this framework. For example, practitioners in Germany have a strong preference to choose a glucocorticoid regimen consisting of ongoing intermittent IVMP therapy in combination with low-to-moderate dose glucocorticoids instead of conventional daily high-dose glucocorticoid regimens . There iThere are several limitations to the PRO-KIND consensus strategies for JDM. First, the concepts outlined here may not represent the optimal way to manage JDM but rather represent current clinical practice among JDM experts in Germany and are based on a low level of evidence. Second, the statements and resulting strategies do not represent treatment protocols or \u201cTo Do\u201d-lists. Management of patients with JDM will remain highly variable. Third, the field is constantly evolving: Some MSAs that were only recently reported in children, for example, anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies, are not included in our list but may be useful to determine in some patients, especially in case of refractory myositis , 46\u201348. In summary, we have developed consensus-based strategies for the diagnosis, monitoring and treatment of JDM by the harmonization of current clinical practice with the overarching goal is to improve the outcome of patients with JDM. Concerning therapy, we are placing an emphasis on a treat-to-target strategy using internationally accepted improvement criteria rather than on individual medications, fitting well into existing frameworks. As others have done previously, we define a minimal dataset to regularly collect in patients with JDM in order to allow comparative effectiveness research. We conclude that, due to the rarity of the condition, international collaboration will be critical in order to improve the outcome of patients with JDM. We believe that this work will add to the armamentarium of providers managing patients with JDM worldwide."}
{"text": "Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3\u2032 end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures. DNA repair is an important biological process that ensures genome stability. Cells rely on an intricate network of DNA repair pathways to counteract various types of DNA damage. One of the most deleterious types of damage is the DNA double-strand break. Double-strand breaks can form when two single-strand breaks are in close proximity, after the attempted replication of nicked DNA or upon replication fork collapse. If double-strand breaks are not repaired, then this can lead to the loss of entire segments of genetic information. Homologous recombination is generally considered an error-free pathway of double-strand break repair because it utilizes a homologous sequence as the template for DNA repair. Homologous recombination invariably involves the generation of joint (branched) DNA intermediates  . The strStructure-selective endonucleases belong to various nuclease families, including the FEN1/XPG, XPF/ERCC4\u00a0and GIY-YIG families. As the name implies, these enzymes cleave specific types of branched DNA structures, as opposed to specific DNA sequences. Most of these enzymes have defined substrate specificities and thus play roles in distinct pathways of DNA repair . For exaInterestingly, some SSEs function in a coordinated manner to utilize their distinct substrate specificities. One prominent example is cooperation between XPF-ERCC1, MUS81-EME1\u00a0and SLX1, which are brought together via their interactions with the SLX4 platform protein ,20. NuclWe previously reported the mechanism by which the Slx4 scaffold activates Slx1 nuclease. In the absence of Slx4, Slx1 adopts a homodimeric architecture that occludes the active site. Conversely, Slx4 disrupts the inhibitory Slx1 homodimer and forms a catalytically active Slx1\u2013Slx4 heterodimer. Guided by structural similarities between the active site of Slx1 and GIY-YIG type II restriction endonucleases, we proposed a possible mode of DNA binding by Slx1 . In contThe present study investigated the mechanism of DNA binding by Slx1 and defined a new DNA-binding interface. The positioning of this interface explains the way in which the enzyme utilizes helical discontinuity in branched DNA substrates as the main determinant for incision point selection. We propose a mechanism that explains the ability of Slx1\u2013Slx4 to cleave various DNA joint molecules that are intermediates during DNA repair.Candida glabarata Slx1 (Cg-Slx1) and the conserved C-terminal domain of Slx4 (Cg-Slx4CCD) (residues 557\u2013726) were codon-optimized for expression in Escherichia coli and obtained from Bio Basic Inc., Canada. Synthetic genes for Thielavia terrestris Slx1 (Tt-Slx1) and a fragment of Slx4CCD (Tt-Slx4CCD3) (residues 834\u2013936) were codon-optimized for expression in E. coli and obtained from GenScript\u00ae. Cg-Slx1 and Cg-Slx4CCD were subcloned into a pET28a vector (Novagen) with an N-terminal 6xHis-SUMO tag. Tt-Slx1 and Tt-Slx4CCD3 were subcloned into a pRSFduet-1 vector (Novagen) using multiple cloning site I with an N-terminal 6XHis tag and multiple cloning site II without a tag, respectively. All point substitutions were introduced by QuikChange Site-Directed Mutagenesis (Agilent Technologies). All proteins (wild type and mutants) were expressed in E. coli BL21 (DE3) Rosetta (Novagen). For protein expression, bacteria were grown in LB medium at 37\u00b0C, induced with 0.4 mM isopropyl \u03b2-d-1-thiogalactopyranoside at OD600 = 0.7\u20130.9, and grown overnight at 18\u00b0C. The cells were harvested by centrifugation, and pellets were washed with phosphate-buffered saline (PBS) prior to purification.Synthetic genes for Cg-Slx1 and Cg-Slx4CCD (wild type and point substitution variants) was purified as described earlier  and lysed by sonication. The lysate was centrifuged at 186 000 \u00d7 g at 4\u00b0C for 40 min, and the supernatant was loaded onto a His-Trap HP column  that was equilibrated with lysis buffer. Protein was eluted using a linear gradient of imidazole from 5 to 500 mM. The fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractions that contained a complex of Tt-Slx1 and Tt-Slx4CCD3 were further purified using a Superdex-200 size exclusion column  in a buffer that contained 20 mM Tris\u2013HCl (pH 8.5), 10% (v/v) glycerol, and 5 mM 2-mercaptoethanol. Fractions that contained the Tt-Slx1\u2013Slx4CCD3 complex were pooled and concentrated using a 30k MWCO Amicon Ultra Centrifugal Filter Device (Millipore).A complex of  earlier . A complTt-Slx1E79Q-Slx4CCD3 at a concentration of 13 mg/ml was crystallized in the presence of either HJs or self-annealing splayed-arm substrate in a buffer that contained 20 mM Tris\u2013HCl (pH 8.5), 100 mM NaCl and 5 mM ethylenediaminetetraacetic acid. The oligonucleotide sequences are presented in The active-site mutant of Tt-Slx1E79Q-Slx4CCD3 was used as the starting model for molecular replacement for dataset 2. Coot was used for iterative model building  were collected at Diamond Light Source at a wavelength of 1.2828 \u00c5. These crystals diffracted to a maximum resolution of 3.2 \u00c5. Diffraction data for crystals with splayed-arm substrate (dataset 2) were collected at beamline 14.1 at Berliner Elektronenspeicherring-Gesellschaft f\u00fcr Synchrotronstrahlung (BESSY)  at a wavbuilding ,27. The lProbity . The dif were col2, 1 mM DTT and 0.5% glycerol. The substrates comprised 200 nM unlabeled DNA substrate and 50 nM DNA that was labeled with fluorescein on X0-1 and Cy5 on X0-4. The samples were then taken out at various time points and stopped by adding equal amounts of 100% formamide that contained Orange G dye and boiling the samples for 5 min at 95\u00b0C. The samples were run on a 20% TBE-Urea polyacrylamide gel at a constant power of 18 W for 40 min. The cleavage sites were mapped using fluorescein- or Cy5-labeled DNA marker oligonucleotides of various lengths , 2.5 mM CaCl2, 100 mM NaCl and 0.1 mg/ml bovine serum albumin at 25\u00b0C. Binding reactions were set in Corning 96 flat bottom black polystyrene plates. Anisotropy was measured using a Tecan Infinite M1000 microplate reader at an excitation wavelength of 635 nm and emission wavelength of 670 nm.The binding of 5\u2032-flap DNA substrate to Secondary structure analysis was performed using a Bruker Tensor 27 spectrometer. Protein samples were used at a concentration of 0.5\u20131.0 mg/ml in 20 mM HEPES (pH 7.5) and 150 mM NaCl. The results were analyzed using OPUS-PRO software.CCD) from several fungal species in the presence of various DNA substrates . We obtained two crystal forms of Slx1\u2013Slx4CCD3 from Thielavia terrestris   of 2.3 \u00c5 over 142 C-\u03b1 atoms . The main difference between them was the connection between the GIY-YIG and RING domains. This region formed a long \u03b1-helix in Cg-Slx1\u2013Slx4CCD3, whereas it lacked a secondary structure and was partly disordered in Tt-Slx1\u2013Slx4CCD3  . One for \u00c5 Table . The ove earlier . The strSlx4CCD3 . The strTt-Slx1\u2013Slx4CCD3 that were grown in the presence of splayed-arm substrate belonged to the P21 space group. These crystals diffracted to a resolution of 2.7 \u00c5. Phases were determined by molecular replacement using the apo structure as the search model. Clear electron densities were observed for DNA that was bound by the protein  and site II . In the new Tt-Slx1\u2013Slx4CCD3-DNA structure, the DNA was located away from the active site and occupied a markedly different position from the position that we predicted earlier. Thus, our Tt-Slx1\u2013Slx4CCD3-DNA structure revealed an additional and novel DNA-binding interface. We designated this newly identified DNA-binding surface as site III.We previously used structural similarities between Slx1 and GIY-YIG type II restriction endonucleases to predict the interface for catalytic DNA binding by Cg-Slx1 . The preapo and Tt-Slx1\u2013Slx4CCD3-DNA structures revealed intriguing structural differences. Two residues that formed important DNA contacts, R111 and R114, were located in a loop that could be visualized in the protein-DNA complex . Further studies will be necessary to confirm this potential mechanism. We also note that the region that comprised residues 97\u2013107 was still disordered in the DNA-bound structure. This region may become ordered when DNA binds to site I around the active site of the nuclease, potentially forming another safety mechanism.Comparisons of the x Figure . In the Tt-Slx1\u2013Slx4CCD3. The DNA molecules from GIY-YIG complexes were used to model nucleic acid binding at and around the active center and site I. This analysis revealed that the two interfaces did not overlap, and the two DNA duplexes were at an angle of \u223c50\u00b0  is related to the previously postulated interface around the Slx1 active site. We superimposed the catalytic structures of GIY-YIG type II restriction endonuclease on our new structure of DNA-bound \u00b0 Figure  and\u00a0B. S\u00b0 Figure .Our models explain the way in which Slx1 is able to accommodate various substrates and cleave them in multiple positions. The key feature of Slx1 is that it introduces cuts near the branch point. Our models suggest that this is achieved by bending the DNA substrate, such that Slx1 recognizes the branch point as a flexible discontinuity in the DNA. By interacting with two arms of the DNA substrate, Slx1 is able to bind and cleave various branched substrates.CCD. These experiments were performed using C. glabrata protein, for which substantial structural and biochemical information is available .Structure-selective endonucleases are thought to require tight regulation in cells because the unrestrained DNA cleavage of branched DNA structures would destabilize the genome. This regulation can be achieved by protein-protein interactions, post-translational modifications, and cell cycle-dependent activation, among others ,31. AddiCg-Slx1\u2013Slx4CCD3 and Tt-Slx1\u2013Slx4CCD3 did not reveal any features that could be related to the specific binding of particular DNA structures. We could only identify patches of positively charged residues binding DNA substrate in a specific orientation around the catalytic site to facilitate catalysis. Therefore, an intriguing issue is the way in which Slx1 interacts with various DNA substrates.Slx1 is quite different, in which it cleaves many diverse types of branched DNA structures and at different positions near the branch point ,20,21. IArabidopsis thaliana has been shown to bind branched DNA structures that represent homologous recombination intermediates. No nuclease activity has been observed for the GIY-YIG domain of Arabidopsis MSH1 (Our structural and biochemical work identified three positively charged patches on the surface of Slx1, designated sites I, II and III ( and pressis MSH1 , but thesis MSH1 .Cg-Slx1, which otherwise exists as an inactive homodimer (Slx1 is active only in the presence of Slx4. We previously reported the role of Slx4 in the activation of omodimer . Our stromodimer ,38.Slx1 is a very promiscuous endonuclease and needs to be tightly regulated. Our present and previous  work ideTt-Slx1E79Q-Slx4CCD3 alone and in complex with the DNA were deposited in the PDB under the accession codes 6SEH and 6SEI, respectively.The structures of gkz842_Supplemental_FileClick here for additional data file."}
{"text": "Retroperitoneal sarcomas (RPS) include a heterogeneous group of rare malignant tumours, and various treatment algorithms are still controversially discussed until today. The present study aimed to examine postoperative and long-term outcomes after resection of primary RPS.Clinicopathological data of patients who underwent resection of primary RPS between 2005 and 2015 were assessed, and predictors for overall survival (OS) and disease-free survival (DFS) were identified.P\u2009=\u20090.030) was significantly associated with diminished OS in univariate and multivariate analyses. When assessing DFS, histologic high grade , positive surgical resection margins , and vascular involvement , were significantly associated with inferior DFS in univariate and multivariate analyses.Sixty-one patients underwent resection for primary RPS. Postoperative morbidity and mortality rates were 31 and 3%, respectively. After a median follow-up time of 74\u2009months, 5-year OS and DFS rates were 58 and 34%, respectively. Histologic high grade (5-year OS: G1: 92% vs. G2: 54% vs. G3: 43%, High-grade tumours indicated poor OS, while vascular involvement, positive surgical resection margins, and histologic grade are the most important predictors of DFS. Although multimodal treatment strategies are progressively established, surgical resection remains the mainstay in the majority of patients with RPS, even in cases with vascular involvement. Soft tissue sarcomas (STS) are a heterogeneous group of rare malignant tumours that can occur in almost any anatomic region . WhereasThe retroperitoneum represents a sophisticated anatomical space with multiple vital structures, and therefore RPS is associated with several therapeutic challenges . EspeciaThe objective of this study was to review our recent experience with RPS and analyse postoperative and long-term oncological outcomes. Besides, we aimed to evaluate factors associated with overall survival (OS) and DFS in this cohort and thus identify patients who may derive the most benefit from a multimodal approach including radical surgery.Retrospective single-centre analysis conducted at the Charit\u00e9 \u2013 Universit\u00e4tsmedizin Berlin, Campus Virchow-Klinikum in Berlin, Germany. Following permission from the local institutional review board (EA1/361/14) clinicopathological data of patients who underwent resection for primary RPS between 2005 and 2015 were collected in a prospective database and further reviewed. We excluded recurrent RPS from this analysis, due to divergent management approaches and outcomes. An interdisciplinary tumour board indicated all resections and all patients obtained written informed consent.Standard preoperative clinical assessment included physical examination, serum laboratory testing, imaging studies, and an anaesthesia evaluation. Multiphase computed tomography (CT) with contrast agents or magnetic resonance imaging (MRI) were computed to define the dimension and location of the sarcoma and to assess the involvement of adherent structures. A multimodal therapeutic approach was individually formulated and scheduled by a multidisciplinary tumour board, which consisted of surgeons, medical oncologists, specialised radiation therapists, and radiologists, for every patient. Due to the high recurrence rate and aggressive nature of dedifferentiated liposarcoma, neoadjuvant chemotherapy with/without radiation therapy was individually considered for treatment, depending on the individual patient presentation. Therefore, preoperative tissue diagnosis (89%) of retroperitoneal liposarcoma included percutaneous biopsy (image-guided core needle biopsy (CNB) or fine-needle aspiration (FNA)) in order to facilitate an accurate subtype-specific consideration for neoadjuvant therapy.Perioperative antibiotics [Metronidazole 500\u2009mg (i.v.) and Cefuroxime 1\u2009g intravenously (i.v.)] were routinely given. All procedures were performed in an open surgical approach according to international standards at that time . A midliAll patients were administered and monitored at a specialised surgical intensive care unit for at least 1 day. The Clavien-Dindo classification was used to grade postoperative complications . ComplicP\u2009<\u20090.05 in univariate analysis. A P value below 0.05 was considered significant, and all statistical analyses were conducted using the SPSS software package, version 24.0 .Unless otherwise specified, qualitative and quantitative variables are constituted as medians (range) and numbers (frequencies). Postoperative morbidity, mortality, OS, and DFS were defined as primary outcomes. Survival analysis was determined using the Kaplan-Meier method, calculating OS from the date of resection to the date of death or the last follow-up. DFS contained the period from the date of resection to the date of first recurrence or last follow-up. Log-rank tests estimated the significance of univariate analyses. To identify factors associated with survival after resection of RPS, the following clinicopathological characteristics were recorded and analyzed: patient sex, BMI, patient age at resection, tobacco use, tumor entity, histologic grade, staging according to the AJCC 8th Ed., chemotherapy, radiotherapy, surgical resection margin, vascular involvement, ASA physical status, and need for intraoperative transfusion. Furthermore, a Cox multivariate regression model was executed, including all variables associated with survival with 2 (16\u201342), and 15 (25%) patients acknowledged consistent tobacco usage. Leiomyosarcoma and dedifferentiated liposarcoma had the highest incidence with 19 (31%) and 14 (23%) patients, respectively. Twelve patients (20%) suffered from well-differentiated liposarcoma, while undifferentiated sarcoma, not otherwise specified, were identified in 13% of all resected tumours. Pleomorphic liposarcoma, malignant peripheral nerve sheath tumours, were detected in 5% of patients, respectively. Histologic grading showed low-grade tumour tissue in 21 (34%) patients, while intermediate grades and high grades were verified in 11 (18%) and 29 (48%) cases, respectively. In terms of the AJCC 8th Ed., the majority of patients (41%) were classified to be Stage IIIB, while Stage IA, Stage IB, Stage II, Stage IIIA and Stage IV were discovered in 3, 28, 7, 13, and 8%, respectively. Forty-nine patients (80%) received no chemotherapy, while ten patients (16%) obtained adjuvant chemotherapy. Two patients (3%) received neoadjuvant chemotherapy. Based on the recommendations of an interdisciplinary tumour board, 44 patients (72%) did not require additional radiotherapy. Four patients (7%) received neoadjuvant radiotherapy, while 13 patients (21%) received adjuvant radiotherapy. In 33 patients (54%) complete tumour removal was achieved. The histological examination discovered persistent tumour cells on the surgical resection margin in 18 patients (30%). In two patients (3%) only an R2 resection could be achieved, while in 8 patients (13%) histological examination did not state sufficient information whether complete tumour resection with surgical margins microscopically negative for tumour cells was successful. Seventeen patients (28%) revealed vascular involvement. In this context, we planned vascular resection in 12 cases already preoperatively. In five cases, preoperative imaging studies underestimated the dimension of RPS and diagnosis of vascular involvement, appeared intraoperatively. The vena cava inferior was the most common vessel involved. In eight cases, segmental resection required a reconstruction by PTFE prosthesis. Within the remaining four cases, direct suture or patch plastic occluded the area of resection. Five patients revealed infiltration of the right renal vein. In all cases, nephrectomy facilitated radical tumour removal. The local recurrence rate of RPS was 41%. ASA physical status included ASA I in 20% of patients, ASA II in 52% of patients, ASA III in 23% of patients, and ASA IV in 5% of patients. In 32 patients (52%) there was no necessity for intraoperative red blood cell concentrate (RBCC) transfusion during the surgical procedure.Between 2005 and 2015, 61 patients with primary RPS underwent open resection at our institution. Table\u00a0Thirteen patients (21%) required up to two RBCCs during the operation, while 26% of patients demanded more than three RBCCs. Postoperative morbidity, according to the Clavien-Dindo classification \u22653, was 31%, and the 90-day mortality rate was 3%.After a median follow-up time of 74\u2009months 2\u2013131), the median survival of patients who underwent resection for RPS was 38\u2009months. The 5-year OS rate was 58% , and histologic tumour grade (P\u2009=\u20090.015). In multivariate analysis, only histologic tumor grade  independently associated OS (Table Table\u00a0P\u2009=\u20090.006), histologic tumor grade (P\u2009=\u20090.018), classification according to the AJCC 8th Ed. (P\u2009=\u20090.004), surgical resection margin (P\u2009=\u20090.047) and vascular involvement (P\u2009=\u20090.044). In multivariate analysis, histologic grade , positive surgical resection margins  and vascular involvement , were independently associated with DFS (Table P\u2009=\u20090.044)  program of patients undergoing resection for primary RPS from 1988 to 2005 and reported similar patient characteristics to those reported in our study . GiulianP\u2009=\u20090.015) independently influenced OS. Equivalent to previous findings it might reflect the more advanced nature of tumours with high-grade transformation [Tumour entity and histologic tumour grade of RPS significantly impact on OS in our univariate analysis. However, in multivariate analysis only histologic tumour grade  compared to survival in patients having biopsy only (17%) . It seemTo date, data regarding vascular involvement and oncological outcomes after resection of RPS is still limited. Previous studies established the feasibility and safety of en-bloc vascular resection for RPS and recommended vascular resection and reconstruction to achieve radical tumour removal when needed , 31. HowChemotherapy and radiotherapy did not demonstrate a significant impact on survival. While neoadjuvant therapy is an established treatment for a variety of other tumour entities, evidence on preoperative treatment for RPS is still undetermined, and high-quality trials are needed , 34. TheThe present study is limited by common biases that are mainly due to the retrospective character of this analysis. Furthermore, treatment regimens are still rather heterogeneous. While extensive surgery remains the mainstay of treatment in RPS, conflicting data on the benefit of neoadjuvant and adjuvant therapies exist, and thus interdisciplinary treatment plans are mainly based on individual preference and expertise of the treating institution .In conclusion, in our analysis of 61 patients, we found that patient characteristics of RPS is similar to those of other extensive studies, with liposarcoma and leiomyosarcoma being the most common histologies. High-grade tumours indicated poor OS, while histologic grade, positive resection margins, and vascular involvement are the most important predictors of DFS. Although preoperative and postoperative radiotherapy and chemotherapy did not significantly affect survival, procedures tailored to the individual needs of our patients are the current advancement of choice. While effective adjuvant treatment regimens are continuously developed, surgical resection, even in cases with vascular involvement should be individually attempted in selected patients with RPS. Furthermore, international collaborations are mandatory in order to enhance the management of RPS and guide clinicians in their daily decision-making."}
{"text": "Pharmaceuticals and personal care products (PPCPs) are abundantly used by people, and some of them are excreted unaltered or as metabolites through urine, with the sewage being the most important source to their release to the environment. These compounds are in almost all types of water  at concentrations ranging from ng/L to \u00b5g/L. The isolation and concentration of the PPCPs from water achieves the appropriate sensitivity. This step is mostly based on solid-phase extraction (SPE) but also includes other approaches (dispersive liquid-liquid microextraction (DLLME), buckypaper, SPE using multicartridges, etc.). In this review article, we aim to discuss the procedures employed to extract PPCPs from any type of water sample prior to their determination via an instrumental analytical technique. Furthermore, we put forward not only the merits of the different methods available but also a number of inconsistencies, divergences, weaknesses and disadvantages of the procedures found in literature, as well as the systems proposed to overcome them and to improve the methodology. Environmental applications of the developed techniques are also discussed. The pressing need for new analytical innovations, emerging trends and future prospects was also considered. The production and consumption of pharmaceutical and personal care products (PPCPs) is considered an important environmental risk ,2,3,4. IThese considerations on the sources of PPCPs shows the key role that the analysis of water plays to fight against contamination. In fact, water\u2014the most affected environmental compartment\u2014may be considered as a mirror of the pollution status of an area, and also a scarce resource that must be preserved with optimal quality and zero pollution. In addition, water contaminants, depending on their physicochemical properties, may also (bio)accumulate in sediments and biota, consequently harming human health . TherefoDetermining and quantifying PPCPs in different types of water provides considerable interesting information not only related to pollution status. For example, the analysis of wastewater samples, divided into influent and effluent waters, could offer information on the PPCPs consumption of a community, estimate the wastewater treatment plant efficiency and establish the most recalcitrant compounds difficult to eliminate. River, lake, and seawater samples could give us an idea of the more persistence substances. The detection in irrigation channels could identify food quality issues. In addition, drinking water is certainly another matrix that should be monitored to assess the potential risks on human and animal health for long-term use ,19.For an accurate analysis of PPCPs in different types of water, it is fundamental to consider all water characteristics that could influence recovery of the contaminants. The pH could affect the structure of molecule, promote ionization according to pKa, or activate a prodrug with a change of structure. Many substances were thermolabile and photosensitive, for this reason, sample temperature must always be considered. The salinity of water could increase or decrease extraction efficiency due to different ionic strengths of the media, or the formation of molecular complexes between PPCPs and multivalent metal cations present in the samples that are soluble in water . Other wIn addition to the matrix, it is also important to consider the structural variability of PPCPs, designed to interact with specific targets. The presence of distinct functional groups  or the existence of nucleophile/electrophile substituents contribute to all chemical-physical characteristics of each active substance. Influencing stability, reactivity, and solubility in water are all parameters that need to be considered before a sample\u2019s preparation for analysis. Despite the variability in the PPCPs\u2019 chemical structures, for most laboratories specialized in the analysis of these compounds, the use of multiresidue methods is very attractive because not only attains a reduction of cost and time, but also offers global patterns of contamination with only one analysis. Moreover, these methods easily facilitate an eco-friendly analysis with decreases in waste. In these multiresidue methods, the sample preparation becomes the heart of analysis that influences any other procedural steps from sample collection and storage to the specific instruments selected for final quantification (high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), etc.). The choice of a liquid or gas chromatography (LC or GC), to analyze the final extract, is guided by the analytes\u2019 polarity. Generally, compounds with polar characteristics are more suitable for LC, and those with non-polar properties are more amenable to GC; most PPCPs are polar or moderately polar . SurprisTherefore, the goal of this review was to critically analyze the status of sample preparation to determine PPCPs in an all-water matrix. Each step of sample preparation, including all analytical variants  used to detect these contaminants were considered. Furthermore, each sample preparation method was critically analyzed, highlighting advantages and disadvantages. This review performs an examination of all studies published from January 2018 to May 2020. The search was conducted on the database Scopus (Elsevier), with two different inputs: \u201cextraction pharmaceutical environmental\u201d/\u201cextraction personal care products environmental\u201d and \u201cextraction pharmaceutical water\u201d/\u201cextraction personal care products water\u201d. More than one thousand one hundred works have been viewed. The selection criteria to choose the studies were based on (i) the presence at least of 10 PPCPs to include attractive multiresidue methods; and (ii) water compartmentation, in all variants , was chosen. In addition, some reviews outside the interval of time were also chosen.PPCPs are organic compounds, and traditionally this type of compound has been extracted by solid-phase extraction (SPE). This technique was commercialized in the late 1970s and rapidly replaced the liquid-liquid extraction that was previously used . Table 1Only 16% of the studies use other types of methods, such as direct injection, dispersive liquid-liquid extraction (DLLME) (based on liquid-liquid extraction), polyether sulfone microextraction (PES) or buckypaper devices. It is important to note here that many of the methods classified as \u201cother\u201d are based on the basic principles of SPE, but using new phases or formats.This technique involved the use of a small amount of sorbent (commonly hundreds of mg) in a cartridge or syringe barrel. After activation of the sorbent, a water sample of hundreds of mL was passed through the sorbent, which retained the analytes of interest (in this case PPCPs) whereas the water was discarded. Then, the analytes retained in the sorbent were eluted using a few mL of organic solvent. This technique has some advantages, such as the minor investment in reagent and materials, and rapidity.\u00ae HLB, Strata\u00aeX and Cleanert\u00ae PEP). Another polymeric sorbent applied was marked by the presence of polystyrene-divinylbenzene  that was applied to polar analytes, or the presence of octadecyl endcapped silica RP (Supelclean\u2122 LC-18 SPE) that was used for nonpolar to moderately polar analytes from aqueous samples.There are different marketed sorbents that work principally with two distinct separation mechanisms: classical reversed phase chromatography (RP) or ion exchange chromatography (IC). Characteristics of these sorbents are summarized in \u00ae MCX, Strata\u00aeX-CW or X-Drug B and UCT XTRACT\u00ae XRDAH) were the most prevalent.The IC sorbents were used in the so-called mixed-mode cartridges that combine the polymeric sorbent with an ionic exchanger that could be weak or strong. The IC can be of cations or anions, and this affects the target specificity. Cationic exchange sorbents (weak or strong) were designed to extract basic PPCPs, and anionic exchange sorbent (weak or strong) were to extract the acidic ones. However, weak polymeric cationic-exchangers (OasisN-vinyl pyrrolidone-co-divinylbenzene) (NVP-co-DVB) that improved the average absolute recovery for 44 PPCPs, with respect to the use of HLB sorbents. Alternatively, Caban et al. [Different studies applied attractive modifications to these traditional cartridges, to obtain the best recoveries for a large spectrum of compounds. Zhu et al.  developen et al.  studied n et al.  mixed fon et al.  combinedThe format of the cartridge and the volume of samples that pass through the cartridge were other elements to take into consideration. The sorbent weight (mg), capacity (mL) and pore size (\u00b5m) can influence the efficiency of the columns. These parameters have a key role on the surface area, on which analytes interacted. The amount of sorbent ranged from 60 to 600 mg, and the most used was a quantity of 200 mg. The capacities most used were 3 and 6 mL. There is a global consensus on this point.In SPE, the volume of water processed generally involves hundreds of milliliters. The capacity to detect lower amounts is one of the advantages of SPE. The matrix effect (ME) could be an element to take in consideration in the choice of volume because it is directly proportional to volume as well as the organic matter content of the water; in this case, clogging of the cartridge slows the process too much. For this reason, some studies chose different volumes of water (depending on its characteristics) with the same method. For example, influent wastewater samples were generally analyzed with smaller volumes compared to effluent samples ,48.In SPE, the first step is the conditioning of the sorbent in order to favor the interaction of the sorbent with the analytes by a mechanism namely \u201csolvation\u201d. RF and IC sorbents are usually activated by filling the column two or three times with a solvent miscible with water  followed by the solvent in which the analyte is dissolved . Methanol and water are most frequent solvents to activate cartridges. Water used for activation can be pH-adjusted or spiked with salt, counter ions or metal sequestrators . The wash was followed by cartridges air-drying by vacuum or pressure to remove the remaining water, although in few cases, the cartridges can be dried with nitrogen instead of air [Once the sample passed through the sorbent, another important step is to remove the impurities retained on the SPE packing. For this reason, a wash solution strong enough to remove these impurities, but weak enough to leave the analytes of interest, is passed through the cartridge. These solutions are water, pH-adjusted water, or in a few cases methanol-water , PTFE, RC, GHP, PP, etc. The size used were 0.20 and 0.22 \u03bcm. This procedure was an ulterior clean-up to remove the particles with a large size to optimize analysis, for example by reducing the presence of obstruction phenomena in column. For this step, it was fundamental to remember which combinations (solvent-filter) are safe and which are corrosive.Regarding the characteristics of the water sample, the preservation of its integrity from the sampling point to the moment of the analysis was vital. To this end, in many cases, the water samples were spiked by preservative compounds or solutions. As described in the U.S. Library of Medicine (2017), a pharmaceutical preservative is referred to as \u201csubstances added to pharmaceutical preparations to protect them from chemical change or microbial action\u201d . In the To ensure the proper quantification of the analytes is always very important. Therefore, samples were spiked by a solution of internal standard (IS) in more than 83% of the studies to obtain more reliable results, taking matrix effects into consideration. It was not always possible to use an isotopic reference for every compound, because the cost of the ISs are high, and are not available for some of the target analytes .Another pretreatment widely used in water sample preparation was filtration, the role of which was to remove suspended substances, such as suspended particles, colloids and microorganisms from samples to prevent obstruction of the SPE cartridges or significant interferences in subsequent treatment processes . In variSPE can be used offline  or online (directly connected to the chromatographic system). However, there are few difference in the components of the techniques between the two formats, with the exception of the valves system used to connect SPE online with the determination technique (commonly any type of HPLC-MS). In both, the main factors that affected the results of these technique were the formats and sorbents of stationary phase (cartridge) and the solvent(s) used for activation, washing and elution. Online SPE can be coupled to both, LC or GC. However, the preferred technique is online SPE liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS), PPCPs and the SPE eluents are much more compatible with the mobile phase of LC than with that of GC.To be functional, online systems require the use of 6 or 10 port valves to automate extraction and connection to the instrument. A schematic illustration of the analytical system is presented in The columns for online-SPE mode were characterized by similar polymeric RP sorbents. The main differences, with offline-SPE, were the length of columns . OASIS HLB  ,50,78, MThe online SPE systems present the advantage to provide automatic and efficient sample loading, clean-up, desorption, separation, and detection at the same time, to reduce the sample volume, save time and solvents, prevent sample contamination and PPCP loss, and improve the method performance. The reduction in sample volume that could decrease sensitivity is normally compensated for by the increase in sensitivity as all the analyte retained in the sorbent passes to the chromatographic column.The disadvantage of these methods is that they are not very versatile to be adapted to different types and conditions of analysis, because sample pH, injection volume, and valve-switching time needs to be very carefully optimized to ensure appropriate method performance for target PPCPs . Once esThe disks are a variant of cartridges that follow the same principle to retain an elute PPCPs. The disks have a higher diameter (commonly ca. 45 mm) and low height (a few millimeters). This format attempts to address several disadvantages of the cartridges, such as plugging due to the suspended particulate matter, high back-pressure that reduces flow rates, and improve retention kinetics of the analytes by using lower particle size. Generally, it was used with higher volumes of samples than SPE. The passage of the sample was much quicker (up to 100 mL/min). Moreover, the use of disks was advantageous for handling dirty samples. Only four studies used this approach . There aHydrophilic divinylbenzene (DVB) disks have been compared with Oasis HLB cartridges. Although the most apolar analytes (LogP \u2265 4) attained higher process efficiencies following Speedisk extraction, it could be noticed that in general, process efficiency was lower than for Oasis HLB extraction: 16 versus 59 analytes having a process efficiency >60% for Speedisk and Oasis HLB, respectively . HoweverThe combination of different disks in order to improve recoveries has also been tested for GC-MS amenable analytes using, in sequence, a glass microfiber disk , a styrene-divinylbenzene disk , and an active carbon disk  . HoweverThe main disadvantages, as evidenced in the studies, are related with highest waste of samples and reagents used to active, wash and elute the sorbent.Other approaches have been reported to extract the PPCPs from water, and even though they are not as used as SPE, can be advantageous for some applications. These approaches are commonly focused on more environmentally friendly alternatives that reduce the use of materials, organic solvents and reagents; the so-called green chemistry. The simplest process is direct injection without any pre-concentration steps ,83. ThisThe microextractions, both solid and liquid, are an attractive alternative to SPE. The Dispersive Liquid-liquid Microextraction (DLLME) ,47 has bOther approaches are based on testing new phases consisted of nanomaterials as more efficient sorbents. Tomai et al.  performeThe last approach was the use of a PASSIL sampler . The acrSeventy-six studies have been selected . Nine st\u22121), TDS , DO , TSS  and BOD . Influent and effluent wastewater samples from hospitals or wastewater treatment plants (WWTPs) were the most analyzed (46% of studies). This matrix was marked by complexity due to the presence of numerous interferents. Some studies investigated the presence of PPCPs in raw wastewater at the treatment plant, some just the effluents, and some investigated both [Different types of water were collected. The principal parameters monitored were temperature, pH, EC . The 40% of works analyzed and studied in this group investigated a large spectrum of water sources: streams, rivers, estuaries, lakes, seas, ground water, and urban and agricultural storm waters. Drinking and tap water (DW) constituted the third group, with an occurrence of 12% in the studies selected. DW was regulated by \u201cThe Drinking Water Directive 98/83/EC\u201d that supervises the quality of water (for human consumption). It provided a general framework and a minimum value of 48 specific parameters that must be monitored regularly .Lastly, two works included other two aqueous matrices: thermal and swimming pool water (SP). Chemicals in these matrices can come from different sources, such as bathers, who continuously release organic matter mainly through sweat and urine .The review article focused on the extraction methods for PPCPs. Although these extraction methods are clearly dominated by offline solid-phase extraction using cartridges, there are significant knowledge gaps in accurately understanding the extraction mechanisms for PPCPs, including some metabolite and/or degradation products. Many of the most recent and innovative methods are based on the combination of sorbents with different chemical-physical properties either in the same cartridge, in parallel, or even in series. These modifications are considered to be small steps, but nevertheless, they represent a great advance by improving the extraction of a group of compounds with very different polarities. Multiresidue methods able to cover more than 100 compounds are already a reality. Rapid, lower cost, and eco-friendly sample preparation techniques are urgently needed. Automated, online preconcentration, and clean-up steps prior to the instrument analysis would be the future of the technique. Solvent-free microextraction methods are the trend of extractions such as DLLME. However, these techniques are rarely used, as they work especially well with non-polar PPCPs, but most are polar.Most of the environmental studies carried out so far have two aspects, (i) analytical including validation of the methods that mainly improves the accuracy in the quantification and the elimination of the matrix effects, and (ii) the environmental aspect in which the whole cycle of the water is covered, including the identification of the sources of these compounds to the environment, the efficiency in the elimination, and the influence of environmental factors such as seasonality. All these studies have contributed to an important advance of knowledge about the distribution and hazards of PPCPs. A gap detected in these studies is the lack of knowledge about the mixtures of PPCPs found in the environment and on the different metabolites and/or degradation products that can be present. It is expected that, in the near future, there will be an increase in knowledge in these fields."}
{"text": "Drosophila suzukii Matsumura has become one of the most important invasive agricultural pest insects across climate zones in much of Asia, Europe, North America, and South America. Populations of D. suzukii have demonstrated notable behavioral and physiological plasticity, adapting to diverse environmental and climatic conditions, interspecific competition, novel food sources, and potential predators. This adaptability and plasticity have enabled rapid range expansion and diversified niche use by D. suzukii, making it a species particularly suited to changing habitats and conditions. This article reviews factors and evidence that influence plasticity in D. suzukii and promotes this species\u2019 invasiveness.After its initial discovery in California in 2008,  Plasticity is a driving force behind the spread of numerous invasive species. Plasticity refers to the degree to which traits of individuals or populations can rapidly adapt to new or changing environmental conditions . AdaptatResistance to or tolerance of stressful conditions and a short generation time increase the risk of unintentional transportation and facilitate introduction . Newly iGrapholita molesta (Busck) (Lepidoptera: Tortricidae), which is able to use many apple varieties as oviposition substrates, or the ability to outcompete local species, as in the Asian ladybird, Harmonia axyridis  (Coleoptera: Coccinellidae), which develop more quickly than the North American native ladybird, Adalia bipunctata L. (Coleoptera: Coccinellidae) , available diet, or pressure from predators or competitors . This plellidae) .Drosophila suzukii Matsumura was first described in Japan , acclimation (long-term exposure in a laboratory setting), or acclimatization   . Adult Ds \u22127.5\u00b0C . Acclimas \u22127.5\u00b0C . Acclimas \u22127.5\u00b0C . Thus, es \u22127.5\u00b0C . Five dae levels .D. suzukii to survive temperatures below 0\u00b0C for longer periods, withstand chill coma symptoms at lower temperatures, and recover from cold exposure more quickly  to volatiles produced by ripening fruit, there is evidence to suggest D. suzukii also uses differences in leaf tissue volatiles produced during fruit development as a supplemental means to locate potential feeding and oviposition sites  had no oviposition preference as adults between blackberry and American pokeweed , flies reared on American pokeweed preferred to oviposit on blackberry , skin toughness, and acidity (pH) have been investigated most commonly (Drosophila suzukii are able to use a wide variety of host fruits by selecting for characteristics which signal health and ripeness of host fruits and a lack of potential competitors, pathogens, or predators. Other plant characters, such as leaf odors, leaf color, and overall health of plant, have received less attention, but are important supplemental cues used to locate host fruits of healthy plants (Characters associated fruit species have been correlated with suitability for feeding and oviposition behavior of commonly . Other fcommonly . Drosophy plants . Foraginy plants .D. suzukii larvae with critical nutrients, including protein, vitamins, and minerals (Drosophila species, yeast quality affects development, fecundity, and lifespan of D. suzukii (Drosophila species, D. suzukii larvae develop most successfully on foods containing lower ratios of protein to carbohydrates, developing more quickly into larger adults with greater potential fecundity (Larval development time varies depending on larval diet. Larvae feeing on fruits such as cherry, blueberry, or raspberry, develop more quickly than those that feed on standard diet media . Larvae minerals . As with suzukii . Howeverecundity .D. suzukii have been documented feeding on floral nectar, tree sap, and honeydew when other food sources were unavailable (D. suzukii will also oviposit and larvae can successfully develop on less ideal materials, including mushrooms and chicken manure (D. suzukii to novel dietary choices, for both the adult and larval stages, contributes to its invasion success and interact with other aspects of its plasticity.In temperate regions, fruit can be of limited availability or quality as a food source for adult flies and as an oviposition site. In laboratory studies, adult vailable . In the n manure . Thus, tD. suzukii and in behavior of larvae. Chemical cues or signs of previous oviposition by heterospecifics, such as D. melanogaster, can deter female D. suzukii from ovipositing in the same fruit (D. melanogaster sex pheromone, cis-vaccenyl acetate, transferred to fruit during oviposition. In direct interspecific competition situations, D. melanogaster larvae have greater survival than D. suzukii larvae (D. melanogaster larvae than fruit containing D. suzukii larvae (D. suzukii females prefer to oviposit in ripe fruit, they are able to shift ecological niches and use ripening fruit to avoid competitive pressures and reduce potential ethanol exposure to their larvae.Interspecific and intraspecific competition can induce changes in oviposition behavior of female adult me fruit . This mai larvae . This isi larvae . AlthougD. suzukii were not deterred from ovipositing in fruit containing eggs from other D. suzukii females (D. suzukii larvae are less likely to remain within their original host fruits throughout their development. In the absence of competition, larvae remain on or in host fruits throughout pupation. However, as the number of intraspecific competitors within a fruit host increases, larvae move greater distances away from the fruit to pupate (In contrast, female  females . In respo pupate .D. suzukii select fruit for oviposition that contain atropine compounds to infer prophylactic protection to the next generation (D. melanogaster\u2019s use of ethanol laden oviposition sites following detection of parasites and parasitoids (In the presence of parasitoid populations, neration . This isasitoids .Success of an introduced species in a novel environment depends primarily on its ability to adapt and explore its surroundings . SpeciesD. suzukii makes it easily overlooked or misidentified in field settings. Without close inspection, it is easily mistaken for native Drosophilids in each of its invasive regions. Although no voucher specimens exist in mainland American entomological collections prior 2008, it is highly likely that D. suzukii was introduced to North America or Europe long before its first recognized detection in California, but it was not present in sufficient numbers to cause significant crop damage or invite notice (D. suzukii invasion (The relatively small size and inconspicuous nature of e notice . Effectsinvasion .Drosophila suzukii has demonstrated a notable ability to adapt behaviorally, physiologically, and morphologically to new environments. Although each of these responses is of themselves modest in scope, the nature of its adaptive responses is arguably more important to its success as an invasive species than is the extent of any one variable response (Drosophila suzukii has shown itself to be highly opportunistic and it has been able to adapt to a wide range of host plant fruit for rearing its larvae and for adult feeding. As a result of its willingness to explore and test novel fruit species, it has become extremely polyphagous. In addition, D. suzukii has adapted to a wide range of temperature and humidity conditions, through its behavior and through physiological, developmental, and morphological plasticity. As D. suzukii has expanded its range into temperate regions, its ability to adapt to changing environmental conditions and then reverse those adaptations as conditions change through successive seasons as been vital to its success. Plasticity within multiple aspects of behavior, physiology, and morphology has allowed D. suzukii to move from a localized introduced species to an established invasive species over a global range.response . Drosophieaa034_suppl_Supplementary_TablesClick here for additional data file."}
{"text": "Drosophila suzukii, and the anthracnose pathogen Colletotrichum fioriniae are an important insect pest and fungal disease of highbush blueberries, respectively, in the United States. However, whether C. fioriniae infection affects D. suzukii preference and performance remains unknown. Here, we conducted choice and no-choice studies to determine the repellent, oviposition-deterrent, and insecticidal effects of C. fioriniae on D. suzukii. In choice tests, blueberry fruit treated with anthracnose solutions containing spores from either field-collected infected fruit (\u2018fruit\u2019) or a laboratory C. fioriniae culture (\u2018colony\u2019) were less attractive to sexually mature D. suzukii females, but not males, than untreated fruit. The plant tissue (fruit or leaves) did not influence C. fioriniae repellency effects on D. suzukii. In no-choice tests, 55% fewer numbers of eggs were laid on, and 65% fewer adults emerged from, blueberry fruit treated with either the \u2018fruit\u2019 or \u2018colony\u2019 anthracnose solution than untreated fruit. Egg-to-adult D. suzukii survival was also 12% lower on C. fioriniae-infected fruit. No repellency or negative effects on survival were observed when C. fioriniae spores were filtered out of the solution. These findings will help efforts towards the discovery of microbial-derived repellent/oviposition-deterrent compounds that could be used in behavior-based management strategies for D. suzukii.Spotted-wing drosophila,  Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), an insect native to Southeast Asia, has become a major pest of soft- and thin-skinned fruit crops, including raspberries, strawberries, blueberries, blackberries, and cherries1. This frugivorous pest has rapidly expanded its geographic range across multiple continents, which now includes many Asian, North and South American, and European countries4. In the United States, this insect was first detected in the Western states  in 2008 and quickly spread to other states; by 2011, it was found in most Northeastern states3. Several behavioral and morphological features in D. suzukii, such as its attraction to odors from ripening fruit6 and an enlarged, serrated ovipositor, enable gravid females to be attracted to, and oviposit on, fresh fruit in addition to overripe fruit7. These features, among others, have contributed to its worldwide pest status compared with most Drosophila species that prefer rotten fruits and are, thus, not considered pests.Spotted-wing drosophila, 8, understanding complex plant-pathogen-insect interactions could help in the discovery of novel pest management strategies in agroecosystems. Drosophila spp., including D. suzukii, have associations with microbes  that can be mutualistic (where both species benefit)9, commensal (where only one of the species benefits)10, or antagonistic (where one species has a negative effect on the other)11. For example, D. suzukii has a strong relationship with mutualistic yeasts that improve larval survival and development and increase oviposition on fruits12. One example is Hanseniaspora uvarum, the most abundant yeast present in the alimentary canal of wild D. suzukii populations13. This may partially explain the greater attraction of D. suzukii to H. uvarum odors than to odors from other yeasts9; this yeast when present on fruits also increases feeding by gravid females14. Additionally, Drosophila suzukii can facilitate infection by pathogens that often results in fruit rotting, reduction of yield, and loss of marketability15. Indeed, a variety of yeasts and bacteria that cause sour rot in grapes gain access to the fruit\u2019s carbohydrate-rich mesocarp through the feeding and oviposition activities of Drosophila spp.16. However, fruit infection by fungal pathogens can also negatively affect Drosophila spp. For example, D. suzukii larval survival and adult size were significantly reduced when they were reared on raspberries infected by Botrytis cinerea Pers., and fruit infection by this fungus also reduced fly attraction and oviposition11. Therefore, odors from microbes associated with D. suzukii could serve as sources of attractive and repellent compounds to manipulate the fly\u2019s behavior in agroecosystems18.Because plant\u2013insect and plant-pathogen interactions share many features including adaptations and counter-adaptions and negative effects of invasive species on humansVaccinium corymbosum L.) agroecosystem, anthracnose fruit rot is an important disease mainly caused by the fungal pathogen Colletotrichum fioriniae (Sordariomycetes: Glomerellaceae)20, which can result in pre- and postharvest fruit losses23. The primary anthracnose symptoms are rotting of ripe fruit, both in the field before harvest and during storage after harvest, and the appearance of distinctive salmon-colored  droplets on the surface of rotting fruit24. If left untreated during bloom and if favorable  conditions persist, C. fioriniae infestation can result in significant crop losses26. Colletotrichum spp. infection is thought to speed the conversion of acids to sugars as a defense response in blueberries27 because there is a positive correlation between sugar concentration in fruit and fruit rot resistance28. This \u201coverripe\u201d effect of the pathogen on blueberry fruit could stimulate avian and mammalian herbivory31 and also have a positive effect on D. suzukii by increasing adult survival, longevity, and oviposition34. In fact, D. suzukii adults have a strong bias towards carbohydrate\u2010rich foods35. However, the Chinese chestnut anthracnose pathogen can cause high mortality of the Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu36, indicating that interactions with anthracnose might not be beneficial to insects.In the highbush blueberry . Alternatively, if C. fioriniae competes with D. suzukii for resources, we hypothesized that females are repelled by pathogen-fruit-associated odors and avoid ovipositing on infected fruits (hypothesis 2). To address this, we conducted choice and no-choice experiments to investigate the effects of C. fioriniae-infected fruit on D. suzukii attraction, oviposition, and immature performance. Specifically, we asked the following: (i) Are D. suzukii female and male responses to an attractive odor (blueberry fruit) affected by odors emitted from anthracnose (C. fioriniae)? (ii) Do anthracnose solutions from either infected fruit or a laboratory-grown C. fioriniae culture differentially affect D. suzukii adult attraction, oviposition preference, and offspring performance? (iii) Is the effect of C. fioriniae odors on D. suzukii influenced by the plant tissue it infects (fruit or leaf)? In other words, is the effect tissue-specific? (iv) Is there a relationship between D. suzukii adult preference and offspring performance on C. fioriniae-infested fruit? An understanding of this blueberry-C. fioriniae-D. suzukii association constitutes the first step towards the development of behavioral control strategies for D. suzukii based on odors from plant pathogens.In this study, we tested two opposing hypotheses. First, given that D. suzukii adults  were likely driven mainly by chemical rather than visual cues. Symptoms of fruit infection were seen after 7.23\u2009\u00b1\u20090.28 and 7.05\u2009\u00b1\u20090.26\u00a0days in the 'fruit\u2019 and \u2018colony' treatments ; no symptoms were observed in the 'filtered' and 'control' treatments.No symptoms, such as the distinct salmon-colored droplets on the surface of fruits, were observed prior to experiments; thus, any behavioral effects of anthracnose on D. suzukii females and males to anthracnose-infected blueberry fruit odors. When given a choice between blueberry fruit  versus an empty vial (without fruit), 100% of the responding D. suzukii females and males were attracted to the fruit odors   versus untreated blueberry fruit, sexually mature D. suzukii females were 3 times more attracted to the untreated fruit than fruit treated with \u2018fruit\u2019 and \u2018colony\u2019 solutions . In contrast, no differences in D. suzukii female attraction were found when they were given a choice between fruits treated with a \u2018filtered\u2019 C. fioriniae solution (without conidia) and untreated fruit  , \u2018fruit\u2019 , or \u2018filtered\u2019  solutions compared with the untreated fruit  plus leaves (nonfood tissue) treated with C. fioriniae and fruit plus untreated leaves. Drosophila suzukii females were 2 times more attracted to the fruits with the untreated leaves than to fruits with the leaves treated with C. fioriniae      C. fioriniae odors reduce the attraction of sexually mature D. suzukii females  to attractive blueberry fruit odors, showing that these females are repelled by odors from anthracnose-infected fruit; (ii) the \u2018fruit\u2019 and \u2018colony\u2019 anthracnose solutions had similar repellent, oviposition-deterrent, and insecticidal activity on D. suzukii females, indicating that the fungal pathogen C. fioriniae is mainly responsible for these effects; (iii) removing the fungal spores (conidia) from the anthracnose solution (\u2018filtered\u2019 solution) eliminated the repellent effects of C. fioriniae on female D. suzukii; and (iv) the effect of C. fioriniae on D. suzukii was independent of the plant tissue (fruit or leaf).The current study documents four important features of the interactions between the invasive insect pest D. suzukii females were less attracted to odors from blueberry fruits when treated with the anthracnose solutions (\u2018fruit\u2019 and colony\u2019). There are three possible, not mutually exclusive, mechanisms for these inhibitory effects. First, volatiles emitted from C. fioriniae could directly be responsible for the observed repellency effects. In fact, fungal odors such as geosmin and 1-octen-3-ol38 have been shown to negatively affect D. suzukii host-seeking and acceptance behaviors. Using video observations, Wallingford and collaborators38 found that 1-octen-3-ol prevents D. suzukii flies from coming into contact with the fruit , whereas geosmin inhibits egg-laying behaviors . Second, anthracnose infection could indirectly induce changes in volatile emissions from blueberry fruit, making them less attractive to D. suzukii. Many pathogens successfully exploit their host plants by inducing chemical changes to avoid, circumvent, or silence the hosts\u2019 detection and/or defense systems41. These pathogen-induced chemical changes could affect volatile emissions from the host plant43. Although it needs verification, the fact that we observed repellent effects of C. fioriniae on D. suzukii females regardless of the plant tissue (fruit or leaf) suggests that these effects might not be due to changes in fruit odors but to odors emitted from C. fioriniae itself. Third, C. fioriniae odors could camouflage the blueberry odors, i.e., masking effect, thus preventing D. suzukii flies from detecting  and/or responding to these otherwise attractive odors44. Although testing these mechanisms was beyond the scope of this study, work is underway to isolate and identify the behaviorally active compounds from C. fioriniae-infected blueberry fruit on D. suzukii. Despite that both D. suzukii males and sexually mature females were attracted to noninfected fruit odors, only females were repelled by C. fioriniae odors, indicating that these odors provide information to host-seeking females on the suitability of oviposition sites. Although not tested in this study, we expect these fungal odors to affect (repel) mated females more strongly than virgin females, since the former likely utilize fruit odors for oviposition17. According to Revadi et al.45, D. suzukii females start producing offspring 2.5\u00a0days after emergence when kept together with males of the same age. We kept female and male flies together for at least 3\u00a0days prior to bioassays, which means that most females were likely ready to start egg laying. Future studies are needed to investigate whether physiological status  of D. suzukii females influences their response to anthracnose odors.Sexually mature C. fioriniae on D. suzukii females, about 20% of the tested flies were not affected by the fungal odors. In our studies, two variables that were constant included the time of anthracnose incubation and its concentration. We used a C. fioriniae incubation period of 48\u00a0h because this time of exposure was previously used to test the susceptibility of blueberry species (Vaccinium spp.) to anthracnose46. Previous studies have also shown that fruit rot pathogens, Colletotrichum spp., can successfully infect blueberry fruit after only 10 to 12\u00a0h postinoculation47 and induce chemical changes in fruit as early as 24\u00a0h after incubation27. Moreover, in preliminary studies, we showed that field-collected anthracnose-infected fruit had levels of spore concentrations similar to those used in our experiment (1.0\u2009\u00d7\u2009107 per mL). Although further studies are needed to determine if increasing the time of anthracnose incubation or reducing the spore concentration affects D. suzukii behavior, our study documents for the first time the repellent effects of C. fioriniae under realistic conditions.Despite our choice assays showing clear repellent effects of D. suzukii oviposition was reduced in fruits infected by C. fioriniae. Anthracnose infection is known to increase sugar concentrations in fruits, possibly as a defense mechanism27 because there is a positive correlation between fruit sugar concentration and resistance to Colletotrichum spp.28. Given that D. suzukii females prefer to oviposit on fruit with high sugar concentrations due to their nutritional benefit7, we expected female oviposition preference for anthracnose-infected fruits (hypothesis 1). However, D. suzukii also prefers ovipositing on ripe fruit in which sugar concentrations are typically lower than in rotten fruit48. It is, thus, likely that D. suzukii oviposition preference is not determined solely by the sugar content in fruits49. Infection by many fungal pathogens, including Colletotrichum spp., typically leads to significant changes in the phytochemical composition of not just sugars but also alcohols, pH, and antimicrobial compounds50. These changes may be collectively perceived by D. suzukii females when assessing the quality of fruits during oviposition. It is also possible that females used visual cues to avoid oviposition on anthracnose-infected fruits; however, fruits did not show any signs of spoilage 48\u00a0h after infection.Under a no-choice scenario, C. fioriniae that were of lower quality for offspring survival. Certain fungi, such as the yeast H. uvarum, have a mutualistic association with D. suzukii13 and have positive effects on D. suzukii larval development and survival12. In contrast, our study showed an antagonistic  effect of the fungal pathogen C. fioriniae on D. suzukii. Previously, Graziosi and Rieske36 had also shown that infection by Colletotrichum acutatum increases the mortality of the Asian chestnut gall wasp D. kuriphilus. Raspberries infected by the fungus B. cinerea also repelled D. suzukii and reduced larval survival and adult oviposition and size11. Interestingly, our results showed that both anthracnose solutions (\u2018field\u2019 and \u2018colony\u2019), but not the \u2018filtered\u2019 solution, have repellent and insecticidal effects on D. suzukii, indicating that removing C. fioriniae conidia eliminates these negative effects. However, some yet unknown components of the \u2018filtered\u2019 solution also had some negative effects on D. suzukii oviposition. Compared to the \u2018colony\u2019 solution that was simply C. fioriniae, the \u2018fruit\u2019 solution was composed of more than one fungal species, although C. fioriniae was by far the most abundant of all species . In fact, C. fioriniae is highly competitive and typically reduces the abundance of other fungal species51. Therefore, it is likely that C. fioriniae competes with D. suzukii larvae for resource when co-exiting in blueberry fruits, which supports our hypothesis 2. The fact that disease symptoms were visible in fruit treated with the \u2018fruit\u2019 and \u2018colony\u2019 solutions, but not with the \u2018filtered\u2019 solution, about a week after infection suggests that fruit became a less suitable host for D. suzukii, which takes\u2009~\u200910\u00a0days to develop from egg-to-adult at 25\u00a0\u00b0C52, as the infection progressed.There was a positive correlation between adult preference and immature performance: females avoided fruit infected by C. fioriniae and the insect pest D. suzukii. We showed that C. fioriniae odors, likely emitted from the spores (conidia), elicit a repellent response in sexually mature D. suzukii females and that C. fioriniae infection of blueberry fruits reduces both oviposition and immature survival in this fly. Altogether, these findings open new opportunities for the potential use of odors from the fungus C. fioriniae to manage D. suzukii in fruit crops once the behaviorally active compounds are identified.In conclusion, this study is the first to demonstrate the antagonistic association between the fungal pathogen V. corymbosum; cv. \u2018Bluecrop\u2019) from a field located at the Rutgers P.E. Marucci Center, Chatsworth, New Jersey (USA), were used for laboratory experiments. Three fruit clusters per bush were bagged at fruit set  to prevent pesticide exposure and natural insect infestation and considering that fruits are susceptible to D. suzukii once they start to color7. Fruit was harvested during maturation  from mid-June through mid-July, placed in clear polyethylene bags, transported to the laboratory, and used for the experiments. For experiments with leaves, green, healthy, mature highbush blueberry leaves  were collected from greenhouse-grown potted plants. Prior to experiments, the surface of fruits and leaves were thoroughly cleaned with sterile deionized water and then dried at room temperature (~\u200925\u00a0\u00b0C).Highbush blueberry fruit  with\u2009~\u200915\u00a0mL of diet and plugged with BuzzPlugs (Fisher Scientific) and were kept in an incubator  set at 25\u2009\u00b1\u20091\u00a0\u00b0C, 65\u2009\u00b1\u20095% relative humidity (RH), and 16:8 light:dark cycle (L:D). Male and female flies used in the experiments were 3\u20137\u00a0days old and, thus, were sexually mature45. Flies were removed from the colony\u2009~\u20095\u00a0h before the start of each experiment.C. fioriniae culture consisted of a single spore isolate (isolate BB#10)19 collected in 2005 from infected blueberry fruit at the Rutgers P.E. Marucci Center and was stored on corn meal agar . High-density sporulation of the isolate was induced by streaking conidia onto clarified V8 juice agar54 with no calcium carbonate and a higher concentration of agar (28\u00a0g/L)19. Cultures were incubated at 25\u00a0\u00b0C in the dark for at least 7\u00a0days before spore suspension preparation.The laboratory C. fioriniae culture . For the \u2018fruit\u2019 treatment, blueberry fruit showing symptoms of anthracnose, i.e., salmon-colored  droplets on the surface of rotting fruit, were collected at the Rutgers P. E. Marucci Center and incubated in plastic containers (15\u2009\u00d7\u200915\u2009\u00d7\u20095\u00a0cm) at room temperature (~\u200925\u00a0\u00b0C) to promote sporulation. Once conidia were abundant , fruit were placed into a plastic screen mesh filter and repeatedly submerged into sterile deionized water. Once conidia were liberated, the spore concentration was estimated using a hemocytometer and adjusted to 1.0\u2009\u00d7\u2009107 spores per mL of deionized water immediately before the beginning of experiments. This concentration was chosen because it was similar to the concentration of spores from the field-collected infected fruit samples. For the \u2018colony\u2019 treatment, C. fioriniae spore suspensions were prepared by washing conidia from the high-density cultures into deionized water and adjusting the spore concentration to 1.0\u2009\u00d7\u2009107 spores per mL of deionized water, as described for the \u2018fruit\u2019 treatment, immediately before their use in experiments. In addition, to remove the conidia from any extracellular enzymatic compounds, a \u2018filtered\u2019 solution was prepared by sterile filtering  the \u2018colony\u2019 spore suspension described above immediately before use . Treated fruit were left to incubate at room temperature for 48\u00a0h before use in choice and no-choice assays (see below) to allow the solutions to settle on the fruit surface; this anthracnose incubation period is sufficient to activate defensive genes and induce secondary metabolite production in blueberries27. A group of 5 fruit from each treatment was set aside to confirm C. fioriniae infection by checking for symptoms (only fruit treated with the C. fioriniae \u2018fruit\u2019 and \u2018colony\u2019 solutions were symptomatic).Anthracnose spore suspension treatments were prepared using two sources: field-collected infected fruit (referred to as \u2018fruit\u2019) and a laboratory C. fioriniae-infected fruit on D. suzukii female and male behavior. Flies were given a choice between untreated fruit (control) and fruit treated either with the \u2018fruit\u2019 solution, \u2018colony\u2019 solution, or \u2018filtered\u2019 solution. In addition, flies were given a choice between untreated fruit and a blank control to determine their attraction to fruit in the absence of other odors. The experimental setup followed similar methods used by Feng and collaborators55. Choice arenas consisted of clear plastic cups . The lid of each cup had an 80-mm diameter circular hole cut in it, which was covered with a nylon mesh  to provide ventilation while retaining flies. Polystyrene tubes  were labeled for the treated and untreated fruit. Equal quantities (~\u20095\u00a0g) of blueberry fruit (N\u2009=\u20095) were used for all treatments, and fruits were placed inside of each tube. Fruits were sprayed with their corresponding treatment until run-off (approx. 1\u00a0mL of solution per fruit); untreated fruits were sprayed with deionized water only. Each pair of tubes in each choice test was sealed with Parafilm\u00ae , with a 4-mm-diameter hole in the center of the Parafilm to provide an entrance for the flies, and placed vertically on opposite sides of a cup. A cotton wick moistened with deionized water was placed on the bottom of each cup as a water source for the flies during the experiment. Ten D. suzukii  were released inside each cup (choice arena), and each choice test was replicated 10 times (N\u2009=\u2009100 flies tested per choice test and sex). Testing flies in groups does not appear to affect their behavior in previous studies using similar choice tests55. The number of D. suzukii flies inside and outside the tubes in each choice arena was counted after 24\u00a0h. The experiment was carried out under a fume hood at 25\u2009\u00b1\u20091\u00a0\u00b0C, 60\u2009\u00b1\u20095% RH, and 16:8\u00a0h L:D.Choice assays were conducted to investigate the effects of odors from D. suzukii to C. fioriniae-infected fruit odors was mediated by changes in fruit odors. To address this, females were given a choice between blueberry fruit (food tissue) plus leaves (nonfood tissue) treated with the \u2018colony\u2019 solution versus fruit plus untreated leaves. Note that C. fioriniae primarily affects fruits but can also attack all other aboveground parts, including leaves. Only females were used in these experiments because males did not respond to C. fioriniae odors (see \u201cN\u2009=\u20095) plus leaves  treated with \u201ccolony\u201d solution and (ii) a tube containing the same amount of fruit and leaves but the leaves were treated with deionized water only. Leaves were sprayed with their respective treatment until run-off. Ten D. suzukii females were placed inside each choice arena, and the experiment was replicated 10 times (N\u2009=\u2009100 flies tested). The experiment was conducted under a fume hood under the conditions described above. The number of D. suzukii females inside and outside the tubes within the arenas was counted after 24\u00a0h.We conducted choice tests to determine if the response of ors see \u201c\u201d sectionC. fioriniae infection on D. suzukii oviposition and adult emergence. Blueberry fruit were placed individually in 118-mL (4\u00a0oz) translucent plastic cups  and simultaneously exposed to one D. suzukii sexually mature female. Each fruit was treated with one of the four treatments described above: fruit\u2019 solution, \u2018colony\u2019 solution, \u2018filtered\u2019 solution, or untreated control. A cotton wick moistened with deionized water was placed on the bottom of the plastic container as a water source. After 24\u00a0h of exposure, D. suzukii females were removed and all eggs laid in the fruit were counted under a stereomicroscope. Note that D. suzukii eggs are visible on the surface of fruit skins by observing the oviposition hole and two white breathing filaments protruding out of the egg7. All fruit containing eggs were subsequently stored in the cups with filter paper at room temperature (~\u200925\u00a0\u00b0C). The number of emerged adults was recorded after14 days, which allowed enough time to complete egg-adult development56. As a measure of immature performance, for each fruit, we calculated the percent egg-to-adult survival by dividing the number of emerged adults by the number of eggs laid multiplied by 100. Each treatment was replicated 25 times.No-choice studies were conducted to determine the effects of D. suzukii flies inside each tube were recorded and the differences in D. suzukii response between treatments compared using paired t-tests. Flies that did not make a choice were considered as nonresponding and were not included in the statistical analysis. Choice data are shown as percentages in Figures.In choice experiments, for each cup (arena), data on the total number of D. suzukii oviposition, adult emergence, and percent egg-to-adult survival (immature performance) was analyzed using generalized linear models (GLMs), followed by Bonferroni post-hoc tests. For oviposition and adult emergence, we used a Poisson distribution with a logit-link function. To analyze the percentage egg-to-adult survival, data were first arcsine transformed, and then analyzed using a normal distribution with identity link function. All statistical analyses were performed using SPSS Statistics 23.0 .In no-choice experiments, the effect of treatments on"}
{"text": "In vitro, adhesion depended on fibrinogen and coagulation factor XIII (FXIII), and supraphysiological FXIII improved adhesive strength. Furthermore, when exogenous FXIII was topically applied into the wound pocket of rats, eleven adhesive failures occurred between eight rats, compared to seventeen adhesive failures between eight untreated rats, whereas the number of cohesive failures remained the same at sixteen in both groups. In conclusion, rebleeding from both adhesive and cohesive failure of clots decreases survival from hemorrhage in vivo. Both endogenous and exogenous FXIII improves the adhesive strength of clots.The adhesion of blood clots to wounds is necessary to seal injured vasculature and achieve hemostasis. However, it has not been specifically tested if adhesive failure of clots is a major contributor to rebleeding and what mechanisms prevent clot delamination. Here, we quantified the contribution of adhesive and cohesive failure to rebleeding in a rat model of femoral artery injury, and identified mechanisms that contribute to the adhesive strength of bulk clots in a lap-shear test in vitro. In the rat bleeding model, the frequency of clot failures correlated positively with blood loss (R\u2009=\u20090.81,  During cohesive failure, the sealant fractures internally from forces that disrupt structural integrity within the clot. Whereas during adhesive failure, the sealant remains mechanically intact, but its substrate interface is disrupted. It is crucial to understand the mechanisms behind the failure of blood clots as it may inform approaches to mitigate rebleeding during severe hemorrhage, but it is not known if adhesive failure contributes to hemostasis and rebleeding in vivo.Blood clots, like all sealants, fail either by cohesive failure or adhesive failure2. Rebleeding typically occurs when blood pressure exceeds the mechanical stability of the clot. This can occur when blood pressure increases during fluid resuscitation2. The failure of clots and volume of blood loss from rebleeds can be exacerbated by trauma-induced coagulopathies, including hyperfibrinolysis and the depletion of coagulation factors3, as well as physical perturbation of the clot during patient transport4. The destabilization of blood clots and subsequent rebleeding correlates with poor survival in trauma patients2. The critical stress a clot can withstand before internal fracture, its cohesive strength, is positively correlated with its elastic modulus, a measure of its stiffness, as measured by a clinically-utilized technique, thrombelastography (TEG)5. While the factors that contribute to clot stiffness have been extensively characterized, such as by using TEG7, the adhesive properties and mechanisms of clots, examined in bulk rather than examining individual components in isolation, are less characterized.Blood coagulation is typically effective at sealing damaged blood vessels to achieve hemostasis, but blood clots can fail during severe hemorrhage, leading to rebleeding and secondary hemorrhage. Rebleeding is common during severe hemorrhage, normally occurring many times before definitive hemostasis is reached8. Fibrin is formed by proteolytic cleavage of fibrinopeptides of its precursor, fibrinogen, during activation of the coagulation cascade. The fibrin monomers then self-assemble to protofibrils, which aggregate into a mesh-like structure10. This fibrin polymer can both directly crosslink and mechanically interlock with extracellular matrix (ECM) proteins in tissue. FXIII is present in plasma as a heterotetrameric proenzyme (FXIII-A2B2) and is converted to the activated transglutaminase  by thrombin in the presence of Ca2+. FXIIIa mediates the covalent crosslinking of fibrin to ECM proteins. FXIIIa also crosslinks fibrin to antifibrinolytic proteins, such as \u03b12-antiplasmin11, as well as fibrin subunits to each other, which increases the density of the fibrin network12. Both of these processes stabilize the blood clot against premature fibrinolysis, and clinical observations indicate that a low FXIIIa concentration in blood is correlated with hyperfibrinolysis in trauma patients13. Platelets are adhesive to materials in the subendothelial space, including collagen and von Willebrand Factor (vWF), through receptors such as glycoprotein (GP) VI and the GPIb-V-IX complex15. While fibrin, FXIIIa and platelets all have adhesive properties, it is not known which of these components contribute to the adhesive strength of the bulk clot. We hypothesized that rebleeding can occur due to adhesive failure, and that supplemental FXIII could increase the adhesiveness of clots during bleeding. To confirm whether the delamination of clots from wound tissue leads to rebleeding, we analyzed the modes of clot failure in a rat femoral artery bleeding model. To test if depletion of FXIII and other clot components would decrease clot adhesiveness, we measured adhesive clot failure of depleted plasma ex vivo, and also tested if reintroducing exogenous FXIII would recover adhesion.Several components of blood are known to have adhesive properties. Fibrin and coagulation factor XIII (FXIII) have been used as surgical sealantsExperiments involving rat models were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance with IACUC guidelines and regulations.Experiments involving human participants were approved by the Human Research Ethics boards of the University of British Columbia (UBC) and the Canadian Blood Services (CBS). All experiments were performed in accordance with UBC and CBS guidelines and regulations. Informed consent was obtained from all healthy human volunteers prior to whole blood donation.All donors were deidentified. The recruitment criteria were inclusive of both sexes.16. Wild-type male Sprague\u2013Dawley rats weighing between 300\u2013450\u00a0g were used. Rats of only one sex were used to reduce sex-related variability in this pilot study, but should be extended in the future to involve both sexes. The rats were anesthetized with ketamine and xylazine and maintained under isoflurane for the duration of the protocol . Before injury, the carotid artery and jugular vein were catheterized to monitor blood pressure (BP) and to infuse saline, respectively. A tracheotomy was performed and a small polyethylene tube was inserted to aid respiration using a mechanical ventilator. The left femoral artery was isolated and clamped proximally and distally. A wound pocket of roughly 2\u00a0mL in volume was made at the incision site to access the femoral artery prior to isolation and incision of the artery to induce bleeding. A 3-mm longitudinal incision was then made in the anterior wall of the femoral artery segment between the clamps to allow for free bleeding. Prior to releasing the clamps, blood was drawn from the carotid artery through the inserted catheter to standardize starting blood pressure to as close to 50\u00a0mmHg as possible  to prevent exsanguination and standardize the initial bleed volume. Following catheter hemorrhage, clamps were removed to initiate bleeding from the wound, and this time was designated t\u2009=\u20090\u00a0min. Solutions containing the test reagents  were applied immediately into the wound pocket and allowed to mix with the forming blood clot while still in its liquid phase. This FXIII concentration was chosen to correspond with a concentration that improved clot adhesion in vitro, accounting for the volume of the wound pocket and potential loss of the exogenous FXIII from the wound pocket during the initial bleeding. After 5\u00a0min of free bleeding to allow clot formation, fluid resuscitation was initiated. Saline (0.9%) was infused intravenously at 3\u00a0mL/kg/min to raise mean arterial pressure to 60\u00a0mmHg as needed. The wound pocket was video recorded continuously starting at t\u2009=\u20090\u00a0min. Each video was viewed by two blinded observers to review each rebleeding event to determine whether the clot failed by adhesion (from the edges) or cohesion . Pre-weighed cotton gauze was used to collect blood spilling over the wound to track blood loss every 5\u00a0min. The blood loss attributed to each clot failure event was estimated as the blood loss within the 5-min interval in which the failure occurred. When more than one failure occurred in the 5-min interval, blood loss was divided equally among the failures. Survivors were euthanized under anesthesia if surviving up to 80\u00a0min or if arterial wave form was lost and mean arterial pressure confirmed to be\u2009<\u200920\u00a0mmHg, whichever occurred first. Animals that did not survive the initial catheter hemorrhage were excluded from the analysis.Test reagents were applied to a rat model of femoral artery injury to evaluate their effects on clot failure mode and hemostatic efficacy. The procedure for the rat model was modified from a previous studyg for 20\u00a0min to separate the RBC from platelet-rich plasma (PRP). The RBC fraction was centrifuged at a higher speed of 500\u00a0g for 5\u00a0min to separate residual plasma from the RBC. The RBC fraction was then washed twice with phosphate buffered saline . In experiments that investigated the effect of RBCs on clot adhesion  (BD Biosciences). The blood was centrifuged at 100\u00d72, 1.1\u00a0mM MgCl2, 2.7\u00a0mM KCl, 137\u00a0mM NaCl, 0.4\u00a0mM NaH2PO4, 10\u00a0mM HEPES, 5.6\u00a0mM D-glucose, pH 6.5) to prevent platelet activation prior to the start of the experiment. The platelets were centrifuged at 250\u00a0g for 10\u00a0min for a final wash. The buffer was removed and the platelets resuspended in fresh Tyrode\u2019s HEPES and counted by a XN-550 Automated Hematology Analyzer (Sysmex). Platelet concentration was adjusted by the addition of more Tyrode\u2019s HEPES as specified in the protocol for each experiment in Supplementary Table Platelets were isolated from platelet-rich plasma (PRP) that was processed by the CBS from whole blood pooled from four deidentified female and male donors. The PRP was stored at 22\u02daC with mild shaking on a small platelet incubator and agitator  and used within five days of the blood draw. To extract platelets, the PRP was centrifuged at 250\u00a0g for 20\u00a0min to pellet the platelets. The platelet-poor plasma (PPP) was removed from the pellet and the pellet was resuspended in citrate glucose saline buffer . The platelets were again centrifuged at 250\u00a0g for 10\u00a0min, the buffer removed, and the platelets resuspended in Tyrode\u2019s HEPES buffer  for 103\u00a0min. The A405 increased with increased turbidity as the clot became insoluble through fibrin formation and then decreased as the clot was lysed17. Clots were measured until complete lysis, defined by when the absorbance of all reactions had reached the baseline level as indicated by the no-calcium negative control.Lysis was measured by transmission spectrometry. Plasma samples (100 \u00b5L) with or without tissue plasminogen activator (tPA)  were added to a 96-well microplate using the recipes listed in Supplementary Table t test was used to calculate the p values in specific comparisons between two groups of interest. SEM denotes standard error of mean, and is defined as the standard deviation of the data set divided by the square root of the number of replicate values recorded.Normality was assumed for all data sets but could not be verified due to low sample sizes. A Pearson (R) calculator was used to calculate the Pearson\u2019s correlation coefficient and p value for correlation analyses. One- and two-way analyses of variance (ANOVA) and the two-stage Benjamini, Krieger, and Yekutieli procedure, with adjusted p values for multiple comparisons, were used to calculate the p values between multiple groups. A Student\u2019s To determine if rebleeding occurs in a rat model of lethal hemorrhage, intravenous fluids were administered following a femoral artery incision to challenge the hemostatic capacity of the formed clot. Blood loss was quantified every 5\u00a0min by weighing the bleed volume soaked by pre-weighed gauze Fig.\u00a0a,b. Fail20. To test if platelets contribute to the adhesive strength of bulk clots to collagen in vitro, PRP clots were prepared by supplementing platelet-poor plasma  with washed platelets for final concentrations that mimic the low (170\u2009\u00d7\u2009109 cells/L), normal (340\u2009\u00d7\u2009109 cells/L) and high (690\u2009\u00d7\u2009109 cells/L) physiological concentrations in blood. Adhesive strengths of PPP clots and PRP clots at low and normal platelet concentrations were not significantly different. At high platelet concentrations, PRP clots were 52% more adhesive than PPP clots  and FXIII on clot adhesion to collagen in vitro, clots were formed between collagen coverslips for 1\u00a0h and subjected to a lap-shear test until adhesive failure occurred. The concentrations of Fg were varied by supplementing purified Fg to Fg-poor PPP (PPP with less than 0.1\u00a0g/L fibrinogen). Supplementing with Fg increased the adhesive strength in a concentration-dependent manner Fig.\u00a0a. This rmin Fig.\u00a0c. Lap shmin Fig.\u00a0. To testmin Fig.\u00a0d. Clot smin Fig.\u00a0e. Whole min Fig.\u00a0f.Figure 2B2 was chosen for this study, rather than FXIIIa, to ensure FXIIIa was only generated at the site of coagulation and minimize off-target crosslinking of proteins by purified FXIIIa. This treatment was compared to the vehicle control of 0.9% saline, using groups of eight rats. Blood in the systemic circulation of the rats was diluted by intravenous administration of saline, reaching approximately 53% and 55% of the original concentration after 20\u00a0min in the control and FXIII groups respectively  was added directly to the wound pocket of rats subjected to femoral artery puncture prior to fluid resuscitation Fig.\u00a0a,b. FXIIBlood clots seal injured vasculature, and thus clots must adhere to the wound site to achieve hemostasis. However, no studies had previously quantified adhesive failure in wounds and correlated them to survival, and it was not clear what mechanisms contribute to clot adhesive strength. This work shows for the first time that adhesive failures are common. We found that adhesive failures accounted for 51\u2009\u00b1\u200914% of rebleeds in untreated control animals. The in vitro results also highlight the important contribution that FXIII plays in preventing adhesive failure of the clot, presumably by enhancing the attachment of fibrin to ECM proteins.5. Our studies that measured clot stiffness and lysis were specifically designed and performed in order to have direct comparisons to clot adhesion. Comparing the results from the lap-shear adhesion assay and the TEG assay showed that the stiffness of a clot was not directly correlated to its adhesive strength. For example, while platelets dramatically increased clot stiffness by approximately 16-fold, consistent with previous reports on the contribution of platelets to stiffness21, the clot adhesive strength to collagen was unchanged by platelets at the same concentration, and only increased by 1.5-fold at twice this concentration. This reflects the importance of platelets in clot cohesion through aggregation and binding of cells and proteins in blood22. While adhesives require cohesion to function, adhesive strength is also affected by the interactions between the adhesive and its substrate23. While platelets had only a mild effect on the adhesive strength of a formed clot 60\u00a0min after initiating clotting, they did contribute to adhesion at a very early stage of coagulation; PRP had higher adhesion than PPP at 2\u00a0min. This may have been due to platelets quickly binding to the adherend and enabling the initial attachment of blood components to collagen, adhering through receptors such as glycoprotein VI and Ia/IIa25. It is important to note that the adhesion test used here was a static system. In microfluidic studies, platelets are important in mediating the initial attachment and subsequent clot formation on collagen surfaces under flow26, and platelets may have a greater impact on mature clots under flow. RBC count did not affect the clot adhesive strength. This was similar to the effect of RBC on clot stiffness. It is known that hematocrit levels do not improve clot stiffness as measured by thromboelastrography20. This could be due to the deformability of RBC27. In conditions where RBC become less deformable, such as sickle cell disease, clot stiffness increases with RBC concentration28.The stiffness of blood clots as measured by thrombelastography correlates with clot cohesive strength and is used often to effectively predict the severity of bleeding30. Weak clot adhesion occurred in PPP deficient in FXIII, but supplementing FXIII increased the clot\u2019s adhesive strength. In particular, a high dose of FXIII (30\u00a0\u00b5g/mL) increased clot adhesive strength by roughly 3-times compared to a lower physiological concentration (10\u00a0\u00b5g/mL). Consistent with previous reports31, clot stiffness also increased by adding FXIII, but the high dose of FXIII only increased clot stiffness moderately (27%) compared to the lower physiological concentration tested. We found that concentrations of FXIII that achieved only modest increases in clot stiffness led to large increases in adhesive strength. This highlights that in addition to increasing clot cohesion through interchain crosslinking of fibrin strands, FXIIIa plays an important role in enhancing the attachment of fibrin to wound tissue. Whole blood clots were more adhesive to extracellular matrix proteins, collagen, fibronectin, and laminin, compared to plain glass, indicating that both the blood composition and the substrate to which the clot is adhered affect clot adhesion.We observed that clot adhesive strength can be affected by both its intrinsic composition and the interactions between the clot and the substrate to which it is adhered. Both fibrin and FXIII improved clot adhesion to collagen, in the absence of platelets, in a dose dependent manner. This suggests that perhaps the primary contributions of fibrin and FXIII on enhancing clot attachment to the collagen surface was independent from the known role of platelets in mediating fibrin-collagen interactions. Unsurprisingly, fibrin was essential for clot stiffness32.After showing in vitro that supplementing FXIII enhanced clot adhesion, we tested if supplementing FXIII directly into the wound site would prevent adhesive failure in our rat hemorrhage model. Of rats that received FXIII, more survived, fewer adhesive failures and lower blood loss occurred compared to control animals. While these comparisons between the control and FXIII-treated groups of rats were not statistically significant with this sample size of eight rats per group, these in vivo results warrant further investigations into the effects of FXIII on clot adhesive strength in future studies with a larger sample size. Models of hemorrhage typically have high variability in survival rate and blood loss. The model used in this study also showed high variability in the mode of clot failures. A larger sample size would be needed to better distinguish the effects of exogenous treatments from intrinsic differences between animals and injuries. Future studies using rats with a genetic knockout of FXIII would also be useful for testing the physiological contribution of FXIIIIt is probable that clot contraction influences adhesion of clots, but this was not specifically tested in this study. Inhibitors of clot contraction, eptifibatide and blebbistatin, did not influence adhesion in the specific in vitro assay used here. In vivo, some shrinkage of the clots occurred even though the wounds were held open, although we did not detect differences in clot contraction between the treatment groups.In conclusion, the bulk adhesion of clots contributes to hemostasis in vivo. Clot failures were associated with blood loss and poor survival, and adhesive and cohesive failures were equally common. Bulk clot adhesion is controlled by fibrin and FXIII, but only minimally by platelets in vitro, demonstrating that clot stiffness and clot adhesion do not have a linear relationship. This highlights the importance of mechanisms of clot adhesion to hemostasis and encourages the development of therapeutics for bleeding disorders or hemorrhage that enhance these innate mechanisms.Supplementary Information."}
{"text": "The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications. Unfortunately, it is difficult to obtain sufficient yields of pure RDase isolated from organohalide respiring bacteria for biochemical studies. Hence, robust heterologous expression systems are required that yield the active holo-enzyme which requires both iron-sulphur cluster and cobalamin incorporation.Bacillus megaterium, Escherichia coli HMS174(DE3), Shimwellia blattae and a commercial strain of Vibrio natrigenes, for cobalamin class-III dependent enzymes expression. The Nitratireductor pacificus pht-3B reductive dehalogenase (NpRdhA) and the epoxyqueuosine reductase from Streptococcus thermophilus (StoQ) were used as model enzymes. We also analysed whether co-expression of the cobalamin transporter BtuB, supports increased cobalamin incorporation into these enzymes in E. coli. We conclude that while expression in Bacillus megaterium resulted in the highest levels of cofactor incorporation, co-expression of BtuB in E. coli presents an appropriate balance between cofactor incorporation and protein yield in both cases.We present a comparative study of the heterologous expression strains  \u2022Reductive dehalogenases and epoxyqueuosine reductases are cobalamin-dependent.\u2022Cofactor incorporation presents a challenge for heterologous expression of these enzymes.\u2022A range of bacterial hosts were assessed as expression host for these enzymes.\u2022An inverse correlation between protein yield and cofactor content was observed.\u2022E. coli yielded best results.Co-expression with the BtuB B12 transporter in  However34U35N36-3\u2032 anticodon sequence , that contain a central cobalamin cofactor bound by a nitroreductase-like fold and 2 [4Fe\u20134S] clusters ,7. Unliksequence . The hettitution .Fig. 1S.E. coli, resulted in soluble but catalytically inactive protein . Be. Be17]. Bmeg (90%), presumably due to the ability of B. megaterium to synthesize cobalamin de novo. StoQHMS with and without co-expression of BtuB contain the next highest cobalamin incorporation, with the co-expressing BtuB strain resulting in a slightly higher cobalamin content than HMS174(DE3) alone (70 and 62% respectively). The cobalamin incorporation in StoQHMS is higher than expected, showing the high efficiency of BtuB and its ability to transport cobalamin, even while under translational control  clusters ,17. DeteVmax\u2212TB, NpRdhAVmax\u2212YT and NpRdhABmeg have high iron:protein ratios of 7.7\u00a0\u00b1\u00a00.1, 6.3\u00a0\u00b1\u00a00.2 and 6.5\u00a0\u00b1\u00a00.1 respectively, indicating the presence of 1.9\u00a0\u00b1\u00a00.1, 1.6\u00a0\u00b1\u00a00.1 and 1.6\u00a0\u00b1\u00a00.1 [4Fe\u20134S] clusters per NpRdhA, respectively. In contrast, NpRdhAShim has a low iron:protein ratio of 4.1\u00a0\u00b1\u00a00.1, suggesting only 1 [4Fe\u20134S] cluster per NpRdhA. As observed with StoQ, NpRdhAHMS (both with and without BtuB) has a low iron:protein ratio of 5.4\u00a0\u00b1\u00a00.2 and 3.6\u00a0\u00b1\u00a00.5 respectively. The [4Fe\u20134S] cluster content for NpRdhA is increased when co-expressed with BtuB . The cobalamin incorporation has previously been implicated in NpRdhA iron-sulphur cluster maturation, due to the close contact between cobalamin and the proximal [4Fe\u20134S] [The iron-content for purified NpRdhA appears generally lower compared to StoQ . NpRdhAV[4Fe\u20134S] .3.4E. coli flavodoxin reductase and spinach ferredoxin) to support the transfer of electrons from NADPH to the NpRdhA  cluster and cobalamin incorporation.Although  require . AnotherE. coli HMS174(DE3) may provide an acceptable compromise between protein yield and specific activity. Not only does this strain result in high protein yields, but also leads to relatively high cobalamin incorporation for both StoQ and NpRdhA . Unfortunately, the iron-sulphur cluster incorporation appears substantially lower when compared to other strains, which will require further study to improve incorporation further, potentially by co-expression of the iron-sulphur cluster pathway operon [Co-expression of class-III cobalamin-dependent enzymes with the cobalamin transporter BtuB in y operon ,40.V. natrigenes, therefore potentially explaining both the increased purified StoQ and NpRdhA obtained under these conditions, as well as cobalamin incorporation due to increased expression of salvage systems [S. blattae (data not shown), under conditions previously successful for expression of other RDases [In an attempt to standardize expression conditions, Vmax\u2122 cells were grown in suboptimal conditions. Upon expression in a recommended enhanced 2xYT medium, both quantities of purified StoQ and NpRdhA and cobalamin incorporation increased in comparison to when grown in TB, therefore showing the importance of not only expression strain utilized, but also conditions under which they are expressed. A potential explanation for the increased protein obtained and cobalamin incorporation could be due to the large quantity of NaCl present in the enhanced 2xYT medium. NaCl has proved essential for optimal growth and also protein synthesis in  systems . The impr RDases .5B. megaterium offers the highest specific activity/level of cobalamin incorporation.In this study we have used two model class-III cobalamin-dependent enzymes, StoQ and NpRdhA, to test a range of heterologous expression hosts for their ability to produce the corresponding holo-enzymes in high yield. Neither isolation directly from the OHRB host, or reconstitution of the apo-protein have been reported as efficient procedures that can yield the required protein amounts to support detailed biochemical and biophysical studies on the mechanism and structure of these enzymes. When keeping most variables, in terms of protein purification and cell growth conditions, as similar as possible, we find that the cobalamin-producing E. coli strain is the best candidate heterologous host for the expression of class-III cobalamin-dependent enzymes, on the basis of the two model enzymes tested.However, protein yield is modest compared to other strains tested under these conditions, and when striking a balance between yield and levels of cofactor incorporation, the BtuB expressing Future work will need to focus on the study of additional members of the class-III enzymes to verify our findings. The NpRdhA enzyme is arguably distinct from respiratory reductive dehalogenases and appears not associated with the membrane. Furthermore, there are the so-called \u201cself-sufficient\u201d reductive dehalogenases, that are fused to the redox module that provides reducing equivalents from NAD(P)H, that provide an attractive target for future bioremediation. To date there are very few examples of these proteins having been expressed successfully and purified to homogeneity ,45.The authors declare that they have no conflict of interest with the contents of this article.T.H. and K.F. grew cells, purified protein and performed experiments. K. F. and S. E. J. R. carried out EPR spectra and analysed the EPR spectroscopic data. K.A.P.P. helped with molecular biology. D.L. conceived and coordinated the study. All authors wrote the paper, reviewed the results and approved the final version of the manuscript.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
{"text": "Optokinetic nystagmus (OKN) is an involuntary eye movement induced by motion of a large proportion of the visual field. It consists of a \u201cslow phase (SP)\u201d with eye movements in the same direction as the movement of the pattern and a \u201cfast phase (FP)\u201d with saccadic eye movements in the opposite direction. Study of OKN can reveal valuable information in ophthalmology, neurology and psychology. However, the current commercially available high-resolution and research-grade eye tracker is usually expensive. Methods & Results: We developed a novel fast and effective system combined with a low-cost eye tracking device to accurately quantitatively measure OKN eye movement. Conclusions: The experimental results indicate that the proposed method achieves fast and promising results in comparisons with several traditional approaches. Research on eye movement has provided a valuable source of information to vision science, psychology, and neurobiology ,2,3. TheOptokinetic nystagmus (OKN)  is an inIn this study, a novel fast and effective system is developed and combined with a low-cost eye tracking device to accurately quantitatively measure OKN eye movement. During eye movement recording, the human eyes often show unwanted noise or baseline drift due to fatigue or distraction, which interfere with the analysis of signals. Our algorithm can detect the position of FP and optimize the result by iterative signal modification. To validate the performance of the proposed system, the results of FP detection were comparable with those of manual reading by experienced physician and of several traditional approaches.In this study, we designed a dynamic stripe simulation program for OKN stimulation that is easy to adjust. We used the low-cost eye tracking device GP3 to collect OKN response data in a simple simulation environment and simply fixed the head to ensure the collected data would be free from interference. The GP3 eye tracker, which is produced by the Gazepoint company, is an easy-to-use, high-performance eye tracker. Gazepoint company produces the eye tracking system technology for everyone. The eye tracker comes with a software suite, Gaze point Control and Gaze point Analysis, which allows you to calibrate a participant, build an experiment, collect data, and analyze data. Finally, the collected data were used to get the position of the scanning moment by the FP filtering method. Six healthy male adults were recruited in the study. The participants had no neurological, ophthalmological, or vestibular impairments. The six subjects underwent complicated tests, and the measured signals collected obvious OKN characteristics for these six subjects. Only participants with best-corrected visual acuity of 6/6 or higher on the Snellen scale were included in this study. The study was approved by the Institutional Review Board at the Ditmanson Medical Foundation Chiayi Christian Hospital and complied with the Declaration of Helsinki . ParticiParticipants sat in a chair in front of the middle of a 23-inch LCD  monitor . Distance between the head of the participant and the LCD monitor was set at 50 cm. The ophthalmologist recommended that the distance between the human eye and the computer screen should be 45\u201360 cm. Therefore, the setting of our environment uses a distance of 50 cm as a fixed value, and the width of the stripe animation is reversed according to this distance. To reduce the signal noise from vertical movement, the participants\u2019 head was placed on a chin rest and a horizontal black bar of 0.5 cm height was placed at 3 cm below the vertical midline of the LCD monitor. The OKN stimulus was generated by showing moving black and white stripes on the LCD monitor at a frame rate of 60 Hz. Eye movements were recorded using a video-based eye tracker  that recorded eye movements with a maximum resolution of 30 arcmin at a sampling rate of 150 Hz, where arcmin is a unit of angular measurement equal to 1/60 of one degree.The eye tracker was calibrated for each participant at the start of each procedure. Calibration was performed by fixating at five different positions on the monitor. The OKN was elicited with a square-wave grating alternative black and white vertical stripes of 4.6 cm width moving horizontally at fixed 20 cm/s speed. The participant was instructed to look at the moving patterns binocularly. Each test lasts for 30 s and was repeated three times. Measurements were recorded on a computer and stored for offline analysis. All programs and algorithms for analyzing data were developed in MATLAB .After completing a series of tests and data collection, the dynamic SlopeThreshold and MoveThreshold were set up to screen out the eligible FP sections based on the data pre-processing and feature determination. The specific slope and length were used as the threshold, where SlopeThreshold represents the filtering range of the slope, which is used to control the OKN state of the eyeball according to the movement direction of the fringe, usually set positive or negative, and MoveThreshold stands for the distance the pupil moves, because the distance the pupil moves in the Fast Phase (FP) is larger, which is our index for screening. Finally, iterative corrections were made to fine-tune each eligible signal to the correct position.Using the slope determination method, we narrowed the selection range of FP in accordance with the occurrence conditions of FP formulated by Kanari et al. 2017) [ [17]. WeAlgorithm 1. Proposed FP position detection.1for i = 1: length (I)2\u2003if (I\u2019s slope meet S)3\u2003\u2003Put Up/Down point to matrix;3\u2003end4end5for j = 1: IC6\u2003for i = length(M)7\u2003\u2003gather points around peak (\u00b11)9\u2003\u2003find maximum/minimum of points10\u2003end11endI:is the input dataS:is the slope thresholdM:is the matrix after select matching data from inputIC:is the iteration Counts (interationCount), through multiple iterations, the signal identified as the FP interval gradually moves to the highest and lowest points of FPSince the GP3 tracking device can only take a maximum signal of 150 Hz and the data received are not stable and complete, even in the SP stage, involuntary eye movement can be incorrectly read as an FP, resulting in the need to dynamically determine the SlopeThreshold and dynamically set the MoveThreshold to filter FPs that are incorrectly read when nystagmus occurs in SP. The detailed processing steps (Algorithm 1) are as follows:We narrowed the selection range of the signal according to the FP occurrence condition formulated by Kanari et al. 2017) [ [17]: ThFor accuracy strategy, generally 2 to 5 saccades occur in an OKN test per second. It varies with different subjects. We performed OKN tests on the left and right three times, respectively (each time about 30 s) for six subjects. According to the definition of FP formulated by Kanari et al. (2017), the highest point and the lowest point of each FP are manually marked as the position where FP occurred correctly. The formula for accuracy is defined as:The detected method is used to detect the FP position and is then compared with the ground truth.The above condition for signal filtering by slopes is relaxed, but it can be dynamically adjusted. In this study, the subjects received the OKN test with a stripe that shifted to the right. For the convenience of sampling, we only sampled at a negative slope.After collecting all the points at which FP occurred, we set a certain length (the maximum of FP between two points in SP) to filter uncontrolled nystagmus. Based on the displacement of length as the threshold, we used an FP length greater than Thr to filter the unsmooth signal caused by nystagmus (Thr (threshold) refers to the setting of MoveThreshold). After multiple tests by several subjects, it was found that the X-coordinate movement of the eyeball in GP3 exceeds Thr = 0.0006, which has a very high possibility for the FP interval. It can be adjusted slightly . It can As shown p-values using paired t-test between each intermediate process of our method and ground truth. More specifically, the paired t-test was used to estimate whether the differences between the performance of each intermediate process of our method and ground truth were significant or not. The results showed that 97% of the FP occurrence points of OKN could be found through our method  at which FP occurred. High-pass and band-pass FIR filtering were adopted for peak detection. After smoothing, an adjustable threshold was set for the screen, and the filtered peak was found. The adjacent maximum/minimum values were found according to the signal position  19](3)(3)19] filtering after the Fourier transform. The reason was that the slope of FP would not affect the threshold judgment with the involuntary eye shift of the subject. Our method could eliminate the need for complex calculations and make the judging simpler and more accurate.p-values using paired t-test between the three traditional methods and the proposed method. More specifically, the paired t-test was used to estimate whether the differences between the performance of three traditional methods and that of the proposed method were significant or not. At first, we thought that we should only use Peak finding [In order to evaluate the performance of the proposed method, the three traditional methods ,8 were i finding  to find  finding  depends Regardless of the normal OKN identification rate, the uncontrolled noise produced by the subjects affected the determination of the correct FP during the prolonged test time. The OKN with noise includes dullness, fixation position deviation, and other phenomena, which further deepen the difficulty of filter setting and greatly decrease the accuracy. Our method simply filtered by the slope and moving distance, which solved many judgment problems, and screened the FP position from an intuitive angle. It reduced the influence of fixation deviation, leaving only the stagnant fixation problem . In addition to the normal FP judgment of OKN and FFT combined with a high-pass filter that can be used well, there were good results on the correct FP judgment of OKN signals with noise.Clinical detection of OKN often requires costly instruments, which poses considerable difficulties in clinical applications. Our main contributions were as follows: (1) low-cost instruments could be used to detect OKN, and (2) simple conditional screening and iteration could be used to obtain the accurate FP position, which was conducive to the calculation and analysis of the subject\u2019s eye disease status.Our method could be used to completely identify the FP position under stable conditions without large eyeball stagnation. However, the reaction of the eyeball cannot be controlled, and abnormal nystagmus and dullness are randomly generated and not included in FP/SP for reference . Our metIn the field of neuro-ophthalmology, many scholars have tried to use OKR to study the physiological and pathological phenomena of eye movements . In the 1940s, scholars studied the relationship between OKR and VOR . Sangi et al. (2015) tested and analyzed the data of children , findingThe data collected by the six subjects in this experiment will be used as a reference index for future clinical eye disease patients. In future work, we will increase the screen distance, stripe animation changes, and other variables to test the patients next to the bed. With diversified tests, OKN factors that may affect patients with eye diseases will have the opportunity to be discovered. The high-accuracy FP recognition rate is of great help to the subsequent calculation of mean slow-phase velocity (MSPV) and other eye movement analysis parameters, and is beneficial to use as a reference index for patients with strabismus and other eye diseases."}
{"text": "We use a low-temperature catalytic-spillover process involving the post-growth incorporation or removal of hydrogen from \u03b1-Fe2O3 thin films. Hydrogenation drives pronounced changes in its magnetic anisotropy, N\u00e9el vector orientation and canted magnetism via electron injection and local distortions. We explain these effects with a detailed magnetic anisotropy model and first-principles calculations. Tailoring our work for future applications, we demonstrate reversible control of the room-temperature spin-state by doping/expelling hydrogen in Rh-substituted \u03b1-Fe2O3.Antiferromagnetic insulators are a ubiquitous class of magnetic materials, holding the promise of low-dissipation spin-based computing devices that can display ultra-fast switching and are robust against stray fields. However, their imperviousness to magnetic fields also makes them difficult to control in a reversible and scalable manner. Here we demonstrate a novel proof-of-principle ionic approach to control the spin reorientation (Morin) transition reversibly in the common antiferromagnetic insulator \u03b1-Fe One major challenge for antiferromagnetic spintronics is how to control the antiferromagnetic state. Here Jani et al. demonstrate the reversible ionic control of the room-temperature magnetic anisotropy and spin reorientation transition in haematite, via the incorporation and removal of hydrogen. Among them, insulators promise efficient magnon-based spin transport with the absence of charge-related Joule losses6. They also host attractive functionalities like spin colossal-magnetoresistance7, ultra-fast dynamics11, high domain-wall propagation velocities13 and spin-superfluidity14. Despite all these advantages, they have so far played only an auxiliary role as pinning or exchange bias layers in thin film spintronic devices as their magnetic order is difficult to detect and hard to control15. Although many recent reports demonstrating clear detection of the AFM-state in insulators by spin-Hall or anisotropic magneto-transport have emerged18, control of the ground-state continues to be a challenge15.Antiferromagnets (AFM) exhibit long-range magnetic order with two or more sublattices aligned so that they produce no net magnetization. There has recently been a surge of interest2O3, which is a well-known antiferromagnetic insulator with a bulk Dzyaloshinskii\u2013Moriya interaction (DMI) that undergoes a spin-reorientation Morin transition between the in-plane and out-of-plane states 21, sizable spin-Hall magnetoresistance18 (when combined with a Pt overlayer) and is also the only reported natural AFM to date that hosts a wide family of topological AFM textures24 at room temperature. Consequently, reversible engineering of the anisotropy, and thereby the Morin transition, is crucial as it would allow control over magnon-transport21 and topological texture dimensions22. Previous reports have shown that strain25, chemical doping30 and meso-structure31 can modify the antiferromagnetism in \u03b1-Fe2O3. However, lack of reversibility and scalability makes these approaches difficult to exploit in practical applications.In this work, we focus on \u03b1-Fe9, but they require table-top lasers and large interaction volumes. Secondly, electric-field control of the AFM-state, which is possible in multiferroic32 and magnetoelectric33 oxides, cannot be replicated in a vast majority of functional materials, where magnetic and electrical degrees of freedom are not coupled. Alternatively, electric-field control via strain coupling to piezoelectric substrates, which has been quite successful in tuning antiferromagnetic metals36, require epitaxial matching or texturing constraints to be satisfied for crystalline systems37. Thirdly, electric-current control of the AFM-state via N\u00e9el spin\u2013orbit torques is also quite useful39, but is limited to materials with strong spin\u2013orbit coupling that break local inversion-symmetry in a specific manner. Finally, spin-Hall effect based spin\u2013orbit torques injected from a heavy-metal overlayer are also quite promising in controlling the AFM-state of insulators43. However, studies in \u03b1-Fe2O3 have so far shown in-plane tuning43, which is insufficient to directly control the Morin transition. Hence, there is a need for developing alternative pathways to directly tune antiferromagnetic properties and spin reorientation in \u03b1-Fe2O3 thin films.In the literature, coherent optical techniques have enjoyed success in manipulating antiferromagnetic insulators2O3 thin films by using spillover. We draw inspiration from pioneering ionic studies, where doping light ions  was used to control anisotropy in ferromagnetic systems45, or transport in strongly correlated-oxides48 and conventional insulators49, although the crystal physics and chemistry underpinning our implementation is completely different. By incorporating or removing hydrogen, we demonstrate a hitherto unexplored ionic approach to control a wide range of antiferromagnetic properties in a reversible and stable manner. H-doping can drive these pronounced changes by delicately tuning the competing contributions to the magnetic anisotropy, as a result of local electron injection and ensuing local distortions. Lastly, we show reversible control of the room-temperature Morin transition by hydrogenation of \u03b1-Fe1.97Rh0.03O3 films.Here, we present a new method of hydrogen (H)-doping to tailor antiferromagnetism in epitaxial \u03b1-Fe2O3 has the trigonal corundum structure, with antiparallel sublattices  stacked alternately along the hexagonal c-axis, as shown in Fig.\u00a0c-axis. Presence of the bulk DMI causes canting of the sublattices within the basal planes19, resulting in a weak macroscopic in-plane canting -oriented \u03b1-Al2O3 single crystals by pulsed laser deposition , with hysteretic appearance/disappearance of the in-plane canted moment, Fig.\u00a025. To confirm the magnetic origin of the transition, we performed X-ray linear dichroism (XLD) spectroscopy at the Fe L2 edge, which is a good element-specific approach to identify the direction of the sublattice magnetization in \u03b1-Fe2O3 (\u2018Methods\u2019)51. The dichroism reverses sign across L to the basal plane from the c-axis. We also performed X-ray magnetic circular dichroism (XMCD) spectroscopy but saw no signal from the weak canted moment (Supplementary-M(H), revealed a hysteretic evolution due to in-plane realignment of antiferromagnetic domains driven by the magnetic field coupling to the canted moment, at Highly-oriented epitaxial thin films of \u03b1-FeTM0 Fig.\u00a0, due to 2O3, catalytic spillover53 of hydrogen into our films was performed through sputtered platinum nano-structures  at low temperatures  was used (see \u2018Methods\u2019). The hydrogen concentration in the H-doped films was found to be in the range ~1.57 \u00b1 0.14\u2009at.% to 2.47 \u00b1 0.14\u2009at.% depending on the H-spillover temperature  performed in tandem with ERDA, which is resonantly sensitive to the O-content of films  curves of the H-doped films revealed a multi-hysteretic in-plane evolution of the antiferromagnetic domains possibly due to the enhancement of local DMI after H incorporation  had little effect on the Morin transition  in a bulk-sensitive fluorescence mode at the Fe K edge. A systematic chemical shift of the Fe K edge and shoulder to lower binding energies was evident in the normalized spectrum and its derivative, of the H-doped samples with respect to the undoped counterparts Fig.\u00a0. Howeverons Fig.\u00a0, unequiv2O3 containing different types of point defects , see \u2018Methods\u2019. In addition, we manually introduced or removed electrons to or from this defect-containing super-cell, to mimic charge-doped regimes of \u03b1-Fe2O3. We found, firstly, that H-dopants prefer to be interstitials, with the formation energy of Hi markedly lower than HO/VO in all charge-doped regimes  spectroscopy of H-doped \u03b1-Fe2O3  calculations of defect formation and charge transfer in a 2\u2009\u00d7\u20092\u2009\u00d7\u20091 super-cell  of \u03b1-FeHi corresponds to the Hi-driven charge transfer. In the neutral and negative-charged cases, we observed that only a small fraction of Bader-charge is transferred from Hi to the nearby O-sites  the classical magnetic-dipolar anisotropy, following a 2O3, these interactions are opposite in sign but similar in magnitude (differing by only ~2% in bulk\u00a0at 0\u2009K), with KSI (KMD) dominating below (above) 50. Hence, the total anisotropy, To understand the observed change of anisotropy and Morin transition, we built a phenomenological model describing their evolution as a function of temperature and H-doping. Based on the discussion in the previous section, the model consists of a matrix of Fesee Fig.\u00a0. EssentiK would evolve approximately linearly26 with the H-concentration, x, as per the relation  concentration (x) into the ratio of Fe2+-species per total Fe cations. The first term in Eq. , and Zi(T) are cation-dependent functions. The latter are obtained from a modified two-sublattice mean-field model , calculated from our phenomenological model for different x values, is shown in Fig.\u00a0x, Hence, the overall effect of hydrogenation is to reduce the single-ion contribution more than the magnetic dipolar counterpart, thereby suppressing the Morin transition. The temperature dependence of the total anisotropy Hi-defects in \u03b1-Fe2O3 . The evolution of T\u2009=\u20090\u2009K. We find that the evolution of 2O3 under H-doping.To test the validity of our model, we also performed DFT calculations of the total anisotropy change, 2O3 with a heavy transition metal increases its total anisotropy, thereby increasing the Morin transition temperature30. Here, we used \u03b1-Fe1.97Rh0.03O3 films which have an elevated Morin transition near room temperature  from stoichiometric targets on single crystalline \u03b1-Al2O3 substrates (CrysTec GmbH), using KrF excimer laser (248\u2009nm). Substrates were cleaned by ultra-sonication in high purity acetone, alcohol and DI Water prior to deposition. The growth was performed at 700\u2009\u00b0C, in oxygen partial pressure of 2/20\u2009mTorr with a laser repetition rate of 3\u2009Hz. Subsequently, in situ high oxygen pressure annealing was performed to minimize oxygen vacancies formed during growth. The films were cooled at 10\u2009\u00b0C/min.\u03b1-Fe2/Ar ratio of 5%/95%) at annealing temperatures in the range 150\u2013270\u2009\u00b0C. Control annealing experiments were carried in high purity 100% Ar or O2 atmospheres under the same temperature conditions.Nano-sized Pt islands used for catalytic H-spillover were grown by sputtering in an Ar atmosphere from a high purity Pt target. The deposition current was 10\u2009mA and duration was 5\u2009s. HAADF mode in STEM identified that Pt formed small and discontinuous nano-structures (NSs) (see Supplementary-\u03c9-scans), reciprocal space mapping (RSM) and \u03d5-scans, and thickness measurements via X-ray reflectivity (XRR) at a four-circle diffractometer in the XDD beamline at the Singapore Synchrotron Light Source (SSLS), see Supplementary-Structural characterization of epitaxial films included high-resolution synchrotron X-ray diffractometry (HR-XRD) involving Atomic positions in the films and the quality of the film-substrate interface were determined through atomic-resolution STEM in a JEOL-ARM200F (with ASCOR aberration corrector and cold-field emission at 200\u2009kV), in HAADF and annular bright field (ABF) acquisition modes. The HAADF and ABF images were acquired with a probe\u2010forming aperture of 30\u2009mrad and collection inner and outer semi-angles in the range 68\u2013280\u2009mrad and 12\u201324\u2009mrad, respectively. While HAADF shows the positions of the heavier elements Fe and Al, ABF shows the positions of O better.2O3 (H-doped and undoped) fine powder samples were placed directly on the crystal for data collection. The spectra were obtained by averaging multiple curves collected using a DTGS detector, with a spectral resolution of 4\u2009cm\u22121, scanner velocity 10\u2009kHz and sampling time of 1\u2009min (68 scans). The bench of the spectrometer was evacuated down to ~1.5\u2009mbar for all experiments. The FTIR spectra were fitted by Lorentzian functions to identify the bond peaks.For the Fourier transform infrared (FTIR) spectroscopy experiments, MIRacle ATR sampling accessory for analysis of solids was used. MIRacle was placed in the sample chamber of Vertex 80\u2009v Bruker spectrometer. \u03b1-Fe67 and molecular bonding changes in \u03b1-Fe2O3, before and after H-doping, were measured by employing Fe K edge X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), respectively, at the XAFCA beamline in SSLS. The measurement was performed in fluorescence geometry and was sensitive to the Fe-signal throughout the films. Normalization, background correction, and analysis were performed in the Athena software. Following the approach in iron-oxide literature, the fraction of reduced Fe2+ species with respect to Fe3+ species in H-doped and control Ar-annealed samples relative to undoped \u03b1-Fe2O3 samples, was estimated by analyzing the\u00a0chemical shifts of Fe K XANES\u00a0 and circularly (XMCD) were carried out at the Fe L edge at BL4.0.2 of the Advanced Light Source (ALS). The measurement was performed in a transmission geometry with the detection in the luminesce-yield mode (which is a transmission measurement using sapphire as a scintillator and is thus sensitive to X-ray absorption through the entire film) in an open-flow liquid nitrogen cryostat which allowed dichroism measurements in the range 80\u2013325\u2009K. X-rays were incident with a grazing angle of 30\u00b0 relative to the surface. For the XLD asymmetry, defined as M(T) curves were measured after field-cooling the samples with an applied in-plane field of 500 or 5000\u2009Oe, and a measurement field of 500\u2009Oe applied during the subsequent warming and cooling scans. Changing the cooling-field magnitude had no discernible effect on the transition temperature. M(H) curves were carried out at a series of temperatures. The diamagnetic \u03b1-Al2O3 substrates contribute a background in the raw M data. This background is removed to extract the final magnetometry curves of the thin films.Magnetic characterization was performed in a superconducting quantum interference device (SQUID) magnetometer of Quantum Design MPMS. 16O16O, to accurately measure the O-content in the \u03b1-Fe2O3 films (see Supplementary-\u22126\u2009Torr. The He\u00a0ions were incident at an angle of 75\u00b0. Backscattered He (for RRBS) and recoiled H (for ERDA) were collected in surface-barrier detectors placed at 165\u00b0 and 30\u00b0 with respect to the incident ions, respectively. A 14-\u03bcm-thick Al absorber-foil was used to stop the forward-scattered He and only allow recoiled H toward the ERDA detector. Both ERDA and RRBS spectra were fit using the SIMNRA software. For RRBS experiments, the energy calibration was performed by using a standard tungsten silicide sample. To calibrate the yield and energy of ERDA measurements, a quantitative analysis of H density was obtained by using an H-implanted Si standard and by performing ERDA measurements at two energies in the range ~2.80-3.04 MeV.The quantitative analysis of H-dopant concentration in samples was performed using elastic recoil detection analysis (ERDA) wherein He ions incident on the sample lead to selective forward recoil scattering of H atoms from the sample. ERDA experiments were performed in tandem with oxygen resonant Rutherford backscattering spectrometry (RRBS) at Inter-University Accelerator Centre (IUAC), using 1.7\u2009MV pelletron Tandem accelerator. The ERDA and RRBS measurements facilitated a direct quantitative determination of Fe/O/H concentrations simultaneously in the samples. RRBS experiments were performed with a resonant energy for oxygen reaction 68 was used for the d-electrons of Fe atoms, and the value of U was set to 4.5\u2009eV, which brings the indirect electronic bandgap of \u03b1-Fe2O3 from 0.56\u2009eV (PBE result) to 2.0\u2009eV, consistent with our experimental bandgap value from UV-Vis-NIR absorption spectroscopy. In addition, spin\u2013orbit coupling (SOC) was also involved in all the calculations to accurately obtain information related to magnetic anisotropy.Density functional theory (DFT) based first-principles calculations were performed through the Vienna Ab-initio simulation package (VASP). The projector-augmented-wave and the Perdew\u2013Burke\u2013Ernzerhof (PBE) method were used for the pseudo-potentials and the exchange-correlation functionals, respectively. The plane-wave cut-off energy was set at 600\u2009eV. The GGA+U approach\u03b1-Fe2O3, respectively, to mimic different doping concentrations. The Monkhorst\u2013Pack grid with 7\u2009\u00d7\u20097\u2009\u00d7\u20093, 7\u2009\u00d7\u20093\u2009\u00d7\u20093, and 3\u2009\u00d7\u20093\u2009\u00d7\u20093, k-point meshes were used, respectively. For each of these cases, defects both of the interstitial (Hi) and substitutional (HO) type were considered. In addition, eight different initial sites for Hi were considered, shown in Supplementary-Etot(X) and Etot(bulk) are the calculated total energies of the system with and without defect X, respectively. nj is the number of atoms of type j (either host atoms or dopants) that have been added to (nj\u2009>\u20090) or removed from (nj\u2009<\u20090) the system to form that defect, and \u03bcj is the corresponding chemical potential of the species. Here, the chemical potential of H and O are\u00a0obtained by considering hydrogen and oxygen molecules\u00a0as their sources, respectively.For hydrogen-doping calculations, one H atom was incorporated into 1\u2009\u00d7\u20091\u2009\u00d7\u20091, 2\u2009\u00d7\u20091\u2009\u00d7\u20091, and 2\u2009\u00d7\u20092\u2009\u00d7\u20091 unit-/super-cells  of 69,q indicates the charged state, EF is the Fermi energy, EVBM the reference valence band maximum (VBM) position, and \u0394V is a correction term added to align the VBM level of the cell containing the defect (in different charge states) to that in undoped \u03b1-Fe2O3, as discussed in ref. 69.Moreover, defects/host atoms can further acquire or lose electrons, forming charged states. This effect is simulated by performing the calculation with excess electron(s) added to or removed from the cell, respectively. The relative stability of each charged state of the cell containing the defect can be measured by modifying the above formation energy in Eq.  as follo\u03b1-Fe2O3, we adopted the Bader-charge calculations as it considers not only the core charge but also the valence charge, serving as a good approximation of the total electronic charge.Lastly, to analyse charge transfer between atoms in H-doped Supplementary Information"}
{"text": "Bartonella. The aim of this study was to investigate the presence of Bartonella spp. in African giant pouched rats (Cricetomys gambianus) and their ectoparasites in Dakar, Senegal. In 2012, 20 rats were caught, and their fleas were identified. DNA was extracted from 170 selected fleas and qPCR was carried out to detect Bartonella spp. Subsequently, a Bartonella culture was performed from the rat blood samples and the isolated strains  were genotyped. A total of 1117 fleas were collected from 19 rats and identified as Xenopsylla cheopis, the tropical rat flea. Bartonella DNA was detected in 148 of 170 selected fleas (87.1%). In addition, Bartonella strains were isolated from the blood of 17 rats (85%). According to Bartonella gene-sequence-based criteria for species definition, the isolated strains were identified as B. massiliensis (four strains) and two potential new species related to the zoonotic B. elizabethae. In this paper, these potentially new species are provisionally called Candidatus Bartonella militaris (11 strains) and Candidatus Bartonella affinis (two strains) until their description has been completed. Cricetomys gambianus and its fleas could constitute a public health risk in Dakar due to the high prevalence of Bartonella infection reported.Bartonellae are bacteria associated with mammals and their ectoparasites. Rodents often host different species of  Bartonella is the only genus of bacteria belonging to the family Bartonellaceae. These microorganisms are Gram-negative and facultative intracellular bacteria. Currently, more than 40 Bartonella species have been described. Many of them can infect a large range of different mammal species, often rodents, which are regarded as the main reservoir hosts of many species of these bacteria. At least 20 species of Bartonella are rodent-associated [Bartonella bacilliformis, Bartonella quintana and Bartonella henselae; these species are not associated with Rodentia. However, many small rodent-associated bartonellae have been reported to cause human diseases. Rattus-associated B. elizabethae, B. washoensis and B. vinsonii arupensis may cause culture-negative endocarditis, B. grahamii is suspected to induce neuroretinitis and endocarditis and B. tribocorum may cause chronic bacteriaemia [Bartonella species [sociated  and moreeriaemia ,4. Blood species .Cricetomys gambianus), also known as the Gambian pouched rat, is a large rodent in the exclusively African family Nesomyidae. This is one of the largest rodents in the world. It weighs between 1.0 and 1.4 kg and grows to approximately 0.9 m long, including the tail, which makes up half of its total length [Cricetomys gambianus is widespread in different natural biotopes and can frequently be found in semi-urban places, near human habitations. This species is nocturnal and eats fruit, seeds and small animal-like insects. In many African countries, especially in West Africa, giant pouched rats are considered as an important source of meat and are hunted by local populations [C. gambianus is a commensal animal and one of the most common rodents encountered in the city of Dakar.The African giant rat , a residential quarter of Dakar, Senegal. Metal cages were placed outdoors, in areas surrounding homesteads, in deep gutters and rainwater harvesting systems. These traps were checked for the presence of rodents every day, early in the morning. Each cage containing a rat was transported to the French military veterinarian at the camp, who identified the rat and collected the ectoparasites.\u00ae, Boehringer Ingelheim, Lyon, France). Whole blood was collected by cardiac puncture in an EDTA-coated tube.A system was used to restrain the animal. After restraining it, each rodent was anesthetised by an intramuscular ketamine injection  were stored  and transported two weeks later to the IHU M\u00e9diterran\u00e9e Infection, Marseille, France. In the IHU, ectoparasites were stored at \u221220 \u00b0C and blood samples at \u221280 \u00b0C until analysis.Within the framework of the Nagoya accord application, we have obtained authorization to access and use, in France, genetic resources from captured rodents .18S rRNA gene [All ectoparasites were morphologically identified using identification keys. Each one was photographed using a Stereo Discovery V20 stereomicroscope. The ectoparasites\u2019 genus and species were then confirmed for some specimens, selected randomly, by using primers targeting the RNA gene .\u00aeDNA tissue kit,  and was performed on a BIOROBOT EZ1  per the manufacturer\u2019s instructions. Prior to DNA extraction, ectoparasites or 200 \u03bcL of rat blood was digested with proteinase K and incubated at 56 \u00b0C overnight.DNA was extracted individually from selected ectoparasites, as well as from all rat blood samples. Extraction was performed using the commercial EZ1Bartonella DNA) and negative (only mixture) controls were added in each reaction.DNA was eluted in 200 \u03bcL of distilled water and stored at \u221220 \u00b0C until analysis. The qPCRs were prepared and performed in 20 \u03bcL final volume containing 10 \u03bcL of Master Mix Roche , 0.5 \u03bcL (of 20 \u03bcM concentration) of each primer, 0.5 \u03bcL of the probe (of 5 \u03bcM concentration), 0.5 \u03bcL UDG, 3 \u03bcL of distilled DNAse- and RNAse-free water and 5 \u03bcL of the DNA sample. The assay was performed in a CFX96 Real-Time system  using the protocol: one incubation step at 50 \u00b0C for two minutes and an initial denaturation at 95 \u00b0C for five minutes, followed by 40 cycles of denaturation at 95 \u00b0C for ten seconds and annealing and extension at 60 \u00b0C for 30 s. Positive  region for the selected ectoparasites and all rat blood samples. Positive samples were screened again using the ITS2 qPCR . Plates were incubated at 37 \u00b0C with 5% COal genes . In paraBartonella isolates were tested by PCRs for species identification using the primers in https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 28 December 2021). The phylogenetic analyses, based on 16S rRNA, rpoB, FtsZ and ITS regions of Bartonella spp., were inferred using neighbour-joining methods, and tree reconstructions were performed using MEGA software version 7 [All of the selected fleas were suggested to the 18S-based standard PCR. ersion 7 . BootstrCricetomys gambianus species, the African giant pouched rat. Overall, 19 of the 20 (95%) rats carried fleas (18S rRNA) confirmed the morphological identification of the fleas. All of the specimens belonged to the same species: Xenopsylla cheopis, the tropical rat flea, also known as the Oriental rat flea.Twenty rodents were captured during the study period. They were all formally identified as ed fleas . The numBartonella DNA by qPCR. Bartonella spp. DNA was confirmed in 148 of the 170 fleas (87.1%) that were analysed. Overall, 17 Bartonella isolates were isolated from the blood of 17 of the 20 captured rats . Using MALDI-TOF MS, four isolates were identified as Bartonella massiliensis with scores > 1.7. The other isolates were not identified to the species level, although their MS spectra undoubtedly clustered within pools of other Bartonella spectra.Regarding the 1117 tropical rat fleas collected, we selected 170 fleas  for screening for 16S rRNA (1400 bp) showed that four almost similar sequences, also identified by MALDITOF-MS, exhibited a 99.9% to 100% identity with Bartonella massiliensis OS09 (HM636440). These strains also showed a 99\u201399.8%, 97.8\u201398.9% and 99\u201399.3% identity in the FtsZ, ITS and RpoB regions with B. massiliensis, respectively. All of the other 16S rRNA sequences of the other isolates were close to each other and showed a 99.57% to 99.64% identity with Bartonella elizabethae (LR134527) and Bartonella kosoyi strain Tel Aviv (MN627780). They had a similarity of 95.3\u201396.9%, 86.7\u201394.5% and 92.8\u201396.8% with these two species at the FtsZ, ITS and RpoB regions, respectively. Some sequences were deleted from analysis because of their low sequencing quality  and in tropical rat fleas (Xenopsylla cheopis) in this country.This study reports the isolation, molecular detection and genetic characterisation of Bartonella strains have been detected in rodents in several countries in Africa: Algeria, Tunisia, Egypt, Democratic Republic of Congo, Ethiopia, Tanzania, Kenya, Uganda, South Africa, Nigeria, Benin and Senegal. The infection rates reported ranged from 4% to 67% [Cricetomys gambianus trapped and examined by PCR detection and culture were Bartonella-positive. A genotype related to Bartonella elizabethae was found in one of these Cricetomys gambianus. Bartonellae, similar to Bartonella massiliensis isolated from a soft tick species Ornithodorus sonrai in Senegal [C. gambianus [Bartonella sp. was detected in 34.7% (69/199) of X. cheopis fleas collected in Cotonou, Benin [Bartonella species were also detected in both C. gambianus blood and X. cheopis carried by the rats, in Nigeria [Bartonella species had already been detected in ectoparasites collected from humans and animals. Bartonella quintana was identified in lice found on the heads of patients in Dakar [Bartonella were isolated from rodents trapped in the Sine-Saloum region of Senegal: B. saheliensis sp. nov from the gerbil Gerbilliscus gambianus [B. mastomydis sp. nov. from the Guinea multimammate mouse Mastomys erythroleucus [B. senegalensis sp. nov and B. massiliensis sp. nov) were also found in the soft tick Ornithodoros sonrai, the vector of relapsing fever [% to 67% ,15,16,17 Senegal , were foambianus . In Westu, Benin . Bartone Nigeria . In Senein Dakar . Two newambianus  and B. mroleucus . Other nng fever .Bartonella is very high, even regarding previous reports on rats and their ectoparasites. In 2017, a study reported a collection of 407 fleas from small mammals trapped around homes in five villages, in north-western Uganda. Among these fleas, 202 (49.6%) were identified as X. cheopis, mainly from Rattus rattus (n = 148), the black rat. The Bartonella prevalence in oriental rat fleas collected from all of the rodents was relatively low . The phylogenetic relationships of Bartonella genetic variants, identified from sequences obtained by analysing fleas, showed the infection by variants partly belonging to the B. elizabethae species complex [Bartonella DNA in 27.5% (53/193) of X. cheopis fleas collected from Rattus rattus specimens. The genotypes were closely related to different Bartonella species, including B. elizabethae [In this study, the prevalence of n = 1117) of Xenopsylla cheopis collected from only 20 C. gambianus suggests a large infestation of the African giant rats by fleas in the location studied and, potentially, in the surroundings. The flea X. cheopis is a well-known parasite of rodents, primarily of the genus Rattus, but can live on many warm-blooded mammals. Moreover, this flea species is a vector for bubonic plague, caused by Yersinia pestis, and can transmit Rickettisa felis, the aetiological agent of murine typhus [Bartonella prevalence in this study can, first, confirm the role of C. gambianus and X. cheopis as reservoirs and vectors, respectively. Second, this high prevalence can be due to the infestation rate of rats (around 55.8 fleas per rat): this facilitates the transmission to and between rats and the reinfection of fleas in return. It seems that the high infection rate in rats conduces to a high detection rate in fleas feeding on these rats. Other arguments are needed to demonstrate that fleas can maintain these Bartonella spp. (such as the persistence of flea infection).In our study, the high number  Candidatus Bartonella affinis (16S, RpoB and ITS, whereas FtsZ did not show this difference, which required the use of more than one target to identify bacteria belonged to the bartonella genus. The high Bartonella infection rate in rodents and their ectoparasites, observed here and in the previous studies, could explain this emergence of new species. However, the role and/or pathogeny of this species in humans or animals remain to be explored. Since our team began studying Bartonella in rodents over the past decade, at least seven species have been isolated and described, not counting these two species: B. florenciae, B. mastomydis, B. massiliensis, B. senegalensis, B. saheliensis, B. gabonensis and B. raoultii [The isolated strains from rats are, in general, close to the finition . Here, w affinis . The genraoultii ,27,28,29Bartonella sp. found in this study may alert local sanitary authorities to the public health risk for hunters or breeders, but also for inhabitants, given that rat holes have been observed in residential areas, especially in semi-urban locations.In addition, due to its size and weight, the African giant rat is considered as a natural resource of animal protein by the local population . The preB. elizabethae, a species that is currently recognised as a zoonotic agent responsible for endocarditis [B. quintana was described in a Senegalese patient [B. quintana in febrile patients visiting a health facility in rural Senegal [The potential new species described in this study are related genetically to carditis , neurorecarditis ,31 and bcarditis  in humancarditis . In Sene patient  in 2002. Senegal .X. cheopis commonly carry the bacteria of the genus Bartonella and potentially play an important role in the transmission of potential zoonotic infections among Gambian pouched rats and from rats to humans. Due to the nature of the Bartonella species detected, this study shows the importance, from a public health point of view, of leading further investigations into the potential zoonotic Bartonella species in Senegal and, more generally, in Africa. Future research may include more epidemiological studies on pests and farm animals due to the close and increasing interaction between humans, animals and the environment. The new potential species merit a complete description, including their genomic features, due to their microbiological, epidemiological and pathological characteristics being unknown to date.This study helps to confirm that"}
{"text": "Their expression profiles were different in the basal and superficial layers. Most of the processes influenced by the UPA in the basal layer were connected to the cell cycle and immune regulation. (4) Conclusion: Specific changes were observed in both layers of the endometrium in the UPA group. However, the miRNA expression in the basal layer was not consistent with that in the superficial layer. Other large studies analysing the long-term impact of SPRM on endometrial miRNA expression are necessary.(1) Background: Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM) widely used for emergency contraception and mid- to long-term leiomyoma treatment. The aim of this study was to identify modifications of miRNA expression in superficial and basal layers of the human endometrium at the end of the UPA treatment for at least 3 months. (2) Methods: Microarray miRNA analysis of formalin-fixed, paraffin-embedded hysterectomy tissue samples was conducted, followed by an Ingenuity Pathway Analysis. Samples were divided into three groups: women having had 3 months of UPA treatment ( Ulipristal acetate (UPA) is a selective progesterone receptor modulator (SPRM). It binds to the major progesterone receptor (PR) isoforms and exerts a wide spectrum of actions, from antagonistic to agonistic effects, depending on the target tissue . It is wUPA, like other SPRMs, has a reversible impact on the morphological aspect of the whole endometrium . EndometAt a molecular level in the myoma tissue, UPA has been shown to modify a number of processes including a decrease in inflammation and induction of cell apoptosis ,10. SpecMicroRNA (miRNA) are small non-coding RNAs. They modify the expression of around 60% of proteins at the posttranscriptional level and promote mRNA destruction. The miRNAs are involved in a broad variety of physiological mechanisms such as morphogenesis, differentiation, apoptosis or cellular metabolism .Some miRNAs have been associated with endometrial receptivity  and flucThere are no data about the miRNA expression regulation in UPA exposed endometrium. Furthermore, comparing miRNA expression in the superficial and basal layers of the endometrium could provide information about the potential persistence of UPA impact on the endometrium. In fact, the superficial layer sheds every month while the basal layer persists.The aim of this study was to identify modifications of miRNA expression, and thus the potential target pathways, in both superficial and basal layers of the human endometrium after 3 months of UPA treatment, before treatment discontinuation, in women treated for symptomatic myomas.Endometrium-containing blocks of hysterectomy samples from non-menopausal women aged 40\u201352 years with moderate to severe symptoms of myomas requiring hysterectomy between 2014 and 2017 were retrospectively collected in three university hospital surgical gynaecology units in Paris, France . Women who had hormonal treatment for at least 3 months before surgery or a history of cancer were excluded. The clinical variables of the women included age, parity and body mass index . The localisation of the myoma according to the FIGO classification  and withThe menstrual phase was assessed on a histological basis and on evaluation of the ER and PR expressions ,18. The The Obstetrics and Gynecology Research Ethics Committee (CEROG\u2014Comit\u00e9 d\u2019Ethique de la Recherche en Obst\u00e9trique et Gyn\u00e9cologie) allowed retrospective sample analysis and approved the study . All women gave pre-operative written informed consent for the participation in the study.Formalin-fixed, paraffin-embedded (FFPE) tissue sample sections of 3 \u03bcm were processed and stained with Haematoxylin and Eosin by standard methods. Immunohistochemistry of the ER and PR were performed on the Ventana Ultra platform using the manufacturer\u2019s recommended settings.Whole tissue sections (4-\u00b5m) were deparaffinized, rehydrated, treated and incubated for 16\u2009min with the pre-diluted anti-ER antibody  and the pre-diluted anti-PR antibody , and then visualized using the ultraView Universal DAB Detection Kit .All evaluations were performed under blinded conditions by a single expert pathologist (CB). Endometrial differentiation was assessed by histological dating according to the Noyes criteria , and oesFFPE tissues were obtained from hysterectomy specimens. Macro-dissections were undertaken with a surgical scalpel on 20 \u03bcm sections under a light microscope to separate the superficial and the basal layers of the endometrium. The superficial layer sampling included the superficial endometrial epithelium and the first row of endometrial glands and scattered stroma. The basal layer sampling included the juxta-myometrial portion of the endometrium with dense stroma. The intermediate layer of the endometrium was excluded from analysis. The samples were stored at 4 \u00b0C and RNA extraction was performed within 2 h.Total RNA was extracted using the miRNeasy FFPE Kit  according to the manufacturer\u2019s instructions. The purity, quality and concentration of all RNA samples were examined by using a microspectrophotometer Bioanalyzer 2100  . The ana\u00ae, Santa Clara, CA, USA) was conducted at the genomic platform of the Institut Cochin, Paris. Each sample was tested once as the microarray hybridization is a robust technology with a broadband analysis. Specific miRNA analysis was performed using Partek\u00ae Flow\u00ae software version 3.0 . CEL files were imported and normalized using robust multi-array averaging (RMA) [p-value \u2264 0.05 were considered to be differentially expressed. The miRNA showing a 2-fold change or more were considered for further analysis. The results were synthetically presented using Venn diagrams. Microarray analysis was conducted on seven samples from seven women treated for at least 3 months with UPA and who were still using it (UPA group), eight samples from eight control women in the proliferative phase (proliferative phase group) and six samples from six women in the secretory phase (secretory phase group). Microarray hybridization on the miRNA 4.0 chips (Affymetrixng (RMA) . Those mhttp://www.ingenuity.com, accessed on 30 April 2018).Evaluation of the impact of UPA on bio-functions and canonical pathways associated with endometrial receptivity was conducted using Ingenuity Pathway Analysis software (IPA: https://www.r-project.org/ (accessed on 19 March 2018).Unless otherwise specified, data were managed using an Excel database and analysed using R version 3.1.3 , available online at n = 7) (n = 8) or the secretory phase . The menstrual phase was assessed on a histological basis and on ER and PR expression evaluations [Three groups of patients were included in the study. The UPA group included women operated on after a 3-month course of UPA, before treatment discontinuation  (n = 7) . Two disluations ,18.The baseline characteristics of the three study groups\u2014the UPA group, and the proliferative phase and secretory phase groups\u2014were comparable . The FIGIn the UPA group, focal and not pronounced PAEC were observed in two cases (29%). The proliferative aspect of the endometrium in the UPA group was observed in six samples and one endometrium was characterised by an early secretory aspect.Endometrial miRNA profiles in the UPA, proliferative phase and secretory phase groups were analysed in each endometrial layer separately using microarray hybridization.The median ratio of the RNA integrity number (RIN) of the analysed samples was 2.3 (interquartile range (IQR) 2.2\u20132.4) and the median concentration of RNA was 66.0 ng/\u03bcL (IQR 36.5\u2013102.8).The expression of miRNA was differentially regulated in the UPA group as compared to the proliferative and secretory phase groups in both the basal and superficial layers of the endometrium .p < 0.05 and fold change > |2| identified two up-regulated and 17 down-regulated miRNAs in the UPA group compared to the proliferative phase group. According to the IPA analysis they were experimentally proven to regulate three and 269 mRNAs, respectively. In the superficial layer the cut-off values for Two miRNAs were up-regulated and 22 down-regulated in the UPA group when compared to the secretory phase group. They were experimentally proven to regulate 23 and 125 mRNAs, respectively. Among these miRNAs, miR-184 was differentially expressed in the UPA group as compared to both the proliferative and secretory phase groups a.In the basal layer, 21 miRNAs were down-regulated and none up-regulated in the UPA group as compared to the proliferative phase group. They were experimentally proven to regulate 444 mRNAs. In comparison to the secretory phase group, three miRNAs were up-regulated and 12 down-regulated in the UPA group. These miRNAs regulate the expression of 11 and 103 mRNA, respectively. Among these miRNAs, miR-1246 and miR-3197 were differentially expressed in the UPA group compared to both the proliferative and secretory phase groups b. Among the 59 miRNAs identified as being modified by the UPA treatment, only 16 showed the same UPA-related profile in both layers of the endometrium. Among these, eight miRNAs were differentially expressed in the UPA group as compared to the proliferative phase group  c and eigUsing IPA, we conducted a functional analysis of the mRNAs regulated by the miRNA previously described in the miRNA expression section. The top 10 statistically most significant biological processes potentially involved in the physiopathology of the human endometrium for each analysis group are presented in IPA analysis of the list of mRNAs of interest  identified the top ten canonical pathways involved. The results are presented in The most significantly influenced canonical pathways were connected to the cell cycle, cell signalling linked to cancer development and inflammation. The miRNAs involved in these processes were down-regulated.The aim of this study was to analyse the effect of the UPA on the endometrial expression of miRNA as compared to the proliferative and secretory phases in superficial and basal layers of the human endometrium. The analysis revealed 59 miRNAs differentially expressed in the UPA group. The identified miRNAs showed differential expression profiles in the basal and superficial layers.Among the identified miRNAs, miR-449a, miR-449b-3p, miR-449b-5p and miR-449c-5p showed the biggest fold change in both endometrial layers. They were down-regulated in the UPA group as compared to the proliferative phase group. These miRNAs are known to be up-regulated in the endometrium of women with repeated embryo implantation failure (RIF) during the implantation window . The miRAmong the identified miRNAs, miR-188-5p has been described to be up-regulated in endometrial cancer as compared to normal endometrium . In our The literature data on the involvement of identified miRNA in embryo implantation and endometrial cancer are resumed in Our IPA analysis of the top ten statistically most significant biological processes regulated by the identified miRNA showed that UPA might potentially indirectly modify the expression of ESR1 (ER\u03b1) and PTEN mRNA. The expression of these mRNAs has previously been found unchanged in endometrium exposed to UPA . HoweverUPA binds to both major isoforms of progesterone receptors and, together with transcription co-regulators, induces or inhibits the signal transduction ,27. Its The impact of UPA on the basal layer of the endometrium suggests a potential long-term impact since the superficial functional layer sheds with menstrual bleeding, while the basal layer persists. The endometrial basal layer may be considered as the \u2018memory\u2019 of the endometrium, as it could be a source of regeneration of the functional layer among others mechanisms, by harboring endometrial stem cells . HoweverThe presence of PAEC in two out of seven samples was observed. This ratio is higher than that described in the literature. However, the changes were focal and not pronounced.The major strength of our study was the large number of patients drawn from a homogeneous population of women with symptomatic myomas included in the analysis. Furthermore, as we analysed hysterectomy samples, we were able to evaluate both endometrial layers. The miRNA analysis was performed using a robust technology with a broadband analysis. The quality control was undertaken for all samples before the analysis.The limitations of this study included its retrospective design and the absence of hormonal assessment of the menstrual cycle phase. However, as symptomatic myomas usually require hormonal treatment, the number of control samples was limited. Another limitation was that all the included patients were experiencing severe myoma-related symptoms and our data cannot be extrapolated to other clinical situations. UPA licence has been discontinued since March 2020 due to severe adverse events and more particularly to liver cell damage, therefore larger studies on UPA in the indication of uterine leiomyoma are impossible in the current circumstances. However, the data may be of scientific interest for other SPRM molecules with mixed agonist and antagonist effects. Finally, the use of paraffin-embedded samples reduced the quality of total RNA (RIN around two). RIN is poorly informative on the miRNA quality obtained from FFPE tissue according to the literature ,31 and mOur results show that UPA specifically modifies the expression of 59 miRNAs, the majority of which have been described as being involved in processes connected to embryo implantation and cancerogenesis. The changes in miRNA expression observed in the basal layer, from which the functional layer renews every month, are different from the molecular changes observed in the superficial layer.While miRNA can act as promoters or inhibitors of cancerogenesis, the regulation of miRNA observed in the UPA exposed endometria was opposite to the modifications described in the literature for tumoral endometria in all but two miRNA. Other large studies analysing the role of miRNAs in the endometrium and the long-term impact of SPRMs on endometrial miRNA expression are necessary.miRNAs are involved in the inflammatory processes playing essential roles in embryo implantation. All but four miRNA were oppositely expressed in the UPA exposed endometrium compared to the published data on the endometria of RIF women.These results deserve further confirmation but suggest that UPA might be used in women willing to conceive without impairing the chances of pregnancy or increasing the risks of developing endometrial malignancies."}
{"text": "Important information about medical laboratory providers is not readily available to all patients, clinicians nor regulators in Kenya. This study was conducted as part of a wider project aiming to improve access to high quality diagnostics by addressing information asymmetries in the Kenyan market for laboratory services.The purpose of this study was to: 1) Gather pricing information for 49 common laboratory tests from medical laboratories in Nairobi, Kenya, noting where these prices were publicly available or withheld. 2) Assess patients\u2019 knowledge of testing information including: turnaround time, price, and test availability.This was a cross-sectional study where a mystery caller approach was used to survey 49 tests for turnaround time, price, and availability across 13 laboratories selected purposively. The mystery shopper survey was complemented by 251 patient exit interviews at two Kenyan hospitals to understand whether patients seeking laboratory tests in Nairobi had access to such information. All 251 patients were selected by convenience sampling.We noted that 85% of the private laboratories did not disclose test prices and turnaround times to their patients. There was a wide range of prices on several key tests, with private in-facility laboratories charging an average test price of 468% of the average test price in public laboratories across all the 49 tests. We also found that many patients lacked key information regarding the tests they needed: 65% did not know the purpose of the test while 41% did not know the test price at all.Under the current system, patients have limited access to information regarding the key criteria required to make a rational decision. This has a significant impact on the quality, price, and turnaround time (TAT) offered by the medical laboratories that operate in this dysfunctional market. Timely access to laboratory diagnosis is crucial to the provision of high quality health care . This isIn some contexts, where patients are not covered by insurance and are paying out of pocket , they tePatients, like their clinicians, also expect high quality diagnostic laboratory services. According to WHO, laboratory quality is defined by the accuracy, reliability, and timeliness of the reported test results . To achiIn summary, research to date suggest that patients make choices based on test price, convenience and their own perception of quality . These pThis study was approved by the Aga Khan university Ethics Committee (Ref No: 2017/REC-103 (V6)).It is regrettable that we were obliged to use a mystery shopper approach to gather test information for many of the laboratories. Such approaches do not allow for informed consent, and it would have been preferable to have communicated openly with the laboratory providers, but this option was not possible unfortunately. Anecdotal evidence would suggest that the laboratories withhold pricing information because they do not wish to compete on price. This issue should be a focus of further study, but for practical reasons during this preliminary research, we adopted the mystery shopper approach.This study was conducted in Nairobi, the capital city of Kenya. According to the 2019 census report, Nairobi has a population size of about 4.4 million people . The medWe collected primary data both on test prices and availability from laboratories as well as from patients seeking tests.A list of all the licensed medical laboratories in Nairobi was obtained from the KMLTTB and purposive sampling was used to generate a list of the top medical laboratory service providers. In the purposive sampling approach, priority was given to laboratories that have a large share of the private clinical laboratory market, operated as chains and had operational licenses as at the time of the survey. We also included an academic (not-for-profit) laboratory, faith-based and public clinical laboratories. Our study sample was made up of 13 medical laboratories consisting of seven private laboratories, two faith-based, three public and one academic (not-for-profit) laboratory. The private laboratories were later categorized into stand-alone and \u2018in-facility\u2019 laboratories. One of the three public laboratories was attached to the National Referral Hospital (NRH). All seven private laboratories operated as part of large commercial chains. The seven chains represented by these seven laboratories have a total of 91 laboratories in their networks within Nairobi. This represents about 18% of the total number of the licensed private laboratories.The cross-sectional survey was conducted between March and June 2019 to obtain testing information for each of the listed 13 laboratories. The information sought for every laboratory included test availability, test prices and the respective TAT. Most of the tests used in this survey were obtained from a list of the top 25 tests that Horton et al found to be the most common by volume across six different international hospital sites .Save for one private stand-alone laboratory, most of the private and faith-based medical laboratories did not display their test price and TAT publicly to their customers. Although test prices and TATs among some public hospitals were not displayed publicly, patients who inquired were informed how much the test would cost and the stipulated TAT. The mystery caller approach was used to collect this information from the laboratories that did not display the information publicly . Two traWe sought to understand if patients had access to relevant information about the tests they needed and how the information they had informed their choices. To achieve this, convenience sampling was used to recruit patients seeking laboratory tests for face to face interviews as we did not want to cause long queues and unnecessary delays . A semi-We set up a station at the entrance of each of the two laboratories for two days at each facility and patients exiting all clinics including antenatal care with test request forms were directed to us by the clinicians. Data were collected in two sessions every day, the morning session and the afternoon session. At least 60 study participants of 18\u00a0years and above were targeted on each day of data collection. This was approximately 30% of all the patients seeking laboratory tests in their respective hospitals. Patients under 18\u00a0years of age were only recruited if they were accompanied by an adult who became the study respondent.A semi-structured questionnaire was then used to obtain information from the patients and their guardians. For example, the patients were asked whether they knew the price of the test they wanted to request, its availability and the TAT. The tests requested were confirmed from the patients\u2019 test request forms. Additionally, we also asked them if they considered test quality when seeking for laboratory tests, how they identified good quality laboratories and if they knew that poor quality tests could result in misdiagnosis. We later informed them of the exact test price, TAT and whether the laboratory in the same public facility was offering the test at that time. All the responses provided by the participants were entered into REDCap and sent to the server of KEMRI Wellcome Trust.Although data were collected from 251 patients seeking laboratory tests, a total of 6 respondents were excluded from the analysis due to incomplete data. The findings reported are therefore based on the responses provided by 245 study participants. The data were analysed both in Microsoft excel 2016 and R statistical software version 4.0.5 [Pricing Survey: The study collected information on 49 tests within 13 central laboratories (described above). We noted that 85.7% of the private facilities and the two faith-based laboratories did not provide information on test price and TAT publicly to the patients. None of the three public facilities displayed their TATs publicly to their clients. However, two of these public laboratories displayed their test prices for their clients to refer and consider. Test availability was highest in stand-alone laboratories as they offered 95.9% of the tests on average, compared with 91.8% in private-in-facility laboratories and 51% in public laboratories. Test availability within public laboratories 8, 9 and 10 was 65.3%, 40.8% and 46.9% respectively . In termThere was great variation in test prices among the laboratories. Mean prices across all 49 tests in stand-alone private laboratories were 300% of the mean in public laboratories and 468% in private in-facility laboratories compared to public. Public laboratories have the lowest mean test prices, with some tests such as HIV viral load and other tests for children under 5\u00a0years being free of charge. Malaria smear test had the largest price discrepancy with the private-in-facility laboratories charging up to 6 times more on average as compared to the public laboratories .Figure A total of 251 patients or their guardians were interviewed from the two public hospitals although our analysis was based on 245 of them who had complete data. Of the 245 respondents, 70.6% were female while 29.4% were male. The average age of the respondents was 33.7\u00a0years and 60.8% of them were employed. The overall number of tests requested was 371 as observed from the 245 test request forms. The highest number of tests noted on a single test request form were six and these included: Liver Function Tests, full haemogram (Complete Blood Count plus a smear), sickling test, Urine chemistry, Oral Glucose Tolerance Test and Syphilis test (VDRL). The full haemogram test, Urine chemistry and Malaria smears were the most frequently ordered tests at 86, 43 and 31 respectively as shown in Of all the 245 study respondents, 64.9% did not know the purpose of the laboratory tests requested by the clinician. Only 86 participants attempted to state exactly what the laboratory test was for and out of these, 75.6% correctly determined the use of the laboratory test requested while 24.4% made an incorrect statement about the purpose of the test.The majority of patients had no information on the test price. Of the total respondents, 41.2% said that they did not know the price of the tests at all while a further 34.7% who attempted to estimate the test price underestimated it. On average, patients that attempted to guess the actual test price underestimated it by 41.5%. Although 59.6% of all the respondents mentioned that they valued quality when it comes to laboratory testing, 45.2% of those who valued quality agreed that it was difficult to identify a good quality laboratory. Generally, 38% of all the study respondents found it difficult distinguishing good quality laboratories from poor quality ones. Nonetheless, some patients (13.1%) trusted government laboratories as good quality laboratories while 15.1% others mentioned that they relied on reviews from other people such as friends and relatives as indicators of quality in medical laboratories. Almost 10% of the respondents trusted that doctors/clinicians always referred them to good quality laboratories .Figure Based on the actual TAT information provided by the laboratories, the average TAT of the 371 tests requested by patients was two hours with a range of 1\u201372\u00a0hours. The longest turnaround time was 72\u00a0hours for a test requiring bacterial culture . At least, 28% of the interviewed patients did not know how long they would wait to get their laboratory results. Almost half of the respondents underestimated TAT while 14.7% overestimated the TATs. Only 11.4% of the laboratory seeking patients correctly identified the TATs of their tests .Figure Our preliminary data revealed a significant lack of transparency in the medical laboratory market in Nairobi, especially in the private sector. While opacity on price is well noted in developed settings , we are As the sole regulator of clinical laboratory services in Kenya, KMLTTB is mandated with five main responsibilities: indexing or examination of college students taking up careers in medical laboratory sciences; registration and licensing of all laboratory staff in Kenya; developing policy guidelines for the implementation of the Continuing Practice Development (CDP); and regulation of all In vitro Diagnostics (IVDs) intended for laboratory testing. Most importantly, KMLTTB regulates the conduct, practices and licensing of laboratory businesses in Kenya . HoweverThe average test price among private-in-facility laboratories was 468% of the average test price in public laboratories. Similar findings on test price variations have also been reported among private laboratories in Kampala, Zimbabwe and the USA . The higSlightly more than half of the participants interviewed 59.6%) said that they value quality when seeking for laboratory tests. Unfortunately, many of them find it difficult to differentiate between good and poor-quality laboratories. At least 13% of the interviewed patients said that they relied on government laboratories for quality tests while 10% mentioned that they trusted laboratories to which their clinicians or doctors referred them. Some clinicians have relationships with laboratories and are incentivized to send their patients to those laboratories while in other cases, they simply want a reliable result for their patient and for themselves. The fact, however, remains that government laboratories have limited test menus 47%-65%) mainly due to lack of proper equipment, reagent stock outs and inadequate personnel 9.6% said. A simil%-65% maiDiagnostic testing is comprised of many processes that happen behind the closed doors of clinical laboratories. Evaluating quality and issuing certification requires both access and expertise. It would be desirable to educate patients and clinicians to seek services from laboratories that have certificates of participation in PT or verified quality accreditations. Laboratories taking part in such programs usually display these certificates publicly at the reception areas of their laboratories and on their websites. The main challenge, however, is that such certifications are not mandatory, and indeed, they come with costs that mean that most clinical laboratories do not have them. Although all the private laboratories that we surveyed were accredited, this is because we only included large private laboratories with bigger market share in this study. Unlike these few bigger laboratories, many of the small and medium private laboratories in Kenya are not accredited. There is an opportunity to make this information available to potential laboratory test seeking clients. Given the high literacy and smart phone ownership level in Nairobi city , one posRegulation of laboratory test prices, especially in the private sector, is a complex issue and seemingly beyond the powers of most laboratory regulatory agencies. However, one possible way of ensuring accessibility to diagnostic testing is for the Kenyan government to introduce a policy ensuring that all public laboratories are well equipped to conduct all the essential diagnostic tests as set out by the WHO . Such a Although the mystery shopper approach is an accepted way of collecting withheld information, especially in the business sector, we did not obtain consent from the non-public laboratories from which we collected the pricing data and it would have been preferable to have had their full cooperation. However, this was the only viable approach of collecting the exact test prices charged to patients. The pricing data obtained in this survey were completely anonymized such that they cannot be linked back to the respective laboratories. Separately, we did not physically confirm from the laboratories whether they had the capacity to conduct the tests as informed during the mystery shopper call. This could have led to overreporting of test availability although it is rare for a laboratory to give false information regarding test availability to a prospective client whom they are expecting to visit their premises to have the test conducted. Most importantly, the findings reported in this study were summarized as proportions, percentages and means. The authors acknowledge that further statistical approaches would have been useful in drawing conclusive inferences from this study. For instance, inferential statistics comparing the average test prices across laboratory categories would confirm if the deviations in test prices were statistically significant. Nonetheless, it is clear from the calculated average test prices that different laboratory categories have different price ranges. It is also possible that our finding on low knowledge of test use among the participants could be attributed to literacy levels, but we are certain that cities such as Nairobi have the highest number of literates compared to non-urban settlements. Finally, this was a small study that aimed to collect preliminary data on test availability, test costs and patients\u2019 access to such information within purposively selected facilities. The patients interviewed in the patient exit interviews within the two public hospitals were also selected based on convenience sampling to avoid delayed care consequently resulting to a small sample size. Therefore, the results obtained here may not be a representative of all the medical laboratories in Nairobi and patient responses were only gained from those using the public sector.Under the current system, patients have limited access to information regarding the key criteria required to make a rational decision. This has significant impact on the quality, price, and TAT offered by the medical laboratories that operate in this dysfunctional market. There is a clear need for all clinical laboratories to provide this vital information to their clients. Patients should also be given access to simpler metrics that they could use to distinguish poor and good quality laboratories. The opaque nature of testing information in the current clinical laboratory market in Nairobi limits access to high quality diagnostics and in turn, this limits the potential for higher quality medical care. There is an opportunity to enable this market to work better. In addition to more traditional means of working with the regulator and the ministry to encourage more open information in the market, we are excited by the prospect of digital health interventions. One possibility is to use a digital marketplace app, in the same way that has been done in other sectors where customers choose taxi drivers, second-hand sellers or holiday lettings."}
{"text": "Chitosan belongs to the group of biopolymers with increasing range of potential applications therefore searching for new raw materials as well as new techniques of obtaining of this polysaccharide are currently a subject of interest of many scientists. Presented manuscript describes preparation of chitosan from crickets. Obtainment of final product required a number of processes aimed at removal of undesirable substances such as waxes, mineral salts, proteins or pigments from above-mentioned insects. Chemical structure of fractions obtained after each step was compared with the structure of commercial chitosan by means of techniques such as X-ray diffraction and FT-IR spectroscopy. Final product was subsequently used for preparation of polymer capsules that were modified with active substance characterized by antibacterial and anticancer activity\u2014nisin. Next, sorption capacity of obtained materials was evaluated as well as a release profile of active substance in different environments. Based on the conducted research it can be concluded that crickets constitute an alternative for shellfish and other conventional sources of chitosan. Furthermore, obtained capsules on the basis of such prepared chitosan can be considered as drug delivery systems which efficiency of release of active substance is bigger in alkaline environments. Staphylococcus aureus without negative impact on human dermis cells [Application of materials of natural origin in wide variety of areas is one of the most interesting trends nowadays. Compounds such as polysaccharides, proteins or other bioproducts have become more and more popular. Special emphasis is directed onto chitin and its derivates . One of is cells .Application of chitosan in preparation of systems of drug delivery was also well-documented ,15. FurtGrowing possibilities of medical application of chitosan resulted in increased demand for this polysaccharide. Therefore, new sources as well as techniques of acquiring of this biopolymer are a subject of scientists\u2019 interest ,22. IntePresented publication reports preparation of chitosan from crickets. Multistage chemical processing results in obtainment material that subsequently constitutes a raw material for synthesis of polymer capsules. These can be used as carriers of active substances such as nisin. It is a peptide of natural origin that is characterized by strong antibacterial properties. Additionally, currently scientists conducted studies on its cytotoxic effect on cancer cells ,26,27.Ethyl alcohol , hydrochloric acid , sodium hydroxide , hydrogen peroxide (30%), citric acid (anhydrous) and calcium chloride  were bought in Avantor Performance Materials Poland . Chitosan , phosphate buffer , alginate  and nisin were obtained from Sigma Aldrich S.A. .\u22121  and X-ray diffraction. Additionally, a weight loss of the processed materials was also investigated when selecting parameters of every stage leading to the preparation of the material with the most similar structure to the structure of commercial chitosan.Chitin is one of the components that builds an external skeleton of many insects such as crickets. However, body of these animals consists of many substances including proteins, waxes and pigments therefore an extraction of chitin-chitosan complex from crickets required a multi-stage chemical process aimed at removing all undesirable substances. Here, such multistep process is presented wherein after each stage a chemical structure of the obtained material was compared to the structure of commercial chitosan using techniques such as Fourier transform infrared (FT-IR) spectroscopy via Nicolet iS5 iD7 ATR Thermo Scientific  wherein the spectra were recorded in the range of 4000\u2013500 cmFirst step of the process involved a removal of waxes by Soxhlet extraction wherein an ethyl alcohol was used as a solvent. Waxes belong to the group of hydrophobic substances and their presence in insects\u2019 bodies can substantially hinder further reactions that are carried out using aqueous solutions. Thus, a fraction of dried crickets was introduced into the thimble and subsequently the Soxhlet extraction was conducted for different amounts of cycles. Next, obtained material was dried at room temperature and subjected to the further stages. Parameters of the process were selected considering weight losses of the processed material.In the second step, fraction of crickets devoid of waxes was treated with hydrochloric acid solution to remove mineral salts. Process was carried out at room temperature with constant stirring. Removal of mineral salts was performed for different periods of time and using HCl with different concentrations. Next, obtained material was washed with distilled water and dried at room temperature. Parameters of the process were selected by defining a weight loss of the processed material as well as by comparison of the structure of obtained fraction with the structure of commercial chitosan. For this purpose, X-ray diffraction and FT-IR spectroscopy were employed. In order to remove protein substances, fractions free of waxes and mineral salts were treated with sodium hydroxide. The process was conducted using 5 M NaOH solution (at elevated temperature and with constant stirring). Reaction was carried out under reflux in different periods of time. Obtained material was washed with distilled water, dried at room temperature and subjected to the XRD and FT-IR techniques.2O2 solution at elevated temperature was employed. Process was carried out for 1 h at elevated temperature wherein obtained material was washed using distilled water and dried at room temperature. Below, in Final step of a chemical treatment of crickets involved a removal of natural pigments such as melanin. For this purpose, a HPrepared chitosan was subsequently used for preparation of polymer capsules designed as drug carriers.The deacetylation degree (DD) of chitosan is one of the most important parameters of this biopolymer which determines its physicochemical and biological properties and thus its application potential. The value of DD corresponds to the molar percentage of monomeric glucosamine units wherein it ranges from 0 to 100 .In order to determine the value of DD, the acid-base potentiometric titration was performed. For this purpose, approx. 0.5 g of chitosan extracted from crickets was dissolved in 0.1 M HCl. Then, the titration using 0.1 M NaOH solution was performed with simultaneous pH measurements made using the pH-meter Elmetron CX-701 . The procedure was conducted three times.161\u2014molar mass of monomeric unit of chitosan which is fully deacetylated, g/mol.The deacetylation degree is subsequently calculated using the following equation:Such an equation allows one to determine the number of moles of amino groups titrated in reference to the total number of moles of monomeric units (both deacetylated and acetylated ones).2 were prepared. Next, alginate solution and chitosan solution were mixed in suitable proportions. Then, nisin solution was added and the whole mixture was dropped slowly to the saturated solution of CaCl2 under continuous mixing conditions. Additionally, capsules based on alginate were also prepared and modified with nisin as reference materials for comparison with alginate/chitosan-based capsules with and without active substance. The procedure of their preparation was the same\u2014the only difference was that the reaction mixture did not contain chitosan solution.Obtained chitosan of crickets\u2019 origin was subsequently used for the preparation of capsules containing active substance. For this purpose, 3% solution of chitosan  in 0.05% acetic acid solution, 0.5% aqueous solution of alginate, 0.1% aqueous solution of nisin and saturated solution of CaClIn Next, prepared capsules were subjected to the investigations aimed at determining their chemical structure, sorption properties and a release profile of nisin from such carriers. A presence of functional groups in the structure of prepared materials was verified using FT-IR spectroscopy. The equipment and applied conditions were the same as in the case of FT-IR analysis of fractions obtained after each step of the chemical treatment of crickets.Sorption capacity of prepared capsules is very important in viewpoint of the potential application of such materials as drug carriers. In order to characterize this property, obtained materials were immersed in distilled water. After these periods of time, swelled capsuled were placed on a moisture analyzer  wherein the drying process was conducted at 37 \u00b0C. The percentage loss of mass was determined after 15 and 30 min and corresponded to the amount of absorbed solution.Release profiles of nisin from the interior of chitosan/alginate-based capsules and alginate-based capsules were determined in two different environments. First environment simulated conditions occurring in human stomach\u2014study was conducted in 2% citric acid solution (pH ~ 2); second one resembled pH which is characteristic for duodenum\u2014such conditions were simulated by phosphate buffer (pH = 7.4). Study was carried out as follows: a suitable amount of capsules containing the highest amount of nisin solution (5 mL) were introduced into sterile vessels containing 200 mL of 2% citric acid and PBS solution and placed on a shaking incubator (Hanchen ES-60E Temperature Controlled Incubator and Shaker Scientific Incu-Shaker Shaking Incubator). Vessels were tightly closed with parafilm to limit the possible impact of the external environment. The release process was performed at 37 \u00b0C so at the temperature simulating conditions occurring in human body and at a shaking speed of 80 rpm. After every 15 min 3 mL of tested solution were taken, introduced into the cuvette and concentration of active substance in tested sample was determined using UV-Vis spectrophotometry  wherein maximum absorbance characteristic for nisin was verified at approx. \u03bb = 230 nm. Importantly, in each case after sampling the medium, in which the release process was performed, was supplemented with 3 mL of 2% citric acid or PBS solution so that the total volume of the release medium remained constant. To sum up, the study was conducted to verify the drug release ability of both chitosan/alginate-based capsules and alginate-based capsules in various environments simulating in pH and temperature conditions occurring in human body. This, in turn, allowed to determine conditions ensuring the most effective release of the active substance.Examples of weight losses resulting from the process aimed at removing waxes including different amount of cycles are shown in Weight losses calculated after extraction conducted for 4 and 5 cycles were very similar and were within the range 33.2\u201335.5%. Thus, it can be stated that four extraction cycles are enough for removal of waxes from processed raw material. Importantly, this is also favorable from an economical viewpoint of view and is in line with the principles of Green Chemistry because lower amount of cycles required lower energy demand. These are the reasons due to which 4 extraction cycles were chosen as a process aimed at removal of waxes from the starting material.Results of the second step of processing of crickets including time, concentration of HCl solution applied, amount of extraction cycles as well as a difference between a mass of starting material and a mass of fraction obtained after demineralization are summarized in Based on the obtained results it can be stated that weight losses of processed materials were significantly lower compared to those ones calculated after the removal of waxes. Thus, it may be concluded that crickets contained a relatively lower amount of mineral salts than waxes. Next, it can be reported that the lowest differences between mass of the starting material and the mass of the fraction after demineralization were observed in the case of processes conducted for 30 min using 2 M HCl (sample 2 and sample 6). Additionally, it was observed that the amount of cycles during the extraction had no significant impact on the course of the removal of mineral salts, so four extraction cycles were again considered as sufficient.Nonetheless, an important aspect of performed investigations was an impact of the conditions applied on the structure of final material thus X-ray diffraction and FT-IR spectroscopy were performed to compare the chemical structure of obtained fractions with the structure of commercial polysaccharide. Obtained XRD patterns are presented in Obtained diffraction patterns allowed to conclude enabled the statement that chemical structure of the fractions obtained by means of the above-mentioned parameters were very similar and relatively close to the XRD diffraction pattern of commercial chitosan. On all XRD diffractograms pattern peaks at approx. 2\u03b8 = 9\u00b0 and 2\u03b8 = 19\u00b0 were observed which correspond to the XRD pattern of standard chitin. Importantly, XRD peaks characteristic for chitosan are at approx. 2\u03b8 = 10\u00b0 and 2\u03b8 = 20\u00b0 (JCPDS # 039-1894) ,29.Additionally, a pattern peak at approx. 2\u03b8 = 26\u00b0 (very slight) and 2\u03b8 = 31\u00b0 was observed for sample 2. XRD peaks characteristic for chitin are also at approx. 2\u03b8 = 23\u00b0 and 2\u03b8 = 26\u00b0  thus it XRD diffractogram of this sample and XRD diffractogram of commercial chitosan were presented separately once again in Next, FT-IR analysis of sample 2 was carried out. Based on the obtained spectra it was possible to determine a presence of characteristic functional groups in the structure of obtained material and compare its spectrum with spectrum of commercial compound. In \u22121 corresponding to the stretching vibrations of -NH2 and -OH groups can be observed. Moreover, bands at 1650 cm\u22121 can also be noticed for both materials which corresponds to the stretching vibrations of -CO group in the acetylamide. When comparing the intensity of these two peaks, it can be concluded that the material obtained has more acetylamide groups compared (band with higher intensity) to the commercial chitosan. It is probably caused by the fact that intensity of this band decreases with the increase of the degree of N-deacetylation of the chitin and commercial chitosan is deacetylated form of chitin so has a higher N-deacetylation degree. In turn, tested fraction at this stage of the chemical treatment resembles chitin more than chitosan which confirms results of XRD analysis. On the spectrum of commercial chitosan as well as on the spectrum of the tested sample band within the range 1100\u20131000 cm\u22121 derived from stretching vibrations of the -C-O-C- group in the glycosidic linkage was also observed. In the case of analyzed fraction this peak has a low intensity compared to this one of commercial polysaccharide. This may be a result of the presence of a number of organic compounds such as proteins or pigments in the tested material (they will be removed in the course of further processing), which probably cause a signal weakening.Considering results of FT-IR analysis it can be reported that spectrum of fraction obtained after removal of waxes and mineral salts is very similar to this one deriving from commercial chitosan. On both spectra, peak characteristics for the same functional groups were observed. In both cases a broad band at 3250 cmResults of deproteination of fractions deprived of waxes and mineral salts are presented below. As in the case of previous stages, a yield of conducted processes as well as a chemical structure of obtained material were considered when selecting the most favorable parameters. Based on the above-presented results it may be concluded that process conducted for 20 h resulted in majority cases in higher yields. However, this may also indicate that a material was not sufficiently purified from proteins. According to the literature reports crickets are characterized by a high amount of proteins in the body therefore such a stage is a very important in viewpoint of their further processing and studies. This was a reason why it was stated that process should be carried out longer so for at least 45 h. Therefore, only fractions obtained after deproteinization performed for 45 h, 55 h and 65 h were subjected to the XRD analysis.In As it may be noticed on all XRD diffractograms peaks at approx. 2\u03b8 = 19\u00b0 and 2\u03b8 = 9\u00b0 may be observed so at values corresponding to peaks characteristic for chitin. Nonetheless, on XRD patterns of samples treated with 5 M NaOH for 55 h and 65 h second peak is slightly closer to 2\u03b8 = 20\u00b0. Moreover, the first visible peak is much more like a peak at approx. 2\u03b8 = 10\u00b0 visible on XRD pattern of commercial chitosan. Importantly, this peak is relatively wide for commercial chitosan. Additionally, on XRD pattern of sample treated with NaOH for 45 h a small wide peak at approx. 2\u03b8 = 23\u00b0 is also visible which is characteristic for chitin\u2014it was not observed on XRD patterns of samples treated with NaOH for 55 h and 65 h (longer time of treatment with NaOH resulted in more effective deacetylation of chitin to chitosan). Thus, the sample subjected to the deproteinization performed for 55 h and 65 h was considered for further experiments. However, when selecting the most favorable conditions of each process it is significant to consider principles of Green Chemistry. That is, when there is no difference between fraction after removal of waxes for 55 h and 65 h then the shorter time of the reaction should be selected. This, in turn, provides lower energy demand and thus is more ecological and economical. Therefore, the sample after 55 h deproteinization was subsequently analyzed using FT-IR spectroscopy.In It may be observed that FT-IR spectra in \u22121 which can be attributed to the stretching vibrations of -OH and -NH2 groups was observed. Next, bands at 2870 cm\u22121 corresponding to the stretching vibrations of the methylene groups assigned to the chitosan pyranose ring were also visible. Bands at 1650 cm\u22121 corresponding to the stretching vibrations of C=O group as well as deformation vibrations of the -NH group were also observed. Additionally, bands at 1580 cm\u22121 characteristic for bending vibrations of -NH deriving from the amide group were noticeable. What is more, the band at 1347 cm\u22121 corresponded to the deformation vibration of C-H group in the methyl group of the N-acetyl group of chitosan. The broad band in the range of 1125\u20131025 cm\u22121 resulted from the stretching vibrations of the glycosidic linkage C-O-C. Moreover, bands occurring at 880 cm\u22121 corresponded to the stretching vibrations of C-N group.On both spectra a wide band at 3350 cm2O2 solution. Reaction was carried out for 1 h at an elevated temperature while a yield was 95.17%. Obtained fraction was analyzed in the same way as previously\u2014by means of FT-IR spectroscopy. In In the last step, the sample deprived of waxes, mineral salts and proteins selected, based on the previously performed processes\u2014the sample after four extraction cycles, demineralization for 30 min using 2 M HCl and 55 h deproteinization\u2014was subjected to the reaction with HIn On both spectra vibrations, characteristics for the same groups were observed. Thus, it may be tentatively concluded that material obtained as a result of multistep processing of crickets can be defined as chitosan and, importantly, the mentioned insects can be a source of this polysaccharide. Differences in the intensity of observed peaks can be a result of a presence of slight residues of other organic substances that were not removed during the processing. Next, XRD analysis was performed for obtained final fraction. In Any significant differences between obtained XRD diffraction patterns of samples before and after depigmentation were not observed. Two slightly shifted peaks in comparison to peaks visible on diffractogram of commercial chitosan were observed. Thus, it can be concluded that processed insects contained slight residues of pigments.In Chitosan derived from crickets was used in the next part of presented studies as a raw material for synthesis of polymer capsules that could be considered as drug carriers.Results of the performed titration are presented in + ions (derived from HCl) by OH\u2212 ions (from NaOH) according to the following reaction:In 2 groups deriving from glucosamine residues of chitosan. Firstly, -NH2 groups are protonated (-NH2 + H+\u00a03+). Next, when NaOH is added, -NH3+ ions are neutralized by OH\u2212 ions and therefore in such a manner the concentration of -NH2 groups may be determined.The second equivalent point may be assigned to the protonation of -NHAnalyzing the titration curve obtained and two equivalent points visible on the curve the deacetylation degree (DD) calculated using the Equation (2) was In 2 solution and subjected to the further studies.Prepared capsules were separated from the CaClPrepared chitosan/alginate-based capsules modified with nisin were subjected to the FT-IR analysis. Additionally\u2014for comparison\u2014spectroscopic analysis was also performed for alginate-based capsules without an active substance. In \u22121. This band in the case of chitosan/alginate-based capsules showed higher intensity compared to the same band visible on FT-IR spectrum of alginate-based capsules. This, in turn, may indicate intense interactions between these two reagents  due to numerous hydrogen bonds and this is a reason why this band is wider for capsules consisting of two reagents compared to the alginate-based capsules [Functional groups characteristic for analyzed materials were identified at specific absorption bands and showed in capsules . Furthercapsules ,32. Simicapsules ,34.Results of studies on sorption properties of capsules with special emphasis on impact of introduced additive on the tested sorption capacity is presented in Obtained capsules were characterized by swelling properties. Such properties grew over time because volume of absorbed solution was higher after 30 min of swelling compared to this one observed after 15 min. Introduction of nisin into analyzed capsules resulted in an increase of their sorption capacity. It was probably caused by the fact that nisin contains in its structure many hydrophilic groups that can form hydrogen bonds with absorbed medium. Groups such as amino or carboxylic dissociate under the influence of water and resulted ions from the same groups repel electrostatically. Then, structure of the tested material becomes less compact and therefore it can absorb more water. Additionally, additive was added in the form of an aqueous solution therefore absorbed liquid can also interact with the solvent.3+ groups (protonated amines) from chitosan and as a result a three-dimensional hydrogel network formed via a physical crosslinking was obtained [It may be reported that alginate-based capsules showed slightly higher swelling ability than chitosan/alginate-based capsules. Nonetheless, the differences were slight and probably due to the higher crosslinking density of capsules consisting of two components, i.e., both chitosan and alginate. It may be concluded that carboxylate groups present in the structure of alginate probably electrostatically interacted with -NHobtained . The intobtained . Consideobtained . FurtherResults of a release of nisin from alginate-based capsules and chitosan/alginate-based capsules are presented below. In \u2212 ions present in tested medium enhance electrostatic repulsion occurring between COO\u2212 anions . Therefore, a structure of capsules becomes less compact and introduced substance has more free space to get out from the polymer system can be released to the tested environment.Analyzing above-presented results it may be concluded that release of nisin is significantly more effective in slightly alkaline environment both in the case of alginate-based capsules and chitosan/alginate-based capsules. In In the case of release process observed for chitosan/alginate-based capsules at the beginning nisin was released gradually while the release of the largest amount of this substance occurred after 75 min of the research. After 2 h of the measurements a total amount of nisin was released from tested materials. Importantly, it is worth mentioning that during the first 45 min of the research only a little substance was released and its sudden ejection was observed even after 75 min. Compared to alginate-based capsules, a maximum release of nisin from chitosan/alginate-based capsules occurs more than twice as late (is long-term) that constitutes an important information in viewpoint of the application of such systems as drug delivery systems.Thus, it may be concluded that the application of chitosan extends the time of release of nisin from the developed systems. The pKa of the primary amino group of chitosan is approx. 6.5 and exactly this functional group affects the solubility of this polysaccharide in acidic environments. On the other hand, in neutral and alkaline environments a partial neutralization of this group as well as an aggregation of chitosan are observed that limits a release of nisin . Similar2 groups present in chitosan bind with H+ ions forming NH3+ cations. Such ions repel each other and as a result structure of a capsule also becomes less compact. However, the mentioned interactions are significantly less intense compared to the interactions between OH\u2212 and COO\u2212 that take place in an alkaline environment. It is caused by the fact that during the synthesis applied volume of alginate solution was four times bigger than volume of chitosan solution. Therefore, release of an active substance from the tested capsules was significantly better in an alkaline solution. It is important information in the viewpoint of the potential application of such materials as drug delivery systems. It may be reported that a release of a drug from such prepared materials is more effective in organs such as duodenum  compared e.g., to stomach (acidic conditions).Due to the solubility of chitosan in an acidic environment a nisin release was possible in such medium however a percentage amount of a released active substance was only 19.16%. A slight signal deriving from the analyzed substance was noticed only after 75 min of the research. It was probably caused by the fact that in such conditions NHCrickets can be classified as a good raw material for preparation of chitosan\u2014polysaccharide, due to its properties, is increasingly used for biomedical purposes. However, multistep chemical processing is necessary to remove from the mentioned insect substances such as waxes, mineral salts, proteins and pigments. Obtained material was characterized by properties and structure almost the same as those ones of commercial chitosan that was proved by means of the following techniques: FT-IR spectroscopy and X-ray diffraction.\u2212 and COO\u2212 ions that resulted in the loosening the structure of polymer capsules. Furthermore, it was proved that a release of nisin from chitosan/alginate-based capsules occurred significantly longer than a release from alginate-based capsules (without chitosan). Importantly, a possibility of achieving an extended release profile of an active substance is undoubtedly a big advantage of the developed systems. Obtained results indicated that a maximum release of nisin from chitosan/alginate-based capsules was observed after approx. two-times longer period of time than in the case of alginate-based capsules.Obtained chitosan of crickets\u2019 origin was used in the next part of the research as a raw material for preparation of polymer capsules modified with nisin. Prepared capsules were characterized by good swelling properties. Introduction of nisin into the capsules resulted in an increase of sorption capacity. Based on conducted studies on release of active substance in different environments from prepared materials it can be concluded that release is significantly more effective in alkaline solutions. It is caused by the electrostatic interactions between OHPolymer capsules based on chitosan of crickets\u2019 origin and containing nisin constitute an interesting material that can be considered for application for biomedical purposes with special emphasis on application as delivery systems of drug with antibacterial or anticancer activity."}
{"text": "In multivariate analysis, the risk of venous thrombosis was independently associated with previous venous events  and NLR values \u22655 . Moreover, the relative risk in both low- and high-standard risk groups was almost doubled in the presence of NLR\u2009\u2265\u20095. These findings were validated in two Italian independent external cohorts  of contemporary PV patients. Our data support recent experimental work that venous thrombosis is controlled by innate immune cells and highlight that NLR is an inexpensive and easily accessible prognostic biomarker of venous thrombosis.We investigated the neutrophil-to-lymphocyte ratio (NLR) as a predictor of thrombosis in polycythemia vera (PV). After a median follow-up of 2.51 years, of 1508 PV patients enrolled in the ECLAP study, 82 and 84 developed arterial and venous thrombosis, respectively. Absolute counts of total leukocytes, neutrophils, lymphocytes, platelets, and the NLR were tested by generalized additive models (GAM) to evaluate their trend in continuous scale of thrombotic risk. Only for venous thrombosis, we showed that baseline absolute neutrophil and lymphocyte counts were on average respectively higher (median: 6.8\u2009\u00d7\u200910 JAK2 genes (JAK2 V617F and exon-12 mutations). Typically, PV displays an elevated red-cell mass, usually along with leukocytosis and thrombocytosis [Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by uncontrolled clonal proliferation of multipotent bone marrow progenitors, sustained by acquired genetic mutations in ocytosis .Thrombosis can be the first presenting sign preceding the diagnosis of MPN in 20% of cases, with a persistent risk during the follow-up, where the incidence is the highest in patients with PV and similar in essential thrombocythemia (ET) and primary myelofibrosis (PMF) , 5. In aThe relationship between leukocytosis and thrombosis has been extensively investigated in several experimental studies , 13 baseThus, no definitive prognostic role could be ascertained as regards leukocytosis, thus preventing it from being included in the thrombotic risk score of PV patients.However, new data are emerging on the role of nonmyeloid inflammatory cells, such as T lymphocytes and monocytes in the process of immune thrombosis; in particular, experimental work has consistently showed that T-reg lymphocytes are involved in the regulation of the prothrombotic action of activated neutrophils in the process of fibrin formation and dissolution . Based oIn the present work, we investigated whether NLR can identify PV patients at higher risk of both venous and arterial incident events by analyzing the ECLAP database that involved PV patients prospectively followed for a median of 3 years and includes a large number of formally validated arterial and venous thrombotic events.The present study included 1508 of the 1638 PV patients enrolled in the ECLAP study, selected on the basis of the availability of differential blood counts at baseline that allowed to calculate the NLR.9/L. Data regarding clinical outcomes, treatments, and laboratory values during the prospective follow-up were recorded at follow-up visits at 12, 24, 36, 48, and 60 months.All patients with new and old PV diagnoses (according to Polycythemia Vera Study Group (PVSG) criteria) were included prospectively with no exclusion criteria with respect to age, therapy, or duration of disease. Treatment strategies had to comply with the recommendation of maintaining the hematocrit value at less than 0.45 and the platelet count at less than 400\u2009\u00d7\u200910The study protocol conformed to good clinical practices and to the Declaration of Helsinki on medical research in humans.The primary endpoint was the incidence rate of arterial and venous thrombosis , and major and minor thrombotic complications as defined above. These events were objectively diagnosed as previously reported .Univariate analysis was performed to evaluate differences in proportions by the chi-square or Fisher exact tests where appropriate. Differences in continuous variables were tested using the nonparametric Wilcoxon rank-sum test.In order to investigate the role of NLR in the time-to-thrombosis prediction, we overcame the traditional Cox proportional-hazard model, which restricts the log hazard ratio (HR) to be linear in the covariates, by applying the generalized additive models (GAM) . GAM proNLR values were dichotomized using the best threshold found according to Liu\u2019s method. Thrombosis-free survival of the resulting two groups was compared using the Kaplan\u2013Meier (KM) estimator and tested with the log-rank test.Cox proportional-hazard models were applied for multivariable analyses. The Harrell\u2019s C-statistic was calculated to measure the incremental discriminatory ability of a new predictor .During a median follow-up of 2.51 years, 166 thromboses  were recorded in 160 of 1508 patients (10.6%). Arterial thromboses were mostly cerebral, while venous thrombosis mostly affected lower extremities (DVT), with or without pulmonary embolism (PE) (Table p\u2009=\u20090.001) and previous thrombosis (p\u2009=\u20090.003), especially if arterial (p\u2009<\u20090.001), and with the presence of at least one cardiovascular risk factor (p\u2009=\u20090.009). No association with baseline leukocytosis or the NLR was found. In patients with venous thrombosis, not age but history of thrombosis (p\u2009<\u20090.001) was also a risk factor for incident events, especially in cases with prior venous events (p\u2009<\u20090.001).Table p\u2009=\u20090.041), and above all, it was greater in categories of patients with higher NLR values (p\u2009=\u20090.002), which resulted from a simultaneous increase of neutrophils (p\u2009=\u20090.002) and a decrease of lymphocytes (p\u2009=\u20090.001).Differently from arterial, venous complications significantly occurred for progressive increase of BMI (p\u2009=\u20090.002) emerged, leading to increase the NLR values (p\u2009=\u20090.005)  Fig. . The hazTherefore, since the NLR integrates these two opposing trends, it was more representative of the association of lymphocyte and neutrophil counts with venous thrombosis than individual counts alone, where confidence intervals resulted wide at higher counts.Conversely, neither absolute values of neutrophils and lymphocytes nor NLR were found associated with arterial events Fig. .Total leukocyte and platelet counts were also tested, but they did not correlate with either venous or arterial thrombosis (data not shown).p\u2009<\u20090.001) and NLR values \u22655, a figure representing the best cutoff resulted applying Liu\u2019s method ), the risk of venous thrombosis was independently associated with previous venous events  and previous arterial thrombosis  retained the prognostic statistical significance for arterial thrombosis, together with an effect of CV risk factors of borderline significance  and one that added to the latter the NLR value above 5, looking for any incremental value of the statistic. The probability that predictions and outcomes are concordant measured by the C-statistic for the first model was 59.24%, while after adding the NLR variable, it increased to 65.09%.Kaplan\u2013Meier curves of time-to-venous thrombosis probability stratified by either conventional risk classification or values of NLR are plotted in Fig. n\u2009=\u2009282 and Rome, n\u2009=\u2009175) of contemporary PV patients, whose clinical characteristics are presented in Table The correlation between NLR values and venous thrombosis found in our learning cohort Fig.  was valiIn this series of patients with PV, we sought to investigate for the first time whether the baseline ratio of circulating neutrophils to lymphocytes (NLR) could be a marker of thrombosis as reported in many studies in the general population . To this9/L, p\u2009=\u20090.022) and lower  in patients with incident venous thrombosis, overall resulting in a higher NLR ratio. The most evident finding was the linear association between the absolute number of lymphocytes and the risk of events expressed by the log of HRs in the GAM models. We found that the category of patients presenting at baseline with lymphocyte values less than 2\u2009\u00d7\u2009109/L had an increased risk of venous thrombosis with HR greater than one, whereas the HRs for venous thrombosis progressively decreased as the lymphocyte counts increased. These changes are consistent with an opposite prognostic tendency of neutrophils, whose increments showed an increasingly higher trend of risk .In univariate analysis, we found that the absolute number of neutrophils and lymphocytes was on average respectively higher  and age \u226565 years.In a multivariate Cox proportional-hazard model using NLR cutoff equal to five, found to most efficiently discriminate against the highest risk categories, we found that this cutoff value was associated with major venous events (HR\u2009=\u20092.18), independently of age and previous venous thrombosis, and this was confirmed in the sensitivity analysis after excluding minor venous thrombosis. On the contrary, NLR did not show any significant association with arterial events that were predicted by previous arterial event (Interestingly, the prognostic discriminatory power of conventional risk factors for venous thrombosis was increased in the presence of an elevated NLR as demonstrated by the values of the C-statistic increasing from 59% to 65%, in either low- or high-risk PV groups. This finding could therefore suggest the value of incorporating this inflammatory biomarker in a scoring system for venous thrombosis in PV.JAK2V617F, which has recently been reported to be correlated with venous thrombosis [It will be interesting to investigate the relationship between NLR values and the allelic burden of rombosis . FurtherWe are fully cognizant of the limitations of the current study considering that this is a post hoc analysis of a clinical trial using data that had already been collected for other purposes and referred to patients diagnosed with PVSG diagnostic criteria . HoweverIn conclusion, these clinical findings support recent biological concepts that innate immune cells contribute to venous thromboembolism . Indeed,Reproducibility checklist"}
{"text": "We have observed white turbidity when a midazolam injection is administered from a lateraltube during the administration of a peripheral parenteral nutrition (PPN) solution. The aim ofthe current study was to determine how to avoid compound changes when co-administering amidazolam injection and a PPN solution.Midazolam solutions were prepared by diluting a midazolam injection with a 5%glucose intravenous infusion. We examined the formulation of the midazolam injection and a PPNsolution at the concentrations used in a clinical setting for changes in appearance, pH, andmidazolam content in test tubes and during administration conditions.2=0.9918). The capture rate of midazolam infused with PPN solution was 91.0% ata 1/6 dilution, whereas it decreased to <90% at a 1/4.8 dilution.With a 1/4.8 dilution of midazolam in undiluted solution, clouding occurred. Astrong correlation was revealed between the midazolam content as measured throughhigh-performance liquid chromatography and the mixture\u2019s midazolam concentration(ROur results suggest that the administration of a midazolam injection solutiondiluted by \u22656-fold with glucose solution or saline from a side tube during the administrationof a PPN solution did not cause changes in composition. Guidelines concerning terminal fluid therapy have beenpublished, and peripheral parenteral nutrition (PPN) solutions are now widely used.5 A patientindicated for sedation usually receives a midazolam injection from a side tube during thesimultaneous administration of a PPN solution. However, since a midazolam injection is stableunder acidic conditions, the move toward alkalinity causes precipitating white turbidity orcrystal precipitation. A delay in detecting this turbidity or precipitation may seriously affecta patient\u2019s health. Clinicians must be sensitive to formulation changes because of the injectionroute.6 Although changes in midazolaminjection formulations during total parenteral nutrition infusions have been reported, fewreports comprehensively describe the changes occurring during simultaneous midazolam injectionand PPN solution administration; only visual changes and pH observations were described, notchanges in midazolam content.7 Therefore, weexamined changes in appearance, pH, and midazolam content during administration conditions basedon differences in the composition of a PPN solution and a midazolam injection solution.Midazolam exerts a sedative effect by activating the inhibitory neurotransmitterGABA receptor in the central nervous system, and it introduces and maintains anesthesia as aninjection, as intensive care sedation during artificial respiration, and as sedation duringpalliative care.1. \u00ae injection solution 10\u00a0mg; Maruishi Pharm Inc.) is administeredthrough a side line at 2\u00a0mL/h. To reproduce this situation, we mixed 8.4\u00a0mL of PPNsolution and 0.4\u00a0mL of midazolam injection in a test tube at the ratio of 42:2 at roomtemperature and under normal light conditions. We used an amino acid and glucose injectionwith electrolytes and a vitamin B1 infusion  which is a colorless and transparent infusion agent that contains approximately 7.5%glucose, 3% amino acid (18 types), electrolytes, and vitamin B1 components (pH isapproximately 6.7). We prepared midazolam solutions,  assuming clinicaldoses of 10\u2013240\u00a0mg by diluting a midazolam injection with a 5% dextrose solution  to create a range that included anundiluted solution, i.e., 1/24 (We often treat patients for whom a PPN solution is administered at1000\u00a0mL/day (42\u00a0mL/h) through a main duct, and a midazolam injection; column temperature, roomtemperature, flow rate, 0.8\u00a0mL/min; detector, UV - 2075 Plus intelligent UV/Vis detector(JASCO Corp.); detection wavelength, 254\u00a0nm, mobile phase, 1\u00a0mM citrate-phosphatebuffer (pH\u00a05.0): methanol: acetonitrile=1:1:1 (V/V). The measurement was performed usingan absolute calibration curve method using a midazolam injection solution as a standardsolution. The calibration curve showed linearity in the range 3.1\u201350.0\u00a0\u03bcg/mL. Wedetermined the relationship between the midazolam content as measured through HPLC and themidazolam concentration of the mixture by fitting the approximate curve. We performed aPearson\u2019s regression analysis to determine the correlation.To observe changes in the appearance and pH and for high-performance liquidchromatography (HPLC), we first slowly added 0.4\u00a0mL of each sample dropwise to8.4\u00a0mL of PPN solution, and we observed the visual changes (color tone/turbidity)immediately after the dropwise addition. After recording changes in appearance, we stirred themixture for 30\u00a0sec using a vortex mixer and measured the pH and midazolam concentrationof each sample. The pH was measured using a pH meter . Themidazolam content was analyzed through HPLC with reference to Kobo\u2019s method,We investigated changes in the combined PPN solution and midazolam solution, and inthe combined saline solution and midazolam solution at room temperature and under normal lightconditions. We prepared four midazolam solution samples by diluting a midazolam injection(Dormicum injection 10\u00a0mg) with saline to 1/2.4, 1/4.8, and 1/6, and an undiluted PPNsolution (500\u00a0mL) was added dropwise via an infusion pump at 40\u00a0mL/h with a planectainfusion set filter . The 0\u00a0min time point occurred immediately after thesyringe pump began to push the solution. We collected 3.5\u00a0mL of each mixed solution,having passed through the infusion in-line filter with a 0.22\u00a0\u03bcm aperture, from the tip ofsyringe pump over a 5\u00a0min period after 10, 30, 60, and 120\u00a0min. For the collectedmixture, the pH was measured under the same conditions as in Experiment (A); the midazolamcontent was measured through HPLC, and the content ratio to the theoretical value derived fromthe standard solution was determined. We added 500\u00a0mL of saline  dropwise at40\u00a0mL/h under the same conditions as with the PPN solution, and midazolam solution sampleswere injected from the side tube at 2\u00a0mL/hr. The dose concentration of the midazolaminjection solution and the measurement conditions of the collected mixture were the same asthose used for the PPN solution.2=0.9918, showing a strong correlation  was 6.72\u00b10.03, and thepH of sample O was 6.66\u00b10.01 . Thecoerelation .The pH values of the collected mixture of PPN solution (with a pH of approximately6.7) at 0, 10, 30, 60, and 120\u00a0min after the infusion of midazolam solution diluted to 1/6began were 6.69\u00b10.07, 6.72\u00b10.08, 6.64\u00b10.00, 6.71\u00b10.08, and6.71\u00b10.08, respectively. The pH values of the mixture with saline (with pH\u00a04.5\u20138.0)at the same time points were 5.47\u00b10.04, 5.56\u00b10.15, 5.14\u00b10.42,4.75\u00b10.11, and 4.71\u00b10.01, respectively. For the midazolam solution diluted to1/4.8, the pH values of the mixture with the PPN solution were 6.82\u00b10.10,6.83\u00b10.11, 6. 76\u00b10.07, 6.83\u00b10.13, and 6.81\u00b10.11, and the pH valuesof the mixture with saline were 5.24\u00b10.12, 5.59\u00b10.08, 4.89\u00b10.00,4.83\u00b10.10, and 4.77\u00b10.10, respectively. The pH values of the mixed solution withPPN solution when injecting the midazolam solution diluted to 1/2.4 were 6.74, 6.75, 6.75,6.73, and 6.72. The pH values of the mixed solution with saline were 5.56, 5.37, 4.55, 4.36,and 4.39. The pH values of the mixture with the PPN solution when injecting undiluted midazolamsolution were 6.64, 6.61, 6.60, 6.57, and 6.60, and the pH values of the mixed solution withsaline were 5.52, 5.04, 4.12, 4.00, and 4.08, respectively .The midazolam content ratios to theoretical values at 0, 10, 30, 60, and120\u00a0min of the mixture with the PPN solution when injecting a 1/6 dilution of midazolamwere 3.4%, 3.5%, 0.4%, 83.2%, and 91.0%, respectively. The midazolam content ratio totheoretical values of the mixture with saline were 1.6%, 0.8%, 37.1%, 82.7%, and 100.3% . The mid2=0.999 or more. An appearance change was observed onlyin the mix of PPN solution and midazolam undiluted solution. The mixture was colorless andtransparent under other conditions.The midazolam content ratio to theoretical values of the mixed solution with PPNsolution when injecting midazolam undiluted solution were 0.1%, 33.5%, 33.4%, 54.7%, and 50.2%,and those of the mixed solution with saline were 0.1%, 0.0 %, 69.3%, 99.1%, and 95.0%,respectively . The cal14 The midazolam injection solution used for the sedation of terminal-stagepatients maintains a stable aqueous solution under acidic conditions, and composition changesoccur under alkaline conditions. Mixing injectable drugs is not possible when one or both of thefollowing are observed: (1) a change in the mixture\u2019s physical appearance is recognized, (2)within 24\u00a0h post-compounding, one or more of the compounded ingredients is decomposed by\u226510%. When changes in a formulation are prevented, it remains necessary to observe the reactionproduct, maintain the content of the active ingredient, and ensure that the medicine isadministered to the patient safely and securely.15Our experiments clarified a method of combined administration for a PPN solution anda midazolam injection solution that avoids changes in the mixed solution characteristics.Guidelines regarding infusion therapy for terminal cancer patients have been issued in severalcountries. The importance of nutrition has also been highlighted in Japan, and guidelines wereissued that consider the use of PPN solution preparations.16 Kuramoto et\u00a0al. mixed equal volumes of ahigh-calorie infusion preparation and a midazolam injection solution in two-, three-, four-, andfive-fold dilutions; they examined changes in appearance and conducted a filter-resistance testand reported that a five-fold dilution of the injection can be administered from the side tubeof a total parenteral nutrition infusions line without causing composition changes.7Avoiding formulation changes and reaction products and maintaining the content ofthe active ingredient will guarantee drug efficacy and safety. In an examination of thestability of midazolam hydrochloride in a parenteral nutrient solution with intravenousnutrients containing amino acids at 1.5%, 2.5%, and 5% concentrations and midazolam injectionsat 0.1 and 0.5\u00a0mg/mL concentrations, the midazolam and amino acid content at 5\u00a0h aftermixing did not change, and changes in appearance, pH, and precipitation did not occur.17 The most interesting point revealed in the current study is that althoughvisible white turbidity occurred in the solution with samples E\u2013O, when we stirred the samplesagain, the white turbidity disappeared and 95.27% of the midazolam could be detected throughHPLC (indicating that only 4.73% was lost). These results were contrary to our expectation,because the solutions measured pH\u00a06.6\u20136.7, which is more basic than the value of thechanging point; i.e., the pH value at which crystals are thought to precipitate. To clarify thepH dependence of midazolam, the pH of the critical point at which no formulation change isobserved could be identified using Azuma\u2019s method or predicting a formulation change.18 The critical-point pH can be determined throughAzuma\u2019s method, but the reason for the value being close to the pH value of the PPN solution isthat the mixed solution pH is included in the beaded infusion. The influence of the pH bufferappears to be large. Moreover, although we observed that the midazolam content of the solutionafter stirring does not fall, in clinical settings there is no guarantee that the mixture underthe same conditions will be stirred. Therefore, midazolam administration at a \u22656-fold dilutionis recommended.In Experiment (A), white turbidity was confirmed at concentrations exceeding a 1/4.8dilution of the midazolam injection solution. Hydrochloric acid as a solubilizing agentinfluences changes in the midazolam formulation, leading to large variation in the clinicalformulation. The pH range of midazolam injections is 2.8\u20133.8, and crystals precipitate at themore basic region including the change point 4.72.In Experiment (B), the midazolam content was measured through HPLC, and weinvestigated whether a difference occurred in the composition change between a PPN solutiontransfusion and saline. We measured the pH of unbuffered saline immediately after mixing withsaline and after 10\u00a0min, and the pH fluctuation after 30\u00a0min tended to be acidic dueto the strongly acidic midazolam injection. This is consistent with the detection of a midazolaminjection after 30\u00a0min, as shown in Figure. 4. In 19 Our results clarified that thecloudy midazolam observed in Experiment (A) is a formulation that does not reduce the midazolamcontent if it is redissolved. It is not yet known how the redissolved midazolam solution wouldaffect the body. Although the cloudy component can be thought of as midazolam, an analysis ofthe components captured by the in-line filter is necessary because the cause of the reactionbetween the midazolam solution and the PPN solution that generates this white turbidity is notknown.20 Since the injection of a highconcentration of midazolam was captured by the in-line filter, it is considered that the in-linefilter should be used in a clinical setting. In Experiment (B), we performed a formulationchange test by setting the time for a midazolam injection to reach a patient\u2019s blood vessel fromthe side tube at a maximum of 120\u00a0min. The compatibility of midazolam with morphine,haloperidol, and similar drugs which are frequently used in the palliative field should bedetermined through compound change tests.23Midazolam was reported to be adsorbed by vinyl chloride, and we therefore used aninfusion route with polybutadiene (non-vinyl chloride) which does not adsorbmidazolam.24 Since the midazolam dosethat could sedate terminal cancer patients was 20\u201330\u00a0mg/day, midazolam administrationstarts at 1\u20132\u00a0mg/h and the dosage is increased until sufficient sedation is achieved.Sedation using midazolam is effective at 80%\u201390%; at approximately 5% it causes complicationsleading to death, but midazolam is generally considered to be safe.25 Midazolam is also recommended as a palliative care sedative. Safeadministration is desired; i.e., avoiding changes in formulation, complications such aspulmonary embolism, and problems such as catastrophic blockage or clogging of the in-linefilter. It is important to promote the proper use of injectable medicines and to investigate andsolve problems occurring with their use.In palliative care, sedation is administered to reduce patient pain. Sedation isnecessary in approximately 30% of cancer patients, for the relief of delirium, dyspnea, malaise,and pain. In a systematic review of studies of midazolam doses, the median starting dose was0.5\u20131.7\u00a0mg/h, the median maintenance dose was 18\u201362\u00a0mg/day, and the range was2\u2013450\u00a0mg.In conclusion, our findings demonstrated that the administration of a midazolaminjection solution diluted by \u22656-fold or more with glucose solution or saline from the side tubeof a PPN solution did not cause any change in the solution composition."}
{"text": "Despite recent rises in fatal overdoses involving multiple substances, there is a paucity of knowledge about stimulant co-use patterns among people who use opioids (PWUO) or people being treated with medications for opioid use disorder (PTMOUD). A better understanding of the timing and patterns in stimulant co-use among PWUO based on mentions of these substances on social media can help inform prevention programs, policy, and future research directions. This study examines stimulant co-mention trends among PWUO/PTMOUD on social media over multiple years.We collected publicly available data from 14 forums on Reddit (subreddits) that focused on prescription and illicit opioids, and medications for opioid use disorder (MOUD). Collected data ranged from 2011 to 2020, and we also collected timelines comprising past posts from a sample of Reddit users (Redditors) on these forums. We applied natural language processing to generate lexical variants of all included prescription and illicit opioids and stimulants and detect mentions of them on the chosen subreddits. Finally, we analyzed and described trends and patterns in co-mentions.Posts collected for 13,812 Redditors showed that 12,306 (89.1%) mentioned at least 1 opioid, opioid-related medication, or stimulant. Analyses revealed that the number and proportion of Redditors mentioning both opioids and/or opioid-related medications and stimulants steadily increased over time. Relative rates of co-mentions by the same Redditor of heroin and methamphetamine, the substances most commonly co-mentioned, decreased in recent years, while co-mentions of both fentanyl and MOUD with methamphetamine increased.Our analyses reflect increasing mentions of stimulants, particularly methamphetamine, among PWUO/PTMOUD, which closely resembles the growth in overdose deaths involving both opioids and stimulants. These findings are consistent with recent reports suggesting increasing stimulant use among people receiving treatment for opioid use disorder. These data offer insights on emerging trends in the overdose epidemic and underscore the importance of scaling efforts to address co-occurring opioid and stimulant use including harm reduction and comprehensive healthcare access spanning mental-health services and substance use disorder treatment.The online version contains supplementary material available at 10.1186/s12954-022-00628-2. Stimulant co-use \u20134, speciRecent findings also suggest that reasons for co-use, particularly with methamphetamine, are multifaceted\u2014such as ease of access, helping boost the \u201chigh,\u201d improving daily function , and helping manage withdrawal symptoms . HoweverEstimate the volumes and short- and long-term trends of opioid-stimulant co-mentionsQuantify estimated co-use rates between pairs of specific opioids and stimulants based on these discussionsIdentify when stimulants are initiated evidenced by mentions among PWUO at an individual level based on timelines of postsThe insights we derive in this study are purely data-driven through natural language processing (NLP) methods, as opposed to hypothesis-driven, and our methods account for growth in the number of Reddit users over time. Being data-driven, we did not start our analyses with any underlying assumptions or hypothesis. Our intuition was that the growing number of overdose deaths due to co-use of opioids and stimulants would be reflected on the Reddit data, but this intuition did not affect our approach. A better understanding of the timing and patterns in stimulant co-use among PWUO as evidenced by mentions of these substances on social media can help inform prevention efforts including harm reduction, as well as future research directions. In the remainder of the article, we refer to people who post on specific subreddits of interest as Redditors, people who use or used prescription and/or illicit opioids, people who used an opioid reversal agent , and people with OUD who are being treated with medications for opioid use disorder (MOUD) as PWUO/PTMOUD, people who use stimulants as PWUS, people who use drugs more broadly as PWUD, and people who co-use opioids and stimulants as PWCU.The overarching objectives of this study are to utilize social media conversation data on Reddit to:subreddits and the moderation of forum-specific content by forum users. Reddit communities have also been found to serve as a means of social support for PWUD [Reddit is particularly popular among PWUO and the broader community of PWUD as it can offer anonymity . Reddit for PWUD , 15, 19.for PWUD , Blueligfor PWUD , and Disfor PWUD  for sevefor PWUD \u201325. ReddTo find potential PWUO on Reddit, we identified 14 opioid-related subreddits spanning discussions on prescription and illicit opioids and MOUD . We focused the study on a set of common opioids, opioid-related medications, and stimulants, including both prescription and illicit types . Thus, we next included additional filters for the terms stimulant, meth, and oxy  of the substances. Since drug names and related expressions are often misspelled on social media, we generated commonly used lexical variants of the terms using the LexExp tool . We founn\u2009=\u20095 following the mention of a negation trigger. Any opioid, opioid-related medication or stimulant term was considered negated if it appeared in the context window following a detected negation expression and if an end of sentence marker  did not occur between the negation term and the drug term. Negated terms were excluded from the final count of mentions. We also manually reviewed samples of posts detected by our searches to identify keywords with a large amount of noise  and excluded those from our counts and further analysis. Of the 13,812 Redditors sampled, a subset who posted in these subreddits had no mention of either an opioid, opioid-related medication or a stimulant (N\u2009=\u20091506) and was excluded from the analyses. All analyses except for those examining the total numbers of Redditors (including PWCU) over time were based on subsets of the remaining 12,306 Redditors who posted about at least one opioid, opioid-related medication, or stimulant.The third NLP task involved implementing a negation detection strategy, customized for Reddit data, to exclude negated drug mentions. Specifically, we used a subset of negation terms from the NegEx algorithm   over the same time frame; and the annual ratio of PWCU to PWUO/PTMOUD. Due to the growing user base of Reddit over time, we expected numbers of PWUO/PTMOUD and PWCU to both increase annually. Hence, the ratio of PWCU to PWUO/PTMOUD likely serves as the best indicator of social media trends in opioid and stimulant co-use within this population.We first studied trends in opioid and stimulant co-use between 2011 and 2020 through multiple metrics, including, the number of Redditors posting each year; the number of Redditors who mentioned at least one opioid or opioid-related medication and one stimulant  frequency of co-use of specific types of opioid-stimulant pairs among PWUO/PTMOUD aggregated over the entire study period; (2) proportion of specific opioids co-used with any stimulant in a given year among the total number of PWCU in that year, to study how mentions of specific opioids or opioid-related medications co-used with any stimulant changed over time; and (3) proportion of specific stimulants co-used with any opioid or opioid-related medication in a given year among the total number of PWCU in that year, to study how mentions of specific stimulants co-used with any opioid changed over time. Redditors with mentions of more than one type of opioid or opioid-related medication or stimulant within a given year were separately considered for each specific opioid-stimulant pair.We combined the opioids and opioid-related medications listed in our analytic sample into 5 categories: 1) heroin, (2) fentanyl & analogs (including carfentanil), (3) Medications for Opioid Use Disorder , (4) opioid overdose reversal agents , and (5) prescription opioid pain relievers . We combined stimulants into 4 categories: (1) methamphetamine, (2) amphetamine-type stimulants , (3) methylphenidate-type stimulants , and (4) a combination of methamphetamine and heroin . Categories included generic names, trade names, and common misspellings or lexical variants  versus those who mentioned a stimulant first .We collected 13,812 Redditor timelines from the 14 subreddits, 12,306 of whom mentioned at least one of our selected opioid, opioid-related medication, or stimulant keywords. Redditors mentioning any opioid or opioid-related medication, or any stimulant in their timelines were approximately evenly distributed\u20149329 Redditors mentioned opioids or opioid-related medication at some point in their timeline; 9151 mentioned stimulants  and prescription opioids were most frequently mentioned with prescription amphetamine-type stimulants . This suggests possibly distinct populations within the social media substance use forums we studied with preferences for particular types of substances. Further understanding the distinction between populations focused primarily on prescription drug misuse and those focused on illicit substance use, and the transitions between these preferences, is a future area for exploration of online data that may yield insights for better targeting prevention and harm reduction efforts. Indeed, while there is research on prescription stimulant misuse , less isWe found noticeable increases in the proportion of stimulant mentions among individuals also discussing MOUD over time, consistent with recent reports indicating that stimulant use among people receiving treatment for OUD may be increasing , 3. Co-uWhile relative rates of stimulant co-use among individuals using fentanyl appear to be on the rise, the relative rate for co-use among individuals using heroin showed a gradual decrease. This is perhaps a result of changes in the illicit opioid supply over recent years, as heroin has experienced supply shortages , 35 and Future work can also focus on further utilizing NLP and machine learning methodology to conduct more fine-grained characterization of the data, such as annotation of posts to filter out posts that do not self-report consumption of a substance mentioned \u201343. AdvaOur study has several limitations. Absent contextual information, the primary limitation is the assumption that an individual mentioning an opioid, opioid-related medication, or stimulant is currently using that substance, which may result in overestimation of the number of PWUO/PTMOUD, PWUS, and PWCU, particularly in analyses where these mentions spanned multiple years. Nonetheless, the number of mentions could be a proxy for use and at minimum, mentions of a substance represent some degree of interest or consideration of a particular substance. Also, since the forums from which the Redditors were initially identified are topic-specific, most posts explicitly or implicitly refer to personal experiences.Second, individuals who post on Reddit may not necessarily be representative of all PWUD and caution when generalizing from these findings is warranted. According to a 2019 PEW Research Center report, Reddit has an overrepresentation of males and younger people . Since tThird, not all PWUO, PWUS, and PWCU necessarily mention all the substances they may use. Additionally, we excluded cocaine from our analyses due to concerns around data quality given challenges associated with accurately identifying mentions. Further, our analyses were limited to opioid-related subreddits and excluded any stimulant-related subreddits given our focus on PWUO/PTMOUDs. These limitations may both lead to an underestimation of stimulant and opioid-stimulant co-use. Fourth, we were only able to conduct our analyses on a sample of Redditors due to the time required for retrieving longitudinal Redditor data. Our concerns about this limitation are mitigated by the finding that statistics derived from smaller samples of Redditors early on in our study remained almost identical to those obtained when larger numbers of individuals were included. The exponentially rising number of Reddit subscribers over time also posed a challenge for us, since we could not quantify how much of the increasing opioid or opioid-related medications and stimulant mentions could be attributed to increases in their use in the community versus the growing Redditor numbers. To address this, we examined the ratio of PWCUs to Redditors, which revealed that the growth rate in substance mentions was higher than the growth rate of Redditors. It is possible that other factors contributed to the rising substance mention rates, but we were unable to include such confounding factors in this analysis. Lastly, methodological limitations inherent to all analyses of unstructured text data apply as not all mentions/expressions are detectable by NLP methods , 47. HowOur study included only\u00a0publicly available data that were retrievable via the Reddit API. No private information was obtained. Subreddits that require membership before viewing content were not included. Since our study falls under the category of observational studies conducted on publicly available data, the Emory University Institutional Review Board (IRB) deemed the study to be exempt (category 4). It was determined that no additional harm to individuals was introduced by the use of the publicly available data. In clinical settings, when protected clinical data of patients are used for research, the standard practice is to seek informed consent prior to inclusion. With social media based observational studies such as ours, obtaining consent requires contacting the subscribers directly, and this action requires a higher level of IRB approval as the direct communication with the subscriber may be considered to be intervention. Such contact also presents bigger challenges in determining if the study presents any additional risk or unanticipated harms to the individual. The ethical considerations that need to be taken into account when conducting research using social media data are evolving through discussions and collaborations among active researchers in this space. Currently, there is some consensus that it is appropriate, and perhaps necessary, to utilize all available public data for addressing serious public health crises, such as the ongoing opioid overdose epidemic . ExcludiWe examined real-world, longitudinal social media data from online opioid-related forums to explore patterns in opioid and stimulant discussions and found large increases in co-mentions of opioids or opioid-related medications and stimulants. These findings are consistent with those from other health data sources suggesting growing co-use of these substances and resultant harms. Although a multitude of factors may influence substance use trends, many are difficult to elucidate and explore using traditional data sources such as survey records or health care data. The social media data we explored offer important exploratory insights into which opioids or opioid-related medications and stimulants are most frequently co-mentioned and how these patterns have changed over time. These data derived from real-world conversations may help in hypothesis generation and yield early insights to shape prevention activities addressing health harms associated with opioid and stimulant co-use.Additional file 1: Appendix."}
{"text": "Patient experience with community pharmacy services can be informed by human-centered design principles and approaches. Pharmacy services may benefit from detailed evaluations of consumer experience and patient-centered service design.To use an online journey mapping platform to understand the patient experience with selecting a Medicare Part D plan for individuals that did, and did not, use a free, pharmacy-led, Medicare Part D consultation service.U tests. Open-ended survey responses were coded by the research team using an inductive approach.This was a two-group cross-sectional survey study in a single, rural community pharmacy. Surveys consisted of 7 demographic items, 30 Likert-type items, and 7 open-ended response items. The pharmacy used purposeful convenience sampling to distribute a paper survey to individuals 65 years of age and older currently enrolled in Medicare Part D between June and August 2019. Surveys were distributed to 36 patients currently enrolled in a Medicare Part D plan, with 18 surveys distributed to patients who had previously used a pharmacy-led Medicare Part D consultation service and 18 surveys distributed to patients who did not use the service. Surveys were uploaded to an online journey mapping platform, producing data visualizations for each group. Multiple choice survey items were analyzed using descriptive statistics, wth service user and nonuser groups compared using Mann-Whitney Persona, Empathy, and Current Journey outputs, which mapped Good Experiences and Bad Experiences within the Medicare Part D plan selection experience. Personas differed in their median household incomes . Empathy and Current Journey outputs showed that service users had a wider variety of emotions compared to non-users. Mann-Whitney U tests yielded 5 items with statistically significant differences  in the plan-selection experience, with both groups similarly uncertain about their plan decision. Qualitative responses indicate patient trust was universally important to a complex decision-making process.In total, 36 surveys were returned to the community pharmacy for a response rate of 100%. The journey map platform generated An online journey mapping platform provided insight into how patients experience a pharmacy service that extends beyond satisfaction. For community pharmacies providing Medicare Part D plan consultation services, pharmacies should consider how they can improve the service experience through communication style and patient-centered service design. \u2022Design Think practices like journey mapping may aid in patient-centered service design.\u2022The elaboration likelihood model (ELM) may help to explain patient experience with a Medicare Part D consultation service with Medicare\u2022Information processing ability and heuristics may influence patient decisions. Previous studies have identified that while patients frequently report positive experiences and high levels of satisfaction with community pharmacy Medicare Part D consultation services, they inconsistently use the information gathered from consultation services to switch Part D plans and infrequently use the services in successive years despite potential cost-savings.18.The objective of this study was to use a journey mapping methodology to depict and analyze patient experiences with selecting a Medicare Part D plan for two groups: pharmacy patients who used a pharmacy Medicare Part D consultation service and those who did not. This study makes an important contribution to the literature, using an innovative methodology to assess the patient experience with a community pharmacy service designed to assist patients with a challenging Medicare Part D plan-selection decision.22.1This was an exploratory study using a two-group cross-sectional paper survey combined with a third-party online journey mapping analysis platform. Surveys were administered between 6 and 8 months after participation in the pharmacy-led consultation service. The study was reviewed and approved by The University of Iowa The Institutional Review Board.2.2The study pharmacy is independently owned and located in a small rural town in the Midwest United States. The pharmacy offers various services like vaccinations, medication synchronization, and medication therapy management (MTM). Medicare Part D consultations are available at the community pharmacy year-round for individuals 65 years of age and older or for those looking to enroll in a Medicare Part D plan for the first time. The pharmacy provided information and resources to be used for Medicare Part D plan-selection decisions during the open enrollment period: October 15th to December 7th, 2018 All consultations were provided by one of two individuals: the supervising pharmacist or one certified technician with Medicare Part D plan-selection experience and training. Data on who provided the consultation could not be linked to the individual survey response.Patients eligible for Medicare Part D were mailed a letter in early October 2018, notifying them of the upcoming open-enrollment period and offering free Medicare Part D consultations. For interested parties, consultations were scheduled in 30-min blocks at the community pharmacy. If patients were unable or unwilling to receive the consultation in-person, the same information was offered via a telephone consultation. Before the scheduled consultation, the pharmacist completed a comprehensive medication review (CMR), with third-party software  used to compare Medicare Part D plans based on the current medication regimen. The pharmacy provided each patient with a statement disclosing that the consultations were conducted with the patient's best interests in mind.The pharmacy staff member conducting the service verified the patient's current Medicare Part D plan before printing off the plan-comparison output generated by pharmacy software. This software evaluated patients' chronic medications and pharmacy preferences to identify plans that offer improved insurance coverage. The software generated a print-out that can be shared with the patient. This printout contained information on the patient's current plan, with comparisons to the three lowest-cost Medicare Part D plan alternatives. Pharmacy staff informed patients that several plans existed, and the ones shown were associated with the lowest yearly patient expenditure. After explaining the components of benefit design , the patient was informed on how to change plans. Patients were encouraged to make plan-switching decisions on personal devices but were allowed to use pharmacy computers to make same-day plan-switching decisions at the request of the patient.2.3The online journey-mapping platform started technical development during the first quarter of 2017. The software team that built the platform regularly design and develop applications for projects where data sets are both collected and visually displayed via a web interface. The first journey-mapping beta project took place during the summer of 2017 and informed several changes related to the proprietary algorithm as well as updates to the user interface. As the beta program progressed, updates were implemented in collaboration with both the leadership team experienced with journey map deliverables and the development team familiar with valid approaches to data collection and visualization. The journey-mapping platform entered full production in November 2019 after multiple rounds of iterative updates from beta projects.Discover, Search, Assess, Decide, Assist. These domains focus on the entirety of the Medicare Part D selection experience. Using the standardized items, a study author with Medicare Part D consultation used an iterative process to refine items to reflect the Medicare Part D experience. In total, 7 demographic items, 30 Likert-type items, and 7 open-ended response items were initially included in the survey. Community pharmacy personnel providing the Medicare Part D consultation service reviewed the survey for initial validity and reliability. After revisions suggested by community pharmacy personnel, the survey was piloted with two Medicare Part D beneficiaries currently enrolled in a Medicare Part D plan but unaffiliated with the study pharmacy to identify issues with survey clarity. After piloting, modifications were made based on suggestions to improve readability. Journey mapping domains and example items from the specific journey-mapping platform are included in Appendix A.Survey questions were based on standardized items from the online journey mapping platform and tailored to fit the Medicare Part D plan selection experience. Survey items corresponded to predetermined journey mapping domains: Persona, Empathy, and Current Journey outputs. The Persona outputs can be described as an avatar representing generalized characteristics of each survey group based on median responses. The Empathy outputs are a visual depiction of four emotion domains: Think & Feel, Hear & See, Pain, Gains. Multiple choice survey response items were mapped to each emotion domain and validated by the journey mapping platform development team. The Empathy output also procured an overall emotional experience score, quantifying the overall experience based on the range of emotions felt in the Medicare Part D plan selection process. Scores may range in values from 0 to 10, with larger values associated with better experiences. The Current Journey outputs provide a visual depiction of the comprehensive Medicare Part D plan selection process, progression through 5 decision-making domains: Discover, Search, Assess, Decide, Assist. The Current Journey outputs mapped Good Experiences and Bad Experiences on the Medicare Part D plan selection journey. These experiences are often denoted as Touch Points in journey mapping best practices.,Bad Experiences) or depict moments of experience success (Good Experiences). During Good Experience moments, patients and customers may be further engaged with the service or experience and are more receptive to additional experiences.,Data from the returned paper surveys were entered into the online journey-mapping platform. The software used in this project includes algorithms and analytics that produce visual representations of the Medicare Part D journey for pharmacy service users and nonusers. The platform generated 2.4The pharmacy used purposeful convenience sampling to distribute a paper version of the journey mapping survey to patients between June and August 2019. Recent work developing patient journeys with healthcare services has considering data collection from as few as 8 participants sufficient to develop a patient persona.2.5U tests to identify significant differences in responses between service users and nonusers. Open-ended survey responses were coded by the research team using a qualitative content analysis approach.Journey map outputs were reviewed and discussed with the platform developer. Scaled survey items were analyzed using Mann-Whitney 3,Empathize, Define, Ideate, Prototype, Test.Empathy domain to better understand the patient and their needs. As a result, journey mapping may be a useful tool to focus efforts on defining problems patients may experience throughout their experience, exploring potential solutions to these problems, and pragmatically testing these solutions. The Design Thinking framework and practical applications of the framework's domains like journey mapping may be a viable option in future research for assessing patient-experience with pharmacy services and developing interventions and services using a patient-centered approach.Journey mapping was used as a practical application of the Design Thinking framework.4In total, thirty-six journey map surveys were distributed, completed, and returned to the community pharmacy, for a response rate of 100%.4.1Personas were different in their household incomes. Individuals who did not use the service reported a median household income of $50,000\u2013$74,999 compared to $25,000\u2013$39,999 for those who used the service. Individuals who used the pharmacy consultation service were older than those who did not use the pharmacy consultation service. Journey mapping software outputs for Persona are included in The two patient 4.2Think & Feel category. Patients who used the service had a variety of experiences with the service, ranging from Frustration and Uncomfortable to Good Feelings and .Favorable Comparison Nonusers reported only positive experiences, including Good Experience and Good Feelings. Service users had more Pain and Gain responses than those who did not use the service. For individuals who used the pharmacy service, their overall emotional experience score was 5.8, while individuals who did not use the pharmacy service generated an overall emotional experience score of 6.7. Empathy outputs generated by the third-party journey mapping software are included in Individuals who used the pharmacy service experienced more empathy points in each of the four empathy domains, with the largest visual difference between the groups in the 4.3Current Journey outputs, service users had more extreme Good and Bad Experiences. Service user journeys had more frequent and larger fluctuations in their experience compared to service nonusers. Areas with visual differences in the plan-selection journey appeared to involve collecting and assessing information related to Medicare Part D plans, the need for Medicare Part D assistance late in the decision-making process, and plan selection/decision-making processes. Despite different journeys, both groups appear to end up at similar places, with both groups reporting low optimism regarding their decision. Service users appeared to have more Touch Points during their Medicare Part D plan-selection journey based on the visual depiction of their Current Journey depicted in From the 4.4U tests showed that five survey items had responses with statistically significant differences between service users and nonusers. Most notably, individuals who used the service had a greater appreciation for the features or benefit designs of other plans  and felt they had more opportunities to compare Medicare Part D plans . General item categories and outputs are included in Mann-Whitney 4.5After thematic analysis, the authors agreed on two major themes that describe patient plan-selection experiences: \u201ccomplexity and uncertainty in the decision-making process\u201d and \u201cthe value of trust in the plan selection process.\u201d Representative quotes from service nonusers [NU] and service users [U] may be found in Appendix B.4.5.1You must look at every detail to get the best deal for you-company, pharmacies, medications. [NU9].It is hard to decide if you want a higher deductible or cheaper drug cost. [NU1].Just went to Farm Bureau and let them decide. [NU14].Went with AARP suggestions, overwhelmed with doing it on my own. [NU2].Patients who did not use the pharmacy service appreciated how challenging and complex the plan selection process could be. These individuals expressed varying degrees of frustration with the plan-selection process but often used non-pharmacy resources to help make their plan decision. In some instances, the individuals who did not use a pharmacy service seemed more comfortable deciding on their own and appeared more aware of the various components of benefit design and different components of their specific insurance plan, often engaging in independent research and plan comparison. Individuals who did not use the service used a wide variety of information sources to make their plan decisions, including family, friends, and insurance agents. When individuals sought help, they often let the individual make their plan selection for them.There are so many options for plans [U4].Least enjoyable: comparing what they did or did not cover. So much paperwork to go through [U7].Comparison left me frustrated, went back to my original plan because my agent can help me. [U12].The most enjoyable part was having my options presented to me and explained in an understandable form. [P10].For individuals who did use the pharmacy service, they appeared more aware of the number of plans that were available to them. While some patients appreciated the opportunity to compare plans, many found the plan-comparison process to be frustrating, specifically covering details such as medication coverage. In one instance, the comparison process drove an individual to select a plan from a previous year. While some patients felt the pharmacy service presented options in an understandable form, others reported there were aspects of plan information they did not understand.4.5.2Some uncertainty at first, but confidence because my husband talked to his health insurance agent. [NU12].I took the advice of trusted agents. [NU13].Both groups of individuals had a challenging time trusting the Medicare Part D plan information they received but were aware that plan-switching may be beneficial every year. Individuals who did not use the pharmacy Medicare Part D consultation service were often still uncertain of their plan decision but seemed to have high levels of trust with the individual that assisted them with their plan decision and/or the information they acquired. Ultimately, service nonusers readily accepted the recommendations provided by trusted agents.I needed to trust the person selling it. Hopefully she was honest and not just working to make a sale. [U6].Least enjoyable was trusting the decision I made was best for me [U14].Still not confident I have the best plan for me!! [U14].For individuals using the pharmacy service, trust was more challenging to establish. Patients using the pharmacy service had a difficult time trusting the pharmacy staff member providing the consultation and the information they received. In some instances, patients felt like the pharmacy staff member was trying to \u201csell them\u201d a specific plan. Ultimately, service users were often uncertain if they had selected the best plan.5The journey mapping process highlighted important differences in the plan-selection experience for service users and nonusers. These differences in experience occurred over the entire service process and were highlighted by differences for users and nonusers in the two personas and the open-ended comments.Commonalities between pharmacy service users and nonusers were that all individuals found the Medicare Part D plan-selection process to be challenging, highlighting that collecting information and assessing it for trustworthiness was difficult, especially in such a technical domain. Both groups reported depending on additional assistance with their Medicare Part D plan selection, but their access to, and use of trusted experts to help with decision-making varied. Despite the differences in information acquisition and plan comparison, both groups still had doubts about their ultimate plan decision, suggesting a target for improvement.One important difference between pharmacy service users and nonusers was their average income level. Older individuals with lower fixed incomes appear to have sought additional support from their community pharmacy more often than younger individuals with more financial resources. People with higher incomes may already have access to services like a financial planner or an insurance agent who may be providing advice. Less well-off or older adults may have less access to these resources.,,Another difference was that individuals who used the Medicare Part D consultation service reported more opportunities for plan comparison and greater awareness of other plan features than those who did not use the pharmacy service. This points to the pharmacy conferring their unique expertise \u2013 but perhaps doing so at a level that exceeds the patient's desire for and capacity to use complex information. Consistent with other studies, this level of information overload seems to have led to patient's having a more challenging plan-selection experience.Individuals who did not use the service, on the other hand, appeared to already have a better baseline understanding Medicare Part D insurance and the plan-selection process, often highlighting the importance of understanding specific insurance jargon (i.e. deductible) in their open-ended comments and choosing plans based on this understanding. Individuals who did not use the community pharmacy service may not have been as overwhelmed with information if their agent offered a narrower recommendation, but still left their decision up to an individual they had previously trusted with difficult financial decisions. While, it is difficult to disentangle what effect might be due to education and income and what effect is due to the pharmacy service itself, the data point to targets for service improvement focusing on patient-centered information and facilitating trust within the encounter.5.1,,,With patients making their Medicare Part D plan decision based on the extent to which they could trust the information presented by the pharmacy team, it is important to explore how trust influences older individuals and their decision-making processes.5.2Elaboration Likelihood Model (ELM) may be a useful model for conceptualizing the relationship between patient preferences for information and trust during challenging decision-making experiences like selecting a Medicare Part D plan.,elaborate, or the ability to perform issue-relevant thinking. The extent an individual elaborates influences the types of persuasive messaging and information they may be receptive to, specifically central and peripheral persuasive messaging. Central routes to persuasion often are useful when elaboration is high. Individuals who engage in high levels of issue-relevant thinking undergo careful processing of information and message, ultimately using the information to make an informed decision. Peripheral persuasion is less direct, where individuals use simple decision values like communicator credibility or trustworthiness to make their decision. Elaboration for individuals influenced by peripheral persuasion messages is low, with issue-relevant thinking minimally involved in persuasion and the subsequent decision-making process.The In the context of the Medicare Part D consultation service, patients were exposed to a multitude of plans with an emphasis on specific benefit designs. Patients were required to perform high levels of elaboration, processing large amounts of complex information and use it to make informed Part D plan selections. By providing patients with high-level detailed plan information, the pharmacy assumed that patients were willing and able to elaborate. When faced with plan comparison opportunities via the community pharmacy service, patients were consistently frustrated and reported negative experiences, which may be attributed to moments requiring high levels of elaboration such as processing information and comparing plans to make a plan-switching decision. Patients who did not use the service were infrequently required to elaborate, as patients who did not use the pharmacy service were presented with considerably less information and were infrequently required to compare plans, which resulted in a better experiences potentially due to lower elaboration requirements. When patients who used the pharmacy service were unable to elaborate, they defaulted to heuristics, like the trustworthiness of the pharmacy team, to make their decision. Given that the information presented to patients emphasized cost-related features of specific plan information, patients may have defaulted to a trust heuristic which may have worsened the plan-selection experience. Individuals who were able to elaborate, or unable but not required to elaborate, may have had more positive experiences as information and persuasive messaging were aligned with patient ability and preference.5.3Patient-centered design and consumer behavior research methodologies can be used to refine community pharmacy interventions and the associated impacts of these refinements on patient behavior and outcomes. While we used a journey mapping software platform, more traditional ethnography methodology and participatory approaches can be used to evaluate the patient experience and facilitate a better understanding of the patient decision-making process. To corroborate findings, additional methodologies should be employed, such as patient observations, interviews, and focus groups. There may be additional benefit in prototyping other interaction designs for community pharmacy services, specifically focusing on Medicare Part D consultations, to compare across patient-pharmacist interaction evaluations. For example, how should messages about plan cost be framed so the patient does not feel like the pharmacist has a financial conflict of interest? Research should be conducted to explore differences in patient perceptions of services based upon the person providing the service: pharmacist, pharmacy intern, or pharmacy technician. As journey mapping does not formally test patient preferences and needs related to an experience, more work is needed to formally test and evaluate patient preferences for Medicare Part D consultation service delivery, including the types of information patients prefer, how users would like information presented, and the level of information complexity that users need to make informed Medicare Part D plan decisions.5.4There are several study limitations to consider. Data were collected from a single, rural community pharmacy located in rural Midwest USA. Other pharmacies may have alternative ways of delivering Medicare Part D consultation services and more diverse patient populations may have different experiences. The community pharmacy distributed and collected surveys that may have contributed to social desirability bias, however, patients who used the service reported worse Medicare Part D experiences than those who did not. Both a pharmacist and pharmacy technician delivered the service, and as a result, experiences may have differed based on the service provider. Further, there may have been variations in the patient experience based on the method of service delivery (telephonic vs. in-person). These service variations are likely to contribute to variations in the patient experience but were unable to be linked to specific survey responses. As such, patient preference for delivery method and service provider should be explored in future studies.6Using a journey mapping process that included qualitative and quantitative data collection and analysis depicted the entirety of a patient experience, both good and bad, with selecting a Medicare Part D plan, with or without using a pharmacy-provided Part D plan selection service. Quantitative data triangulated these results and identified key differences in experiences and demographics between community pharmacy Medicare Part D consultation service users and nonusers. Qualitative data provided context and themes informing theoretical applications and explanations associated with the patient experience. By integrating these methodologies, consumer trust literature, and ELM, we can identify clear areas of pharmacy service improvement with recommendations for patient-centered refinement informed by existing theory. Based on these findings, community pharmacies offering Medicare Part D consultations would benefit from redesigning interventions to focus on the way information is delivered to patients, what information is provided, and how they can instill trust in the patients they serve.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
{"text": "Anaplastic lymphoma kinase tyrosine kinase inhibitors are standard therapeutic agents prescribed for anaplastic lymphoma kinase-positive non-small cell lung cancer, and treatment with these agents has been shown to contribute to long-term survival in patients. However, there is no consensus regarding the course of treatment after the\u00a0onset of anaplastic lymphoma kinase tyrosine kinase inhibitors related\u00a0drug-induced interstitial\u00a0lung disease.Here, we present a case of successful lorlatinib treatment after the onset of drug-induced interstitial lung\u00a0disease caused by alectinib.A 57-year-old Japanese man was diagnosed with stage IVB non-small cell lung cancer by bronchoscopy, but gene mutation testing could not be performed because of the small amount of specimen. After diagnosis, first-line therapy with cisplatin/pemetrexed was initiated, but the patient developed renal dysfunction. Bronchoscopy was performed again to guide further treatment, and the non-small cell lung cancer was found to be anaplastic lymphoma kinase positive. Alectinib was started after the onset of progressive disease, but it resulted in drug-induced interstitial\u00a0lung disease, necessitating alternative treatments. He subsequently received nanoparticle albumin bound paclitaxel, which was halted in view of the renal dysfunction. Thereafter, lorlatinib was administered, which was continued without drug-induced interstitial\u00a0lung disease relapse.Since alectinib can occasionally cause drug-induced interstitial\u00a0lung disease, as in the present case, lorlatinib may be an option to continue treatment in patients without other treatment alternatives. With the identification of gene abnormalities in anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC), effective ALK tyrosine kinase inhibitors (ALK-TKIs) have been developed for treatment. ALK-TKIs are more effective than cytotoxic chemotherapy, and long-term treatment with TKIs has been shown to prolong the overall survival of patients. Lorlatinib is a novel third-generation ALK TKI that has been shown to be more potent than second-generation TKIs in biochemical and cellular assays and has the broadest coverage of the identified ALK resistance mutations. Additionally, it was designed to cross the blood\u2013brain barrier so as to achieve high exposure levels in the central nervous system (CNS) . BecauseHowever, it is unclear whether lorlatinib can be used after discontinuation of another ALK-TKI owing to adverse events, for example, in cases of drug-induced interstitial\u00a0lung disease (DILD). Here, we present a rare case showing the feasibility of continuing lorlatinib after the onset of DILD caused by alectinib.18F-fluorodeoxyglucose positron emission tomography/CT (18F-FDG PET/CT) was performed, and abnormal accumulation was observed in the longitudinal hilar lymph nodes , vertebral body, and left pleura. The results of the 18F-FDG PET/CT and other findings led to the diagnosis of lung adenocarcinoma . We recommended rebiopsy considering the possibility of a genetic mutation, but the patient refused the examination because of strong coughing during the first bronchoscopy and progressive symptoms of hoarseness. The patient strongly desired an early treatment, so we initiated first-line therapy with cisplatin (75\u00a0mg/m2) and pemetrexed (500\u00a0mg/m2) 3\u00a0weeks after the first visit. However, the patient developed grade 2 renal dysfunction , making it difficult to continue the therapy . In addition, progressive disease was noted  was initiated after treatment with steroids. However, 3\u00a0months later, obstructive pneumonia associated with an increase in the size of the primary lesion was observed . Because of this complication and upon the patient\u2019s request for oral medication, lorlatinib (100\u00a0mg/day) was administered as fourth-line therapy. Treatment with antibiotics and lorlatinib reduced the lesion\u2019s shadow, and no recurrence of DILD was observed. Thirty-three months after the start of treatment, stable disease has been maintained, and lorlatinib has been continued  scan showed a tumor, with its major axis measuring approximately 40\u00a0mm in the left upper lobe of the lung. The hilar and mediastinal lymph nodes were also swollen Fig. a. PrimarIn the search for advanced lung cancer treatment, driver genes have been identified, and several molecular-targeted drugs have been developed and reported to exhibit remarkable antitumor effects. For ALK-positive NSCLC, alectinib was more effective than crizotinib in phase III trials (ALEX study) and is, therefore, widely used for first-line treatment . LorlatiHowever, a molecular-targeted drug with a clear antitumor effect may still have to be discontinued upon the occurrence of side effects. The overall frequency of DILD associated with ALK-TKIs is 2.14% . AlthougAs of August 2022, no previous study has reported serious lung disease induced by lorlatinib, and it can be administered in normal doses even to patients with renal dysfunction . As seenIn summary, in patients with driver mutation-positive NSCLC, continuation of treatment with TKIs to the maximum extent possible is essential to improve the prognosis. Even after TKI-induced DILD, another TKI may be considered as a treatment option."}
{"text": "Primary intracranial neuroendocrine tumors are extremely rare malignancies with very few cases reported in the world literature. We describe a primary neuroendocrine carcinoma arising from the right cerebellopontine angle, the second case that has been described in this location. The possible origin in this place and treatment are described.A 29-year-old male patient, diagnosed with schwannoma of the\u00a0right cerebellopontine angle, and treated with radiosurgery at another institution, came to our hospital six months later, The patient presented with a history of rapid progression of numbness on the right side of the face, diplopia, dizziness, vomiting, and facial palsy. On examination, the right cranial nerves V, VI, VII, VIII, and IX were affected. The MRI showed tumor growth\u00a0occupying the right cerebellopontine angle, with compression of the brain stem and cerebellum. A right retromastoid craniectomy removed the tumor partially and the histopathological examination revealed a high-grade neuroendocrine carcinoma.We describe a primary neuroendocrine\u00a0tumor of the brain that, despite its rarity, must be considered in the differential diagnosis. There are currently no guidelines for the management of these tumors. According to previously reported cases, surgery is the first line of treatment, followed by radiotherapy or chemotherapy. We consider that such a rare case is needed to be reported for a better understanding of the disease and its neurobiology. Neuroendocrine neoplasms (NENs) are a heterogeneous and uncommon group of malignancies with varied histology. Rarely, these tumors can occur intracranially. There are nine reported cases with a primary origin in the central nervous system (CNS) -10. The Because of the wide and even distribution of the endocrine cells, these tumors can arise in almost any site of the body, but the most common origin is in the gastrointestinal tract and bronchi. Brain metastasis has been reported in 1.3-1.4% of the patients , but priA 29-year-old male, presented at another institution, with progressive right facial palsy, vertigo, nausea, and vomiting. A magnetic resonance image (MRI) demonstrated a right tumor in the internal auditory canal, protruding to the cisterna of the CPA. It was assumed to be a schwannoma of the right vestibulocochlear nerve Figures -1B. The A head MRI showed tumor growth, occupying the right CPA, with compression of the brain stem\u00a0and extension to the cerebellomedullary cistern Figures -1D. The Histopathological examination revealed a poorly differentiated carcinoma with solid sheets of large, polyhedral cells with amphophilic and ample cytoplasm Figure , with foA thorough evaluation to exclude any evidence of extracranial NENs was performed, including computed tomography (CT) of the thorax, abdomen, and pelvis, which was negative for other tumors. A positron emission tomography-computed tomography (PET-CT) with 18-fluorodeoxyglucose (FDG) was performed to exclude tumor spread that was negative for extracranial NENs. The patient underwent four cycles of chemotherapy (ChT) of cisplatin\u00a0and etoposide. And trying to strengthen the ChT, a course of monoclonal antibody and an immune-checkpoint inhibitor pembrolizumab was included.\u00a0Despite this, he experienced worsening of his facial symptoms and numbness of the other side of the face, as well as difficulty swallowing. An MRI eight weeks after surgery revealed huge disease progression and a severe displacement of the brain stem Figures -1H. ThirNeuroendocrine tumors (NETs) are relatively uncommon tumors arising from enterochromaffin cells located in many organs such as the gut and lung . They arThese cells are found diffusely throughout different organs and are well described throughout the gastrointestinal tract, the respiratory tract, and the central nervous system (CNS) -17.\u00a0ManyIntracranial involvement by a NEN is most seen as hematogenous metastasis. Its presentation is common 13 months after the initial diagnosis, on average. The incidence of patients with NENs having brain metastases is <5%. Furthermore, only 1.4% of metastatic brain tumors are NENs, and most of these lesions originate from the lung. There is no gender preference for the occurrence of brain metastases. When patients do present with metastatic NENs to the brain, they typically present with other local and distant metastases . Brain mPrimary intracranial NECs are rarely mentioned in the literature, accounting for only 11 cases until the preparation of this manuscript (Table One feature that stands out is that 6 out of 11 primary NENs arise extra-axially. Two cases originated from the pineal\u00a0parenchyma\u00a0,8 and thPrimary brain NENs are rare lesions, particularly at the cerebellopontine angle. The first line of treatment for this type of tumor is surgery followed by radiation and chemotherapy. The prognosis of these tumors is correlated to\u00a0their histological grade. The case we presented was a high-grade tumor with a limited prognosis."}
{"text": "Brain-derived neurotrophic factor (BDNF) has a demonstrated role in promoting memory functions and neuronal survival. A common variant in the BDNF gene, the Val66Met polymorphism, has been found to reduce BDNF expression and increase vulnerability to neurocognitive impairments and age-related cognitive declines. Research suggests that BDNF Val66Met may be associated with hypertension, a well-established risk factor for health and brain functioning across the lifespan. While BDNF is most studied for its effect on learning and memory, its role in moderating other cognitive domains is not as well understood, especially those most affected by hypertension. Furthermore, no study to date has investigated these relationships exclusively with older adults and those at increased risk for cognitive decline. Therefore, the aim of this study was to investigate the effect of BDNF Val66Met and hypertension on the executive function ability of older adults (mean age 71.3\u00b19.2 years) diagnosed with amnestic Mild Cognitive Impairment (aMCI) (N = 108). Results showed that BDNF Val66Met moderated the relationship between hypertension and executive functioning, such that hypertensive carriers of the BDNF Met allele performed significantly worse compared to Val-Val homozygotes, and independent to the effects of aging. These results indicate that genetic and vascular risk interactions can predispose older adults with aMCI to impairments in multiple cognitive domains, and potentially increase susceptibility to further declines or conversion to dementia. These findings have implications for future research on dementia disease pathology, and highlight the importance of strategies that target fixed and modifiable risk factors to promote cognitive resilience."}
{"text": "The authors regret that the names of two co-authors (Jianwei Lu and Li Guo) were spelled incorrectly in the original article. The correct author names are given here.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."}
{"text": "The authors regret that Paulo Ribeirinha\u2019s name was spelled incorrectly in the original article. The correct spelling of all author names is presented above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."}
{"text": "The authors regret that their names were displayed incorrectly in the original article. The correct author names are as presented above.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers."}
{"text": "Clostridioidesdifficile associated disease. Its precise mechanismof action and the origin of limited activity against Gram-negativepathogens are relatively unknown. Herein, treatment with MGB-BP-3alone significantly inhibited the bacterial growth of the Gram-positive,but not Gram-negative, bacteria as expected. Synergy assays revealedthat inefficient intracellular accumulation, through both permeationand efflux, is the likely reason for lack of Gram-negative activity.MGB-BP-3 has strong interactions with its intracellular target, DNA,in both Gram-negative and Gram-positive bacteria, revealed throughultraviolet\u2013visible (UV\u2013vis) thermal melting and fluorescenceintercalator displacement assays. MGB-BP-3 was confirmed to bind todsDNA as a dimer using nano-electrospray ionization mass spectrometryand nuclear magnetic resonance (NMR) spectroscopy. Type II bacterialtopoisomerase inhibition assays revealed that MGB-BP-3 was able tointerfere with the supercoiling action of gyrase and the relaxationand decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichiacoli. However, no evidence of stabilization of thecleavage complexes was observed, such as for fluoroquinolones, confirmedby a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlightadditional mechanisms of action of MGB-BP-3, including interferenceof the action of type II bacterial topoisomerases. While MGB-BP-3\u2032slack of Gram-negative activity was confirmed, and an understandingof this presented, the recognition that MGB-BP-3 can target DNA ofGram-negative organisms will enable further iterations of design toachieve a Gram-negative active S-MGB.MGB-BP-3is a potential first-in-class antibiotic, a StrathclydeMinor Groove Binder (S-MGB), that has successfully completed PhaseIIa clinical trials for the treatment of  MGB-BP-3 also has potent (<1\u03bcg/mL) antibacterial activity against methicillin-resistantand methicillin-susceptible Staphylococcus spp., Streptococcus spp., and vancomycin-resistant and vancomycin-susceptible Enterococcus spp.13 There islimited information in the literature regarding MGB-BP-3\u2032sactivity against Gram-negative organisms, possibly reflecting thewell-known challenges of penetration through the double membrane system.Strathclyde MGBs (S-MGBs), based upon a distamycin template,haveshown remarkable anti-infective properties. Their favorable cytotoxicityprofiles to mammalian cells give them selectivity indices that makethem suitable for development as novel drugs. This has enabled extensive MGB-BP-3 1 has sucStaphylococcusaureus show a steep decrease in bacterial viabilityindicative of catastrophic failure of the biochemical machinery withinthe bacterium, rather than a sigmoidal dose-response curve indicativeof interaction with a single molecular target.13 This is perhaps unsurprising as MGB-BP-3, and S-MGBs moregenerally, are thought to bind to many AT-rich sequences within theminor groove of dsDNA, similar to the template natural product distamycin.MGB-BP-3 is hypothesized to bind as a dimer, with two MGB-BP-3 moleculeswithin each binding site, again, similar to distamycin. This bindingmode has been shown for some early S-MGBs, and also more therapeuticallyinteresting alkene-containing S-MGBs but not MGB-BP-3 itself.14 However, there have been limited studies thatspecifically characterize the interaction of MGB-BP-3 with dsDNA.The biological consequences of MGB-BP-3 interacting with dsDNA haverecently been explored using RNA-Seq and DNase I and potassium permanganatefootprinting. It was demonstrated that MGB-BP-3 binds to and inhibitstranscription from multiple essential promoters on the S. aureus chromosome, and that resistant mutantswere unable to be generated by serial passage experiments.13 However, given the molecule\u2019s abilityto bind to many different sites on the bacterial genome, MGB-BP-3has the potential to interfere with other biological processes involvingDNA, although these have not yet been investigated.Dose-response curves of MGB-BP-3againstHerein,an investigation into the origin of MGB-BP-3\u2032s selectiveactivity against Gram-positive bacteria is presented, including synergystudies and fluorescence microscopy. Furthermore, we expand the evidencebase for MGB-BP-3\u2032s multiple mechanisms of action by providinga detailed account of its interaction with dsDNA and interferenceof the action of type II bacterial topoisomerases. Studies also explorereasons for the lack of significant Gram-negative activity to determinewhether there are prospects of developing S-MGBs for these species.S. aureus and Enterococcusfaecalis) and four Gram-negative  isolates (80 0.2 \u03bcM) but no measurable MICs (up to 100 \u03bcM) againstthe Gram-negative bacteria. Potent activity against Gram-positivepathogens was further demonstrated using an expanded panel of Gram-positivestrains .The selective activity of MGB-BP-3 for only Gram-positive bacteriawas confirmed using a panel of ESKAPE pathogens comprising two Gram-positive to more than 2000-fold (E. coli) compared with MGB-BP-3 alone. This synergismwas confirmed by carrying out checkerboard assays using the same setof Gram-negative bacteria. Fractional inhibitory concentration indices(FICIs) were indicative of strong synergy between MGB-BP-3 and allGram-negative bacteria ,was investigated 1. A highbacteria 1. Checkeynergism 2.Table S2), was used in fluorescence microscopy studies ofthe same panel of ESKAPE pathogens .These results are indicative of the intrinsic resistanceof Gram-negativebacteria to MGB-BP-3 being due to poor intracellular accumulation.To further demonstrate this, a fluorescent analogue of MGB-BP-3, S-MGB-245,which has a similar lack of Gram-negative activity  family pumps (reflecting published PA\u03b2N specificity) normembrane permeation is sufficient in isolation to potentiate activityin these strains. There could also be other factors that contributeto elevated resistance.18 Activity was potentiated in strainsknown to be more permeable, K. pneumoniae M6 (PMBN and PA\u03b2N) and PAO1 (PMBN only). The latter againsuggests that inhibition of RND efflux pumps in P.aeruginosa is not enough to potentiate activity.Poor intracellular accumulation of MGB-BP-3 is likely to be causedby either efficient efflux, poor membrane permeation, or a combinationthereof. To investigate these possibilities, we carried out potentiationstudies on a range of well-characterized Gram-negative bacteria usingthe membrane permeabilizer, polymyxin B nonapeptide (PMBN), and theefflux pump inhibitor PA\u03b2N used in the presence of Mg to minimizeits membrane permeation effects. or PA\u03b2N 2. For A.19 Although the effects are likely to be pleiotropic, a tolC mutant showed a significant reduction in the MIC suggestingthat efflux through AcrAB-TolC, or other membrane transporters thatutilize TolC, is a component of reduced efficacy in Gram-negativebacteria  complex, resulted in significantly higher MICsfor MGB-BP-3 but not for ciprofloxacin, suggesting that the observedreduction in susceptibility is perhaps linked to specific featuresof the \u03b2-barrel protein complement of the outer membrane , suggesting that the membraneitself remains intact in this directed insertion mutant under theconditions tested.The role of efflux in mediating resistance was confirmedusingdirected insertion mutants from the Keio collection.bacteria 3.Simila strains 2. Convermembrane 3. The MIS. aureus as a bioassay, given its high degree of susceptibility. The data of salmon, afluorescence intercalator displacement assay using gDNA of the ESKAPEpathogens, a native mass spectrometry experiment using a short AT-richDNA oligomer, and an NMR investigation of MGB-BP-3 binding to thesame short oligomer.Tm = 11 \u00b0C, Thermalmelt analysis ofMGB-BP-3 binding to salmon gDNA showed significant stabilization ofthe complex, indicative of strong binding .Nano-electrosprayionization mass spectrometry (nESI-MS) was also used to confirm thatMGB-BP-3 binds to dsDNA. Like the natural product distamycin, S-MGBsare thought to bind to AT-rich sequences as a dimer, i.e., two moleculesof S-MGB bind within each binding site on the minor groove of dsDNA.For these experiments, a self-complementary oligomer with an AT-richbinding site (5\u2032-CGCATATATGCG-3\u2032) was used. nESI-MSof the oligomer alone shows the expected DNA duplex in charge states4- and 5- Panel A, 5, and up1H NMR spectrum was acquired on 5\u2032-d(CGCATATATGCG)-3\u2032. The free DNA showed the expectedappearance of five imino proton signals in the region between \u03b41H = 12.50 and 13.50 ppm, integrating to 10 proton equivalentsfor 10 protons  in slow chemicalexchange from a potential total of 12 imino proton resonances  (Table S5). The DNA sample was subsequentlytitrated with aliquots of a concentrated solution of MGB-BP-3 initiallyto a 1:1 ligand/DNA duplex molar ratio (Table S6). Additionally, new 1H NMR signals were observed growinginto the NMR spectrum in the region \u03b41H = 8.50\u201310.00ppm with each ligand aliquot addition and growing to a maximum bythe end point . At the lowest sampletemperature, this region of the NMR data integrated to 10 proton equivalents,consistent with 10 different imino protons. Additionally, six methylsinglet NMR resonances were observed in the same NMR spectrum in achemical shift window consistent with resonances arising from thyminemethyl groups.Low-intensity, broad-lineshape resonances associatedwiththe DNAimino protons of a ligand/DNA complex are characteristic of looserassociation between the ligand and DNA, the formation of a less well-definedcomplex in terms of fit between the ligand and DNA compared with othercomplexes, suggestive of some conformational exchange, or a combinationof some or all of these characteristics.ach step 6d\u2013i. NotWithout further supporting evidence, two possibleexplanationsmay be speculated. These observations may first be explained by thepresence of a completely asymmetric ligand\u2013DNA complex. Sincetwo equivalents of ligand are bound, this explanation would requireassociation of the individual ligands to be bound to the DNA in quitedifferent locations or with quite different orientations or conformationswith respect to each other. Alternatively, the observations may beexplained by the presence of two different symmetrical ligand/DNAcomplexes, each of which gives rise to five imino proton resonancesrepresenting 10 imino protons for each symmetrical complex.1H, 1H] NOESY NMR data of the cooled (1 \u00b0C)2:1 ligand/DNA duplex sample were partially assigned. The DNA proton 1H NMR resonances for the 2:1 ligand/DNA complex indicate twouniquely identifiable sets of linked spatial correlations consistentwith a typical data assignment protocol . Chemical exchange links the two sets of data even at thetemperature used to acquire these NMR data. The data may be interpretedby considering two possible ligand binding models  andcell death.22 Thus, it was important to investigateif this mechanism was relevant to MGB-BP-3.Havingdefinitively demonstrated that MGB-BP-3 interacts stronglywith dsDNA, the ability of this interaction to interfere with criticalDNA processes was investigated. While it has been demonstrated thatMGB-BP-3 can interfere with transcription,S. aureus and E. coli enzymes, further suggestingthat the principal reason for the lack of Gram-negative activity isdue to inefficient intracellular accumulation. The lack of activityin the gyrase and topoisomerase IV cleavage assays suggests that MGB-BP-3does not cause the accumulation of DSBs as is observed in the mechanismof action of fluoroquinolones. This is further confirmed by the limitedeffects (<1.5-fold) of MGB-BP-3 on fluoroquinolone-resistant gyrases(E. coli S83L gyrase and S. aureus S84L gyrase mutants) on the supercoilingassay IC50s.MGB-BP-3 interferedwith the supercoiling action of gyrase and the relaxation and decatenationby topoisomerase IV 6. It is E. coli reporter strains, as used previously and a DNA-damaging agent (mitomycin C (MMC)) were used as positivecontrols for the DNA SOS response, and a compound that interacts withthe 30S subunit of the ribosome (doxycycline) was used as a negativecontrol, all treated at 0.5\u00d7 MIC. A significant fold-inductionof fluorescence for recA and lexA was observed for ciprofloxacin and MMC, while no significant inductionwas seen for doxycycline. MGB-BP-3 did not induce the SOS responsein this assay at 0.5\u00d7 MIC, with no induction seen for eithergene, comparable to doxycycline, which is known to kill bacteria bya mechanism not linked to DNA damage.23 This experiment confirms that, unlike the fluoroquinolones, whileinterference with the action of type II topoisomerase is likely tobe a contributory part of the mechanism of action of MGB-BP-3, itdoes not induce DSBs and the SOS response.To provide further evidencethat the interference of the actionof type II topoisomerases occurred through a mechanism distinct fromfluoroquinolones, the SOS response was assessed in a series of eviously 8.18 MGB-Table S1), confirming the differentmechanism of action.Further evidence comes from theobservation that MGB-BP-3 is equallyeffective with ciprofloxacin-resistant and ciprofloxacin-susceptiblestrains but also for the new class of drugs as a whole, that the best possibleunderstanding of the mechanism of action is obtained.MGB-BP-3 is a memberof a new class of drugs with a new mechanismof action and satisfies the four WHO criteria for genuine novelty.The literaturedata supporting the development of MGB-BP-3 providestrong evidence for its effectiveness against Gram-positive pathogens;however, there is limited information on its effectiveness againstGram-negative pathogens. In this study, we add to the evidence thatMGB-BP-3 is highly active against Gram-positive bacteria while demonstratingits limited activity against Gram-negative bacteria. As is the casefor many Gram-positive only drugs, MGB-BP-3 appears to be ineffectiveagainst Gram-negative bacteria due to poor intercellular accumulation.This may be due to a combination of poor penetration and efficientefflux, but the specific mechanism appears to vary depending on thecharacteristics of the specific strain. In many cases, strong potentiationwith PA\u03b2N and/or PMBN was observed that suggests that MGB-BP-3may be effective against Gram-negative bacteria if an appropriatesynergistic partner can be found, or its structure can be alteredto allow for sufficient accumulation.The full mechanism ofaction of MGB-BP-3 is not well understood,not least because its design adopts a multitargeted approach. Multipleexperimental techniques have confirmed that MGB-BP-3 binds stronglyto dsDNA. Importantly, by confirming that MGB-BP-3 binds to gDNA fromboth Gram-positive and Gram-negative organisms, we have provided furtherevidence that the difference in activity of MGB-BP-3 in these bacteriais due to differential intracellular accumulation, and not a differencein target engagement. The precise interactions of MGB-BP-3 with DNAwere further characterized by demonstrating that the compound bindsas a 2:1 dimer, similar to the natural product distamycin, from whichit is derived. Furthermore, NMR studies strongly suggest the potentialof a dynamic binding interaction with dsDNA, at least for the specificshort, AT-rich dsDNA oligomer used in the experiments.13 Here, MGB-BP-3 has beenshown to interfere with other DNA-centric processes, such as the actionof topoisomerase and gyrase, but it is not thought that MGB-BP-3 interactswith these enzymes directly to achieve this interference. Instead,we suggest that this is likely to be achieved by either MGB-BP-3 directlymasking the enzyme binding site on DNA or indirectly altering thetopology of the binding site through an allosteric mechanism fromproximal binding. Notably, the absence of interference with the cleavageassays and the absence of induction of recA and lexA indicate a different mechanism of action to the fluoroquinolones,which is consistent with MGB-BP-3 binding to DNA and interfering withthe formation of the DNA\u2013enzyme complex.Previousstudies employing RNA-Seq identified \u223c700 transcriptswith drug-altered expression profiles, many of which were associatedwith DNA replication, supercoiling, and primosome formation. Moreover,potassium permanganate footprinting confirmed that MGB-BP-3 bindsto certain SigA-dependent promoter regions, preventing transcriptionof these genes.13 Consequently, the evolution of target-basedresistance for MGB-BP-3, and by extension other S-MGBs, should beunlikely.Our study hascontributed to the understanding of the mechanismof action of MGB-BP-3 and has identified additional DNA-centric mechanismsthat MGB-BP-3 can interfere within bacteria. These findings providefurther evidence that the resilience to resistance of MGB-BP-3 seenin the laboratory may be due to its multitargeted design requiringmany simultaneous mutations at the multiple target sites on the genomicDNA.MGB-BP-3 and S-MGB-245were preparedas previously described in refs , respectSalmonDNA  at 1 mg/mL in 1 mM phosphate buffer (pH7.4) containing 0.27 mM KCl and 13.7 mM NaCl was annealed at 90 \u00b0C for 10 min. MGB-BP-3 at 10 mM in DMSO wasdiluted with the same phosphate buffer, and combined with the salmonDNA stock to yield a single sample with 10 \u03bcM S-MGB and 0.02mg/mL gDNA in 1 mM phosphate buffer containing 0.27 mM KCl and 13.7mM NaCl. Control samples containing only MGB-BP-3 or gDNA were prepared,respectively. Samples were melted at a rate of 0.5 \u00b0C/min from45 to 90 \u00b0C with spectra recorded at 260 nm on a UV-1900 UV\u2013visspectrophotometer fitted with a Peltier temperature controller (Shimadzhu)using LabSolutions  software. The melting temperatures(Tms) of the MGB-BP-3:DNA complexes were determined by fitting a sigmoidalfunction using a Boltzmann distribution in OriginPro. Two independentexperiments were carried out with values quoted with an error no worsethan \u00b10.5 \u00b0C.Bacterial genomic DNA  or salmon genomic DNA  dissolved in 1mM phosphate buffer pH 7.4  to a concentration of 100 \u03bcg/mL inSybrSafe was used as supplied by the manufacturer in DMSO, and MGB-BP-3 wasprepared as 10 mM stock in DMSO. These stock solutions were dilutedappropriately with each other and 1 mM phosphate buffer to give atest solution comprised of 20 \u03bcM S-MGB, 12,500-fold dilutionof SybrSafe and 3.92 \u03bcg/mL DNA. Control solutions of gDNA andSybrSafe, gDNA, and SybrSafe at these concentrations were also prepared.Test and control solutions were heated to 30 \u00b0C and the fluorescenceof each solution was measured using the SYBER filter setting of aStepOnePlus using melt analysis mode (StepOne Software v2.3). Thereduction of fluorescence due to the binding of MGB-BP-3 to the gDNAwas calculated as a normalized percentage based on the fluorescencemeasured due to the control with SybrSafe and gDNA as maximum andthe control with only SybrSafe as minimum. Low normalized percentageindicates a greater ability to displace SybrSafe from the gDNA andsuggests strong binding to gDNA. Three independent experiments werecarried out and the results were presented as average values \u00b1standard error of the mean.DNA oligonucleotidesequence5\u2032-CGCATATATGCG-3\u2032 was purchased in lyophilized form and the purity was confirmed by NMR. Stock solutionsof DNA (100 \u03bcM) were prepared with 150 mM ammonium acetate buffersolution  and2 mM potassium chloride solution . This solution was annealed at 90 \u00b0C for10 min and allowed to cool to room temperature. S-MGB stocks (10 mM)in 100% DMSO  were diluted to 1 mM S-MGBsolution with 150 mM ammonium acetate. Final samples were preparedfrom this solution to yield final concentrations of 9 \u03bcM DNA,100 \u03bcM KCl, and 100 \u03bcM S-MGB, 1% DMSO. DNA solutions containingno S-MGB included 1% DMSO and were used as controls.via a thin platinum wire . The following instrument parameters were used forthe DNA:MGB-BP-3 complex: capillary voltage 1.2 kV, sample cone voltage80 V, source offset 110 V, source temperature 40 \u00b0C, trap collisionenergy 3.0 V, trap gas 4 mL/min. Data were processed using MasslynxV4.2 and OriginPro 2021, and figures were produced using Chemdraw.Nativemass spectrometry (nMS) experiments were carried out on a Synapt G2-Siinstrument  with a nano-electrospray ionizationsource (nESI). Mass calibration was performed by a separate infusionof NaI cluster ions. Solutions were ionized from a thin-walled borosilicateglass capillary  pulled in-house to nESI tip with a Flaming/Brown micropipettepuller . A negative potential inthe range of 1.0\u20131.2 kV was applied to the solution 2O/D2O atpH = 7.4. A concentrated stock solution of MGB-BP-3 was prepared toallow the addition of 5 \u03bcL aliquots of solution up to a maximumtotal addition volume of 50 \u03bcL or until a titration end pointwas detected by virtue of no further changes to the NMR data.DNA samples for NMR spectroscopy were preparedas 1 mM duplex solutions solubilized in 550 \u03bcL of a 50 mM phosphatebuffer solution prepared with 9:1 H1H NMR data were acquired with excitation sculptingfor solvent suppression (Bruker pulse program zgesgp). The 600 MHzNMR data were acquired using a Bruker AVANCE-II+ NMR spectrometeroperating at 600.13 MHz for 1H resonance on a standardgeometry triple-resonance (TBI-z) probehead equipped for z-pulsedfield gradients with the probe temperature regulated and calibratedfor data acquisition at 298 K. The 800 MHz NMR data were acquiredusing a Bruker AVANCE NEO NMR spectrometer operating at 799.43 MHzfor 1H resonance on a standard geometry 5 mm TCI-z cryoprobeprobehead. Sample temperatures were varied within the range of 298\u2013274K and stabilized prior to data accumulations. In both instances, datawere acquired with 128 transients over a 1H frequency widthequivalent to 20.0276 ppm centered at 4.694 ppm into 32 K (600 MHz)or 64 K (800 MHz) data points using a relaxation delay of 2.0 s betweentransients. For the excitation sculpting routine, sinc soft pulses(bandwidth \u223c125 Hz) were used for selective inversion at thesolvent frequency together with smoothed square-shaped gradient pulses(1 ms duration) in a ratio of 31:11. 2D  NOESY NMR data were acquired at 800 MHz on the MGB-BP-3/DNA complexstabilized at 274 K (Bruker pulse program noesyesgpph) into 4 K datapoints for each of 1024 t1 increments over \u03c91 and \u03c92 frequency widths equivalent to 20.1756ppm using 64 transients per increment. A mixing time of 250 ms wasapplied to allow development of the nOe and chemical exchange responses.Equivalent reference data were acquired for the ligand-free DNA sampleat 600 MHz by way of creating a reference data set. All raw data wereprocessed within the TopSpin 4.0.5 environment. Fully processed 2DNMR data were imported into the NMRFAM-Sparky environment (version1.414 powered by Sparky 3.13525) for datainterpretation and reduction.1D These experiments were contracted outto Inspiralis Ltd. . In all experiments, the activityof the enzyme was determined prior to the testing of the compoundand 1 U defined as the amount of enzyme required to fully supercoil,decatenate, or relax 0.5 \u03bcg of the substrate in 30 min. Cleavageactivity was separately determined and 1 U defined as the amount ofenzyme that gave maximum cleavage without degradation of the substrate.S. aureus and S84L gyrase for E. coli wereused.MGB-BP-3 was tested over a range from 0.01 to 100 \u03bcM  or 0.05\u2013500 \u03bcM (cleavage)and added to the reaction before the addition of the enzyme. FinalDMSO concentration in the assays was 1% (v/v) for supercoiling, decatenation,and relaxation, and 5% for cleavage. All enzymes and DNA substratesused were obtained from Inspiralis Ltd. . For fluoroquinolone-resistantgyrase supercoiling assays, S83L gyrase for 2, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5% (w/v) glycerol,and 0.1 mg/mL BSA. Each reaction was stopped by the addition of 30\u03bcL of chloroform/isoamyl alcohol (24:1) and 20 \u03bcL of StopDye , 10 mM EDTA, 0.5 \u03bcg/mLbromophenol blue) before being loaded on a 1.0% TAE . The gels were run at 90 V for 2 h.DNA gyrase (1 U) was incubated with 0.5 \u03bcg of relaxed pBR322DNA in a 30 \u03bcL reaction at 37 \u00b0C for 30 min under the followingconditions: 35 mM Tris\u2013HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 6.5% (w/v) glycerol, and 0.1mg/mL BSA. The reaction was then incubated for a further 30 min with2% SDS and 0.5 \u03bcg/mL proteinase K. Each reaction was stoppedby the addition of 30 \u03bcL of chloroform/isoamyl alcohol (24:1)and 20 \u03bcL of Stop Dye , 10 mM EDTA, 0.5 \u03bcg/mL bromophenol blue) before being loadedon a 1.0% TAE  gel, runat 90 V for 2 h.DNA gyrase (1 U)was incubated with 0.5 \u03bcg of supercoiled pBR322DNA in a 30 \u03bcL reaction at 37 \u00b0C for 30 min under the followingconditions: 35 mM Tris\u00b7HCl (pH 7.5), 24 mM KCl, 4 mM MgClCrithidia fasciculata) in a 30 \u03bcL reaction at 37 \u00b0C for 30 min under the followingconditions: 50 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate,10 mM magnesium acetate, 10 mM dithiothreitol, 1 mM ATP, and 50 \u03bcg/mLBSA. Each reaction was stopped by the addition of 30 \u03bcL of chloroform/isoamylalcohol (24:1) and 30 \u03bcL of Stop Dye before being loaded ona 1.0% TAE gel, run at 90 V for 2 h.Topo IV (1 U) was incubated with 200 ng kDNA (Kinetoplastcatenated DNA purified from E. coli decatenation assayexcept that the substrate was supercoiled pBR322 DNA.As for Topo IV (1U) was incubated with 0.5 \u03bcg of supercoiled pBR322DNA in a 30 \u03bcL reaction at 37 \u00b0C for 30 min under the followingconditions: 40 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate,10 mM magnesium acetate, 10 mM dithiothreitol, and 50 ug/mL BSA. Thereaction was then incubated for a further 30 min with 2% SDS and 0.5\u03bcg/mL proteinase K. Each reaction was stopped by the additionof 30 \u03bcL of chloroform/isoamyl alcohol (24:1) and 30 \u03bcLof Stop Dye before being loaded on a 1.0% TAE gel, run at 90 V for2 h.E. coli gyrase supercoilingexcept that the assay conditions were 40 mM HEPES-KOH (pH 7.6), 10mM magnesium acetate, 10 mM DTT, 2 mM ATP, 500 mM potassium glutamate,and 0.05 mg/mL BSA. The gels were run at 80 V for 3 h.As for E. coli gyrase cleavage exceptthat the assay conditions were 40 mM HEPES-KOH (pH 7.6), 10 mM magnesiumacetate, 10 mM DTT, 500 mM potassium glutamate, and 0.05 mg/mL BSA.The gels were run at 80 V for 3 h.As for 2, 5 mMDTT, 1.5 mM ATP, 350 mM potassium glutamate, and 0.05 mg/mL BSA. Eachreaction was stopped by the addition of 30 \u03bcL of chloroform/isoamylalcohol (24:1) and 30 \u03bcL of Stop Dye before being loaded ona 1.0% TAE gel, run at 70 V for 2 h.Topo IV (1 U) was incubated with 200 ng of kDNA ina 30 \u03bcL reaction at 37 \u00b0C for 30 min under the followingconditions: 50 mM Tris\u00b7HCl (7.5), 5 mM MgClE. coli topo IV cleavageexcept that the assay conditions were 50 mM Tris\u00b7HCl (7.5), 5mM MgCl2, 5 mM DTT, 350 mM potassium glutamate, and 0.05mg/mL BSA.As for 50 data using the followingequation: Exponential Decay, Single, 2 Parameter f = a*\u202fexp(\u2212b*x)Bandswere visualizedby ethidium staining for 10 min, destained for 10 min in water, analyzedby gel documentation equipment  and quantitatedusing Syngene Gene Tools software. Raw gel data (fluorescent bandvolumes) were collected from Syngene, Gene Tools gel analysis softwarewas calculated as a % of the 100% control (fully supercoiled DNA band)and converted to % inhibition. The raw gel data were analyzed usingSigmaPlot Version 13 (2015). The global curve fit nonlinear regressiontool was used to calculate IC5 CFU/mLwere incubated with doubling dilutions of MGB-BP-3, prepared froma 10 mM DMSO stock, in a 96-well plate for 20 h at 37 \u00b0C in trypticsoy broth (S1) orMHB (Table S2). Optical density was read and the MICdefined as the lowest concentration of compound to inhibit 80% ofvisible growth.The minimum inhibitoryconcentration (MIC) was defined using a microbroth dilution method.Briefly, bacteria at a concentration of 1 \u00d7 104  and a stocksolution of 10 mM vancomycin was prepared in sterile water.The reaction was carried out asper the Minimum Inhibitory Concentration procedure except that theinoculum was supplemented with 1 \u03bcg/mL lipopolysaccharide (LPS) of compounds at 25 \u03bcg/mL kanamycin for 20 h at 37\u00b0C with shaking. MICs for each compound were as follows: MGB-BP-3,3.125 \u03bcM; ciprofloxacin, 2 \u03bcg/mL; mitomycin C, 64 mg/L;doxycycline, 4 mg/L. GFP fluorescence and optical density were measuredand the fluorescence was normalized to cell density. The fluorescenceof the treated samples was divided by the fluorescence of the untreatedsamples to calculate the fold-induction of the promoter.The 5 CFU/mL in MHB and incubating with 1 \u03bcM S-MGB-245, orthis and 50 \u03bcM PA\u03b2N for 1 h. For those experiments withsodium azide, 1 or 10 mM of this was incubated for 1 h before incubationwith S-MGB-245. Subsequently, a 10 \u03bcL aliquot was mounted ona glass slide and fitted with a cover slip.A 10 mM stockof S-MGB-245 in DMSO,10 mM stock of PA\u03b2N in DMSO, and 100 mM stock of sodium azidein MHB were also prepared. Samples for microscopy were prepared byobtaining log-phase bacteria at a concentration of 1 \u00d7 10Images were captured using a ZeissLSM 880 confocal laser scanning microscope and a 63\u00d7 objectivebefore processing with associated Zen Blue and Adobe Photoshop software.Detection and acquisition parameters were maintained across each speciessample set."}
{"text": "In the Lifestyle Interventions and Independence for Elders (LIFE) trial, a structured exercise intervention slowed kidney function decline in sedentary older adults. Biomarkers of kidney health could distinguish potential mechanisms for this beneficial effect.Randomized controlled trial.A total of 1,381 sedentary adults aged 70-89 years enrolled in the LIFE trial.Structured, 2-year, moderate-intensity exercise intervention versus health education.Physical activity was measured by step count. Primary outcomes were changes in 14 serum and urine biomarkers of kidney health collected at baseline, year 1, and year 2. We determined the effect of randomization on changes in kidney measures and then evaluated observational associations of achieved activity on each measure.Participants assigned to exercise walked on average 291 more steps per day than participants assigned to health education. The intervention was not significantly associated with changes in biomarkers of kidney health. In observational analyses, persons in the highest versus lowest quartile of activity  had significant improvement in urine albumin ,\u00a0\u22120.37 to\u00a0\u22120.06]), alpha-1-microglobulin (\u22120.18\u00a0mg/L [\u22120.28 to\u00a0\u22120.08]), trefoil factor-3 (\u22120.24\u00a0pg/mL [\u22120.35 to\u00a0\u22120.13]), epidermal growth factor (0.19\u00a0pg/mL [0.06-0.32]), uromodulin (0.06\u00a0pg/mL [0.00-0.12]), interleukin 18 (\u22120.09\u00a0pg/mL [\u22120.15 to\u00a0\u22120.03]), neutrophil gelatinase-associated lipocalin (\u22120.16\u00a0pg/mL [\u22120.24 to\u00a0\u22120.07]), monocyte chemoattractant protein-1 (\u22120.25\u00a0pg/mL [\u22120.36 to\u00a0\u22120.14]), clusterin (-0.16\u00a0pg/mL [\u22120.30 to\u00a0\u22120.02]), serum tumor necrosis factor receptor-1 (\u22120.25\u00a0mg/dL [\u22120.39 to\u00a0\u22120.11]) and tumor necrosis factor receptor-2 (\u22120.30\u00a0mg/dL [\u22120.44 to\u00a0\u22120.16]). In sensitivity analyses, incremental changes in activity were most impactful on urine interleukin 18 and serum tumor necrosis factor-1.The original study was not designed to assess the impact on kidney health. Non-white individuals and patients with advanced chronic kidney disease are underrepresented.Randomization to structured exercise did not improve kidney health at a group level. However, higher exercise was associated with concurrent improvements in biomarkers of glomerular injury, tubular function/repair, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis.In the Lifestyle Interventions For Elders (LIFE) study, randomization to an exercise and physical activity intervention improved the slope of estimated glomerular filtration rate over 2 years compared with health education among older adults. In this study, we sought to determine whether there were specific biomarkers of kidney health that were affected by the exercise and physical activity intervention to investigate potential mechanisms for this positive impact on kidney decline. We found that randomization to the intervention did not improve any of the 14 measures of kidney tubule health. However, in observational analyses, higher activity was independently associated with improvements in several domains, especially tubular injury and generalized inflammation. These results help to clarify the impact of physical activity on kidney health. Recent trials indicate that physical activity is beneficial in elderly patients with chronic kidney disease (CKD)Biomarkers of kidney health can be loosely classified as representing glomerular damage, glomerular function, tubular injury, tubular dysfunction, and tubulointerstitial repair/fibrosis.14CysC). Furthermore, those who achieved higher activity levels derived greater benefits to eGFRCysC preservation from the intervention. Crucially, the association of higher activity with slower eGFR decline was independent of changes in blood pressure or body weight, suggesting that physical activity can directly mitigate the decline in glomerular function in older adults.The Lifestyle Interventions and Independence for Elders (LIFE) trial was a multicenter trial designed to test the effect of a combined exercise and activity intervention on major mobility and disability in community-dwelling, sedentary older adults.The present study aimed to explore the following: 1) the effects of randomization to an exercise and activity intervention on domains of kidney health beyond glomerular function and 2) the associations of physical activity as measured by step counts with these domains of kidney health. To achieve these objectives, we used urine and serum specimens that were stored at the baseline, year 1 and year 2 visits of the LIFE trial to measure multiple biomarkers reflecting various domains of kidney health. We hypothesized that both randomization to the exercise intervention and higher step counts during the LIFE trial would be associated with relative improvements in biomarkers reflecting glomerular injury, tubular function and repair, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis.The LIFE trial was a phase 3 multicenter, randomized controlled trial of a moderate-intensity physical activity and exercise program compared with a \u201csuccessful aging\u201d health education program, involving 1,635 sedentary older adults enrolled between February 2010 and December 2013 across 8 centers in the United States.All participants in the LIFE study underwent informed consent before participation. This study was approved by the University of California, San Francisco Institutional Review Board (18-25182).Participants in the LIFE trial underwent block randomization stratified by field center and sex. The intervention arm underwent a combined-activity and functional exercise intervention. The activity component targeted 150 minutes of moderate-intensity exercise per week, primarily composed of walking, and the functional exercise component included strength, flexibility, and balance training. Participants were expected to attend exercise sessions at their field center twice weekly and to conduct home-based activities 3-4 times weekly throughout the trial duration. Exercise sessions were individualized, and participants were expected to progress toward a goal of 30 minutes of daily walking, 10 minutes of lower extremity strength training, 10 minutes of balance training, and large muscle flexibility exercises. Intensity started low and increased over the first weeks of the intervention. Participants were asked to walk at a self-perceived exertion of 13 (\u201csomewhat hard\u201d) on the original Borg scale and perform lower extremity strength exercises at a self-perceived exertion of 15-16 (\u201chard\u201d). The health education arm of the trial involved weekly workshops over the first 26 weeks followed by monthly workshops. These sessions addressed a variety of health topics relevant to older adults  but did not specifically address physical activity.,Baseline assessments conducted before randomization included self-reported socio-demographic information , medical history , and physical examination (body mass index and systolic blood pressure). Physical activity was assessed using accelerometers (ActiGraph wGT3X-BT) at baseline and at 6, 12, and 24 months of follow-up; on each occasion participants wore the accelerometer on their hip for 7 days. Only data collected at baseline and 12 and 24 months were used for these analyses because they were contiguous with the assessments of biomarkers of kidney health. Details of these measures were described elsewhere.During the trial, serum and urine specimens were collected at the baseline, year 1 and year 2 visits and immediately stored at\u00a0\u221280\u00a0\u00b0C. Urine alpha-1 microglobulin (A1M) and albumin were measured using the Siemens BN II Nephelometer. Urine creatinine level was measured with a commercially available colorimetric kit using the Jaffe reaction from R&D Systems. Urine biomarkers  were measured using multiplex assay kits from Meso Scale Discovery (MSD) according to manufacturer protocols. Serum markers  were also measured using a multiplex plate from Meso Scale Discovery (MSD). For each biomarker, all measures for an individual participant were conducted concurrently to avoid assay drift.t tests, Wilcoxon rank sum tests, and \u03c72 tests were used to compare normally distributed, nonnormally distributed, and discrete characteristics, respectively, between participants with and without accelerometry or biomarker measures. The associations of the intervention on the outcomes of standardized changes in biomarkers of kidney health were analyzed using the mixed effects models with an unstructured covariance matrix. Urine biomarkers were indexed to 1\u00a0g of urine creatinine. We standardized the changes in biomarkers to compare the intervention effects across different outcomes qualitatively. The interpretation of the intervention effect was based on the standardized change in biomarker (unit: SD) when comparing physical activity versus health education. Covariates included baseline biomarker, sex and field center (stratified variables), intervention, clinic visit (year 1 and year 2), and intervention-by-visit interaction. Hypothesis tests for intervention effects at the year 1 and year 2 assessment visits were performed using contrasts of the year 1 and year 2 intervention means. Overall comparisons between intervention groups across follow-up visits were obtained using a contrast to compare average effects across both follow-up visits.Baseline characteristics were summarized by randomization groups using means and standard deviations (SDs) for continuous variables or counts and percentages for discrete variables. Here, We next evaluated associations between measured time-updated step count during follow-up with standardized changes in biomarkers of kidney health. The associations between standardized time-updated step counts with standardized changes in biomarkers of kidney health function were determined using mixed effects models. The covariates in the basic model included baseline biomarker, sex and field center (stratified variables), intervention, clinic visit, and intervention-by-visit interaction. Because these analyses were not comparing randomized groups, we additionally adjusted for potential confounding variables, including age, body mass index, systolic blood pressure, race, education, diabetes, cardiovascular disease, and hypertension. All analyses of urine biomarkers were conducted using the biomarkers indexed for urine creatinine. Step count was also analyzed using quartiles for ease of comparing associations with standardized levels of the biomarkers; high versus low quartiles of activity were compared across the intervention period to ascertain the cumulative effect of activity on biomarkers of kidney health.In our prior study,We next attempted to distinguish associations of incremental changes from baseline in activity, as opposed to cumulative effect of activity over 2 years, with parallel changes in the biomarkers of kidney health across the entire cohort. In these analyses, the baseline step count and the changes in step count from baseline were separate covariates for the outcomes of longitudinal biomarkers of kidney health after adjustment for baseline biomarker, intervention, sex, clinical sites, age, body mass index, time-varying systolic blood pressure, race, education, diabetes, cardiovascular disease, hypertension, visit, and intervention-by-visit interaction. Both baseline step count and outcome measures  are standardized. Change in step count was calculated as standardized follow-up step count minus standardized baseline step count.P value of\u00a0<0.05 was considered to indicate statistical significance. We did not adjust for multiple comparisons because all of the outcomes are interrelated as manifestations of kidney tubule health; our global hypothesis was that physical activity would be associated with improvements in kidney damage and dysfunction. The statistical software used to conduct these analyses included SAS version 9.4 and R version 3.6.1.For all analyses, a 2-sided CysC at baseline was 54\u00a0\u00b1\u00a017\u00a0mL/min/1.73\u00a0m2   .Table\u00a02EIn contrast, when time-updated step count was evaluated comparing high versus low quartiles of activity  or as a Incremental changes in step count from baseline demonstrated significant associations with improvements in tubular injury (IL-18) and generalized inflammation (TNFR1). The coefficients of all remaining biomarkers except for serum KIM1 were directionally associated with improvements in kidney health. Effect sizes of incremental changes in step count were somewhat smaller than the associations with baseline step count .Table\u00a04AIn this analysis of 1,381 community-dwelling sedentary older adults in the LIFE trial, we found no effect of randomization to a structured physical activity and exercise intervention on biomarkers of kidney health, including measures of glomerular injury, tubular function and repair, tubular injury, generalized inflammation, and tubulointerstitial/fibrosis. However, in observational analyses, those participants who had higher time-updated step counts showed improvements in several domains of kidney health. Specifically, participants who walked more had trajectories reflective of less glomerular injury , better tubular function (lower urine A1M and TFF3), better tubular repair (higher urine EGF and UMOD), less tubular injury (urine IL-18 and NGAL), less generalized inflammation (TNFR1 and TNFR2), and less tubulointerstitial fibrosis/repair (urine MCP1 and clusterin). In sensitivity analyses that distinguished the incremental effect of increased activity from baseline on changes in biomarkers of kidney health, we observed that changes in activity appeared most impactful in the domains of tubular injury (IL-18) and generalized inflammation (TNFR1).CysC (as shown previously),,Our findings are somewhat contradictory as randomization to the physical activity intervention did not result in a significant change in any kidney biomarker except for eGFRThere are relatively few prior studies evaluating the effects of exercise on markers of systemic inflammation and/or glomerular health, and they have reported mixed results. Prior studies have differed from this LIFE ancillary study in several ways: smaller sample sizes, restriction to specific stages or etiologies of CKD, shorter exercise interventions , and a narrower profile of kidney health measures. For example, a study of home-based aerobic exercise and resistance training for participants with stage 4 CKD showed no improvement in urine albumin over 6 months, but did show improvement in urinary liver-type fatty acid-binding protein (a marker of tubular injury) and C-reactive protein .,These results help to elucidate the mechanisms by which activity and exercise might affect changes across multiple dimensions of kidney health. Importantly, in addition to a reduction in glomerular filtration rate, the aging kidney has declining proximalIt is important to note that although step counts are a measure of physical activity, this study was geared toward studying the effect of a combined-activity exercise intervention on changes in biomarkers of kidney health and not activity alone. However, the trial only included 2 measures of observed activity that could be used to evaluate a dose-response relationship between components of the intervention and changes in kidney health: step counts and time spent in moderate-intensity activity. In our prior study,There are several other limitations to be acknowledged. This is an ancillary study to an existing trial, and the original study was not designed to test the impact of physical activity and exercise on kidney health. Non-White individuals were underrepresented, and the study included few participants with advanced CKD. Furthermore, given LIFE\u2019s focus on older individuals, these findings may not apply to a younger population. Due to the timing of specimen collection, our analyses were limited to a 2-year follow-up; we do not know whether any beneficial effects of physical activity would be increased or decreased during longer follow-up periods. There are other possible factors that can influence the ability to maintain activity as well as kidney health that this study is not able to quantify, including socio-economic factors affecting access to nutrition, transportation, or caregiving. However, the findings are both thematically and directionally consistent with the randomized effect of physical activity on improvements in eGFR, and the markers assessed here are used to understand potential mechanisms. Nonetheless, the associations of higher step counts with changes in biomarkers of kidney health are observational and are subject to residual confounding effects despite our adjustment for multiple covariates.In summary, in a trial that employed a structured physical activity and exercise intervention for older adults, no differences in biomarkers of multiple domains of kidney health were observed between individuals randomized to structured physical activity intervention and those randomized to health education alone. However, those participants who had higher step counts had relatively improved biomarkers of kidney health, including glomerular injury, tubular function, tubular injury, generalized inflammation, and tubulointerstitial repair/fibrosis. Incremental changes from baseline in activity appeared most impactful on the domains of tubular injury and generalized inflammation. These results help to clarify the overall impact of physical activity on kidney decline. Future research should explore whether these same biomarkers of kidney health change over time as a natural course of aging and, if so, whether physical activity and exercise can slow the natural process of aging in the kidney. Additional studies are needed to evaluate which components of physical activity are most beneficial for kidney health, whether exercise intensity gives a graded benefit in kidney health, and whether persons with advanced kidney disease also benefit from physical activity."}
{"text": "There are limited longitudinal studies on the effects of the COVID-19 pandemic on mental health and well-being, including the effects of imposed restrictions and lockdowns.This study investigates how living in a pandemic, and related lockdowns and restrictions, affected the mental health of people living in Australia during the first year of the COVID-19 pandemic.A total of 875 people living in Australia participated in a longitudinal survey from 27 May to 14 December 2020. This time period includes dates that span pre-, during and post-wave 2 lockdowns in Australia, with strict and sustained public health measures. Linear mixed models were fitted to investigate the effect of lockdown on depression and anxiety symptoms.Symptoms of depression and anxiety improved over time, during and after lockdowns. More adverse mental health symptoms were observed for people with a history of medical or mental health problems, caring responsibilities, more neurotic personality traits or less conscientiousness, and for people who were younger. People who reported being more conscientious reported better mental health.Despite notoriously strict lockdowns, participants did not experience a deterioration of mental health over time. Results suggest a lack of significant adverse effects of lockdown restrictions on mental health and well-being. Findings highlight cohorts that could benefit from targeted mental health support and interventions, so that public policy can be better equipped to support them, particularly if future strict public health measures such as lockdowns are being considered or implemented for the COVID-19 pandemic and other disasters. These occurred in the context of fear and anxiety about how the COVID-19 virus might affect individuals, communities, healthcare services and society. Although public health restriction measures were necessary to \u2018stop the spread\u2019, particularly in the absence of vaccines in the early stages of the pandemic, there have been psychosocial (and economic) costs as a result.2The potential for the COVID-19 pandemic to affect mental health and well-being was recognised early on in the pandemic, as a result of concerns around contracting the virus itself as well as from the measures used to contain its spread.3 Studies have reported increased levels of psychological distress and symptoms of depression and anxiety compared with pre-pandemic levels.8 COVID-19 restrictions have also affected social connectedness, with reports of increased social isolation and loneliness associated with increased psychological distress.9 Other downstream effects of public health measures, including job losses and reduced financial resources, have also adversely affected mental health.11 Furthermore, mandatory contact tracing and quarantine have contributed to feelings of anxiety and guilt about the effects of infection, quarantine and stigma.12 COVID-19 infections and associated restrictions have also shown to contribute to, or exacerbate, existing chronic mental illness, including anxiety, depression, post-traumatic stress disorder and substance misuse.13It is crucial to understand how the virus itself and associated restrictions affected the short- and long-term mental health and well-being of people, and how these factors track over time. For these reasons, an international call to action was made for high-quality data on the mental health effects of the COVID-19 pandemic.8 There are limited longitudinal mental health and well-being studies about people living in Australia during the pandemic, and only one of these reported data collected before the second wave.15 This study reports results of one of the few surveys in Australia that capture data leading into and following the second wave, and the infamously strict restrictions imposed during that time.However, the majority of studies examining the mental health and well-being effects of the COVID-19 pandemic in Australia are based on cross-sectional data collected in the early days of the pandemic, when public health orders were not as strict and threat of community transmission was lower (i.e. the first wave). Those studies, which reported that mental health and well-being was substantively lower than pre-pandemic normative estimates, may have captured the immediate, acute shock of a global pandemic following a severe bushfire season.16 and wave 3 from August 2021 until October 2021 (from the Delta variant of COVID-19).17 In 2020, there were 899 deaths from COVID-19 registered in Australia, and 89% of deaths were in Victoria (n\u00a0=\u00a0800).16In Australia, widespread restrictions of movement, distancing measures and physical isolation, or \u2018lockdowns\u2019, were implemented from March 2020 as a response to Australia's first locally transmitted case of COVID-19. Up until October 2021, Australia had three major \u2018COVID-19 waves\u2019 or distinct peaks of infections that led to widespread lockdowns. The general dates are: wave 1 in March and April 2020 , wave 2 from end of June to October 2020 (mainly affecting the state of Victoria with most infections acquired via community transmission)18 The government's strict lockdown strategy was effective in eliminating community transmissions,19 and restrictions were eased in Victoria from 26 October 2020, when zero community transmissions were recorded in all Australian states. There was an extended period in Australia with no community transmissions until the third wave began. When the last lockdown in Victoria ended on 21 October 2021, the people of Melbourne (a city of 5 million people) had spent a cumulative 262 days under strict lockdown conditions since March 2020, one of the most stringent in the world, leading to the nickname \u2018world's most locked down city\u2019.20 These lockdowns were characterised by long periods of border closures, imposed curfew, childcare and school shutdowns, closure of retail and hospitality, restrictions on movement (\u22645\u00a0km from home) and restrictions of visitors to homes, with only four essential reasons to leave home . Getting a COVID-19 vaccination was only added as a fifth reason to leave home in April 2021, when the first vaccines were available.Most Australians experienced stay-at-home orders during wave 1 of COVID-19 infections. These first wave restrictions were easing in May 2020 (when our survey began collecting data), with single-digit daily case numbers in Victoria at the start of June 2020. However, case numbers were soon to rise as a result of hotel quarantine breaches, resulting in the second wave. The state of Victoria subsequently experienced a large and prolonged second wave of COVID-19 infections, which resulted in approximately 19\u00a0000 recorded cases and 800 deaths, linked primarily to 222 outbreaks in residential aged care homes.22 Our analysis of baseline cross-sectional data from this survey,23 collected from 803 participants during the peak of the second wave (from 27 May to 19 August 2020), revealed that self-rated knowledge about COVID-19 was high, adherence to government recommendations to prevent the spread of COVID-19 was high and trust in the Australian government's handling of the COVID-19 pandemic was high, as were participants\u2019 confidence in the Australian healthcare system during the COVID-19 pandemic. Participants also strongly believed that access to essentials  would be maintained.We conducted a longitudinal survey investigating the impact of the COVID-19 pandemic on the mental health and well-being of people living in Australia during the first year of the pandemic, from 27 May 2020 to 14 December 2020. The survey was modelled on the UCL COVID-19 social study, and had the broad aim of exploring the emotional, mental, health and societal behaviours and experiences of people during the COVID-19 pandemic.In the current study, the primary aim was to investigate the impact of the pandemic and major lockdowns on the mental health and well-being of people residing in Australia across three periods: pre-major lockdown, during major lockdown and post-major lockdown in 2020. As Victoria experienced a prolonged lockdown period in 2020, secondary analyses assessed if changes in depression or anxiety differed by participant location , or according to baseline participant characteristics. We hypothesised that greater depression and anxiety symptoms would be observed during the major lockdown period compared with pre- and post-major lockdown. We also hypothesised that people living in areas with longer and more restrictive lockdowns (i.e. Victorians) would experience poorer mental health.Participant recruitment was conducted by the National Ageing Research Institute, using a convenience sampling approach through a variety of channels, including media, social media, targeted advertising, and personal and professional networks. Participation was open to any person living in Australia during the survey, aged 18 years or above. Because the pandemic had population-wide effects that were occurring in real time, and because of the time-sensitive nature of this research, a convenience sampling approach was used to enable quick collection of data at a crucial time during the pandemic, and to generate a large sample in a short period of time.https://projectredcap.org/), or via hard-copy paper versions, which could be posted to participants with a postage-paid reply envelope. New participants could join the study and complete the baseline assessment at any point during that period. The survey collected sociodemographic information, a basic health profile, occupational and social roles (e.g. key worker or carer status), and information about living situation. The baseline questionnaire required approximately 20\u00a0min to complete and weekly follow-up questionnaires took approximately 15\u00a0min, where participants shared ongoing information about their mental health, loneliness, stressors, adherence to restrictions, confidence in government management, and exercise and social behaviours. Study participants were also invited to participate in a single qualitative interview, and findings will be reported separately.Data collection began on 27 May 2020 and continued until 14 December 2020 via an online platform .https://doi.org/10.1192/bjo.2023.65). Of those with complete surveys (n\u00a0=\u00a0920), 45 individuals had missing or invalid postcode information (see Supplementary Table 2). Excluding individuals with incomplete surveys and incomplete/invalid postcodes resulted in a sample size of 875 participants eligible for analyses. See A total of 1223 individuals began the online survey, with 1215 (99.3%) providing written consent to be included in the study. Among those that provided consent, 295 (24.3%) had incomplete surveys; these individuals did not provide data on the outcome measures or for several key covariates of interest required for the analyses 25 tool. These tools align closely with diagnostic criteria for major depressive disorder and generalised anxiety disorder, respectively.24 In both tools, items ask how often symptoms were bothering respondents in the past week. PHQ-9 scores range from 0 to 27, with scores of 5, 10, 15 and 20 representing cut-off points for mild, moderate, moderately severe and severe depression, respectively. GAD-7 scores range from 0 to 21, and scores of 5, 10 and 15 represent cut-off points for mild, moderate and severe anxiety, respectively.Self-reported mental health was assessed at all time points, using two outcomes: depressive symptoms measured with the nine-item Patient Health Questionnaire (PHQ-9)26 was completed by participants once, at baseline. This measures five key dimensions of personality: openness, conscientiousness, extroversion, agreeableness and neuroticism. Scores for each of the five personality dimensions were determined from the mean score across the three items associated with each dimension. Higher scores for each personality trait represent greater strength for that trait.The Short 15-item Big Five Inventory (BFI-S)Victoria was the only Australian state/territory that entered a prolonged major lockdown in 2020. Metropolitan Victoria (i.e. Melbourne) entered major lockdown at a different time to the rest of Victoria. Participant responses were classified as pre-, during and post-major lockdown, according to both the date the survey was completed and the participant postcode of residence.The pre-major lockdown period was defined as between 27 May (study initiated) and 1\u20139 July 2020 (for Melbourne), and between 27 May (study initiated) and 2 August 2020 . The lockdown period was defined as between 1\u20139 July and 27 October 2020 (for Melbourne), and between 3 August and 27 October 2020 . The post-major lockdown period was defined as between 28 October and 14 December 2020 (study end).There was variation in the metropolitan Victoria lockdown as some hot-spot postcodes were placed in lockdown before others (further details are in Supplementary Table 3). Participants living outside of Victoria were considered as a comparator group. For the comparator group, the pre-major lockdown period was considered to be the period before the whole of Victoria being in major lockdown (i.e. 27 May to 2 August 2020).29 Country of birth was included as an exploratory variable to investigate whether separation from family owing to COVID-19-related travel restrictions affected mental health. Area of residence was included as lockdowns varied according to location.Baseline covariates considered to be potentially associated with mental health outcomes included age (years); gender identification ; country of birth ; caring responsibilities for older relatives or friends, or for people with long-term conditions or disabilities ; history of clinically diagnosed mental health conditions ; currently engaged in paid employment, including casual, part-time, full-time or self-employment ; and area of residence . These covariates were selected based on well-known factors considered to be potentially associated with mental health outcomes in the literature.30 This index ranks areas in Australia according to relative socioeconomic advantage and disadvantage based on information from the 5-yearly Census of Population and Housing. SEIFA quintiles were considered ranging from 1 (most disadvantaged) to 5 (least disadvantaged).Area-level socioeconomic status was derived by linking participant postcodes to the 2016 Australian Bureau of Statistics Socioeconomic Index For Areas (SEIFA) Index of Relative Socio-economic Advantage and Disadvantage (IRSAD).Secondary analysis within the Victorian subsample examined which of the following time-fixed risk factors and personality traits were associated with the change in mental health over time across the three time periods: pre-major lockdown, during major lockdown and post-major lockdown in Victoria: (a) increased risk because of older age ; (b) caring responsibilities (yes/no); (c) history of mental health conditions (yes/no); (d) medical history/comorbidities ; (e) country of birth ; (f) essential/key worker ; (g) having one or more children in the household (yes/no); and (h) personality traits as measured by the BIF-S, i.e. extroversion, agreeableness, openness, conscientiousness and neuroticism.31 Participants were defined as having a medical history of comorbidities if they reported having high blood pressure, diabetes, heart disease, lung disease, cancer or other chronic physical health condition or disability.Participants were considered at increased risk of serious COVID-19 outcomes if they were aged 65 years and above with a medical condition or aged above 70 years, as per the Department of Health's definition.n\u00a0=\u00a0875) were calculated along with missing data proportions , an interaction between area of residence and lockdown period was included in adjusted LMMs. In secondary analyses, an interaction between each potential time-fixed baseline risk factor and lockdown period was included in adjusted LMMs, to assess if these factors were associated with differences in depression or anxiety symptoms over time for Victorian participants. The eight factors listed above were considered in separate LMMs in this exploratory analysis.n\u00a0=\u00a053), as well as any participants who had missing outcome values at all time points for which they provided data . This resulted in a complete-case sample size of 767 (87.7% of the 875 eligible participants) for the PHQ-9 and 764 (87.4%) for the GAD-7. Descriptive characteristics of the full sample, complete-case sample and omitted participants were considered to determine if characteristics were comparable between those who were and were not included in the analysis (see Supplementary Tables 4 and 5). Descriptive characteristics for the complete-case sample and omitted sample of participants were compared to determine if there were any differences in the characteristics of included and excluded participants (Supplementary Table 4). The two samples were generally comparable, although there was a slightly higher percentage of participants who were female (81% v. 74%), employed (56% v. 48%) or had no caring responsibilities (79% v. 74%) in the complete-case sample compared with the omitted sample of participants.A complete-case analysis was conducted, omitting participants with missing data for any of the covariates (n\u00a0=\u00a0767) and GAD-7 (n\u00a0=\u00a0764), 668 (approximately 87%) provided longitudinal data. Each participant on average provided data for 9.7 (s.d.\u00a0=\u00a07.4) and 9.8 (s.d.\u00a0=\u00a07.1) weeks (maximum 25) for the PHQ-9 and GAD-7, respectively.Characteristics of the eligible study sample at baseline are shown in Mean pre-lockdown PHQ-9 score was higher than both the mean scores during and post-lockdown, indicating more depressive symptoms before lockdown  and 3. TThe mean pre-lockdown GAD-7 score was higher than both the mean score during and post-lockdown, indicating more anxiety symptoms before lockdown  and 4. TExamination of the changes in mental health over time by participant location in Australia  and 3 shn\u00a0=\u00a0669; 87%) were living in Victoria and experienced strict lockdown measures in 2020. Descriptive statistics are shown in A large proportion of respondents from the sample , compared with those who had no such history . PHQ-9 sHowever, as in the primary analysis, all groups show that depressive symptoms on average decreased slightly over time . There wSimilar to that found for depressive symptoms, GAD-7 scores on average were higher for (a) younger people  across all time periods compared with those who were older , (b) forAs with depression symptoms, there was no interaction effect for any of the potential moderators considered. For all groups, on average, the level of anxiety decreased, indicating that anxiety symptoms improved modestly in this sample over time . For allThe effect of lockdown on symptoms of depression and anxiety were largely unmodified by personality traits  did not experience poorer mental health as a result of the lockdown. Regardless of where participants lived , results indicated symptoms of depression and anxiety improved over time and during and post-lockdowns. However, the change in PHQ-9 and GAD-7 scores was consistently small, with considerable overlap in the confidence intervals of the mean estimates in all analyses, and likely do not reflect clinically meaningful changes. Finally, we found that the effect of lockdowns on symptoms of depression and anxiety were largely unmodified by personality traits. Irrespective of time period, there was a trend for people who scored highly on neuroticism to report more depressive and anxiety symptoms, and conversely, those who self-reported high conscientiousness reported lower levels of these symptoms.27). Additionally, people with caring responsibilities showed more anxious symptoms compared with those with no caring responsibilities. These results are consistent with other studies; despite greater risks and potential difficulties faced by older people during the pandemic and associated restrictions, this age group has been found to report lower rates of psychological distress and poor mental health and lower scores on mood symptoms scales across multiple COVID-19 studies.32 However, findings from the secondary analyses may reflect characteristics of these particular groups, independent of a pandemic. For example, research conducted before the pandemic indicated that depressive or anxiety symptoms tended to decrease with increasing age.33 Additionally, the negative effects of caring on psychological health have been well established, including on anxiety, depression and burden.29 and an Australian study found that carers with unmet support needs faced a two-fold increase in the odds of psychological distress relative to those with no unmet needs.34Secondary analysis of Victorian participants  indicated that mental health generally improved across all groups, with no change in trajectory based on any of the potential modifying factors. People with a history of medical conditions or clinically diagnosed mental health problems, or younger participants , displayed greater depressive and anxious symptoms on average 36 than the pre-lockdown mean reported for this study , which suggests a general increase in depressive symptoms triggered by the pandemic. However, consistent with our results of improved mental health scores across the three time periods, the UCL COVID-19 social study also found improving mental health over time,22 and findings from China indicated that rates of psychological distress declined in the weeks following the initial outbreak.37 Other findings from Australia also showed that people who were still working early in the pandemic showed improved mental health over time.11 This may reflect some adjustment to a \u2018new normal\u2019 for individuals whose lives and livelihoods were not severely affected by the pandemic, or a gradual return to the pre-pandemic baseline after the initial outbreak.Our analysis is one of the few surveys in Australia that capture data leading into and following the second wave and the infamously strict restrictions imposed during that time. There are limited studies published to date with longitudinal data for mental health and well-being on people living in Australia during the pandemic, only one of which reported data collected before the second wave.38 Another longitudinal survey, conducted between April and September 2020, found increased depressive symptoms and poorer self-reported coping, hopefulness and quality of life during the second wave of the pandemic, compared with the first wave.15 Hopefulness, however, rebounded in September 2020 across their sample, likely because of to the impending easing of government-imposed restrictions. Anxiety, stress and resilience, on the other hand, remained relatively stable. Other research has consistently shown that mental health and psychological well-being have been adversely affected during the pandemic by job loss or reduced work, financial stress, pre-existing mental health disorders, feeling that government restrictions were negatively affecting their daily lives or that they would continue for the long term, loss of social connectedness, or worry about contracting COVID-19.27 The analyses presented in this study controlled for as many of those factors as the available data would allow, to examine the effect of pandemic-associated restrictions on mental health. Taken together, the present results and other findings in the literature suggest that individual experiences of the pandemic and resultant effects on mental health are driven by the ways in which public health measures and restrictions have directly affected the lives of people rather than being in lockdown per se. It is, therefore, necessary to examine individual factors that may influence the way in which lockdown may affect participants\u2019 mental health.In contrast, two cross-sectional surveys administered in April 2020 and July to August 2020 found substantially greater prevalence of clinically depressive and generalised anxiety symptoms in Victoria compared with other states and territories during the second wave.39 which would have affected mental health. Caution should therefore be taken in generalising our results to the wider population. Participants could also both join and leave the study at different time points, meaning that participants that contributed data during the pre-lockdown period may differ to participants that contributed data during the lockdown or post-lockdown periods. This means that differences observed could be attributable to differences between participants rather than within-individual improvements in mental health. Thus, improvements in mental health over time may not be a result of within-individual changes in mental health but could be explained by the number of participants who have provided outcome data at the different lockdown stages. However, our sensitivity analyses of participants that provided data across all time periods provided consistent findings (see Supplementary Table 10).Although significant in our model, the point estimates or the limits of the confidence intervals did not reflect clinically meaningful differences for either depression or anxiety symptoms . Although the PHQ-9 and GAD-7 are well-validated scales for the purpose of assessing depression or anxiety symptoms, it is important to note that these measures were collected via self-report and are not diagnostic data, and as brief self-report instruments, are not as valid as more detailed measures. There are also some self-selection biases to consider. Convenience sampling likely resulted in sampling bias. Most participants were women (80%), very few opted for the option of hard-copy surveys and the survey was predominately conducted online, which may have skewed the sample of participants toward people who were more able and willing to access the necessary technology. Additionally, nearly 50% of the sample were in the most advantageous socioeconomic quintile and were in current employment, which skewed our sample as those in more socioeconomically disadvantaged and diverse circumstance experienced disproportionately higher mortality and morbidity associated with the virus,In summary, this study reports a lack of deterioration in mental health over time in people living in Australia during the first year of the COVID-19 pandemic, even throughout notoriously strict lockdowns and restrictions. These results are meaningful as there are limited longitudinal survey studies on the well-being effects of COVID-19, especially those that consider the ways in which public health measures and restrictions directly affect the mental health of people. Our findings reveal the cohorts that appear to be at greater risk of poor mental health and should be targeted for mental health supports and interventions. These were people with a history of clinically diagnosed mental health problems, younger participants , people with a history of medical conditions, people with caring responsibilities and people with more neurotic personality traits. For example, given that high conscientiousness was associated with better mental health in our study, targeted mental health supports could focus on improving conscientiousness in at-risk groups. This also suggests that public health messaging highlighting social responsibility may resonate with, and thus support the mental health of, highly conscientious people during a pandemic; however, further research is required to investigate this. Other future research should examine the short- and long-term effects of job loss, financial stress and social isolation on mental health, to better understand differential effects of lockdown consequences on mental health trajectories across the pandemic.These results provide insight into how people are navigating the pandemic and tracking mental health across time. This may help researchers, clinicians and policy makers to better understand the effects of COVID-19 and restriction measures on individuals, identify which groups are most at risk, and inform the advice that people are given about how to maintain mental well-being and mental health during a pandemic. As the pandemic and its extended effects continue, investigations such as this study into the impact of the COVID-19 pandemic on mental health are vital for informing service delivery and supports and providing more evidence, as future strict public health measures such as lockdowns are considered for pandemics or other public health reasons."}
{"text": "Mental health problems are a considerable public health issue and spending time in nature has been promoted as a way to access a range of psychological benefits leading to the development of nature\u2010based interventions for people with severe and enduring mental health problems. Less, however, is understood about the potential benefits and efficacy of day\u2010to\u2010day routine access to outdoor green and blue spaces for mental health service users.N\u2009=\u200911) using qualitative interviews and an online general population survey (N\u2009=\u20091791). Qualitative evidence highlighted the restorative benefits of nature and identified a number of barriers associated with fears around personal safety, social anxiety, fatigue and lack of motivation. COVID\u201019 had also restricted access to green and blue spaces. Having social contact and support encouraged people to spend time outdoors. In the quantitative survey, self\u2010report and standardised measures  were used to assess past and current mental wellbeing.Using a mixed\u2010methods design between April and October 2021, we explored the benefits and barriers to spending time outdoors with a purposive sample of mental health service users  and they were involved in the development of the research idea, funding application, design, data collection, analysis, writing up and dissemination activities. Natural environments provide interest and offer opportunities to escape from daily hassles or worriesBeing outdoors creates opportunities to establish social networks and increase social capital that can contribute to wellbeing,Although the evidence base is limited,perceptions of one's healthEven ,p.376 can lead people to reflect on their life, and \u2018one's priorities and possibilities, on one's actions and one's goals\u2019,p.197. Spending time in nature can compound feelings of isolation or be a reminder of how disconnected everyday life can be from the physical world.While the many benefits of green and blue spaces have been explored, it is also possible for individuals to experience negative associations with green and blue spaces. Nature can evoke overwhelming, existential anxieties: climate change, the ruthlessness of survival within nature and the \u2018perspective\u2010making power of nature,N\u2009=\u200911) and a general population survey accessed online (N\u2009=\u20091791) (April\u2013October 2021).We know less about the benefits and effectiveness of routine access to green and blue spaces for mental health users. To explore these issues further, we used a mixed\u2010methods design to identify potential barriers to spending time outdoors with a purposive sample of mental health service users  was no longer possible due to working\u2010from\u2010home restrictions to ameliorate this, we distributed paper questionnaires within Praxis Care's supported living settings.2.2Participants for the qualitative interviews were in receipt of a range of different services  and were invited to participate by the Head of Research in Praxis Care based in Northern Ireland.Participants for the quantitative online survey were recruited through a number of different sources in the United Kingdom and the United States including a targeted sample of people interested in mental health issues accessed through Praxis Care's internal communication network and via the Mental Health Foundation and Praxis Care social media platforms (Twitter and Facebook), and a general population sample using Amazon MTurk's survey tool.2.3A semistructured interview framework was developed to explore a range of themes with a purposive sample of mental health service users recruited through Praxis Care services. The qualitative interviews explored: exposure, frequency and dose of accessing green and blue spaces\u2014time of day, social connections; facilitators and barriers to accessing green and blue spaces; benefits; negative effects and past experiences and future hopes.data familiarisation and writing familiarisation notes; (2) systematic data coding; (3) generating initial themes from coded and collated data; (4) developing and reviewing themes; (5) refining, defining and naming themes; (6) writing the report.Telephone interviews were conducted with 11 participants between February and April 2021. Three members of the research team, including two experts by experience, carried out one\u2010to\u2010one interviews lasting approximately 45\u2009min each. Qualitative interviews were digitally recorded and transcribed for analysis by P. W. Thematic analysis2.4Findings from the qualitative interviews and the research literature were used to develop a quantitative online survey using the Qualtrics\u2122 platform. The short online survey was advertised online and included the following measures.2.5Demographic information: year of birth; gender identity; sexual identity; disability status; relationship status; dependents and carer status; employment status; household financial coping; ethnicity; current or previous mental health diagnosis; current medication; city/country location.2.6Experience of mental health problems was assessed using a self\u2010report measure, endorsement of any of the following statements was coded as a dichotomous variable \u2018Yes\u2019  or \u2018No\u2019: \u2018I have no mental health problems\u2019.2.7\u03b1\u2009=\u2009.82\u2013.85).The PHQ\u20108 is a well\u2010validated self\u2010report measure for assessing depressive symptom severity2.8\u03b1\u2009=\u2009.89\u2013.93).The short version of the Warwick\u2013Edinburgh Mental Wellbeing Scale (SWEMWBS)2.9Participants were asked \u2018over the past month, on average, how often have you spent time outdoors?\u2019 and could choose one selection from seven items: \u2018more than once a day\u2019, \u2018once a day\u2019, \u2018several times a week\u2019, \u2018once a week\u2019, \u2018once every 2 weeks\u2019, \u2018once\u2019, \u2018none\u2019. This applied to any free or leisure time not spent working, job hunting, studying or doing household chores. They were then asked \u2018Is there a time of day you prefer?\u2019 A range of options was available from early morning to night time, participants could endorse multiple items.2.10Questions about facilitators and barriers were generated from the research literature. Participants were asked to indicate \u2018what encourages you to spend time outdoors? \u2018the weather\u2019; \u2018my mood\u2019; \u2018my friends/family\u2019; \u2018the right clothing/shoes\u2019; \u2018conservation/environmental reasons\u2019; \u2018to get a break from other people\u2019; \u2018my pets\u2019 and a free text option of \u2018other\u2019. Respondents were asked \u2018Not everyone enjoys spending time outdoors, are there times when you don't like going outside?\u2019 and could endorse as many items that applied to them from: \u2018no free time/other commitments\u2019; \u2018feeling unsafe\u2019; \u2018it's difficult to get to/access\u2019; \u2018the weather\u2019; \u2018how I look/feel about myself\u2019 and \u2018makes me feel anxious\u2019.33.13.1.1M\u2009=\u200953, SD\u2009=\u200912.61). The use of Praxis Care services was a proxy measure of an existing mental health problem that could range from mild\u2010moderate to severe and enduring.Eleven service users (six women and five men) participated in the telephone semistructured interviews; each interview lasted around 45\u2009min. They ranged in age from 34 to 84 years old . For others who viewed leaving the house for everyday tasks as a source of anxiety, \u2018I do like to go out but it's just that I get butterflies\u2026you panic a bit and then you want to go back to the house\u2026\u2019 .3.1.3Interviewees made a clear association between spending time outdoors and mental wellbeing. The word \u2018lift\u2019 was used to describe the effects of spending time in green and blue spaces, \u2018lift your spirits\u2019 , \u2018lifts your mind\u2019  and \u2018the psychological benefits are it keeps my mood lifted\u2019 . Nature provided a contrast to the built environment, where \u2018concrete almost wears you out\u2026nature\u2026 it's uplifting you\u2019 .Enjoying wildlife, specifically birds and birdsong, was mentioned by half of those interviewed, and functioned as a way of connecting with nature.3.1.4Nature was described as calming, soothing, and peaceful and expressed as pure and cleansing, \u2018I think it clears you and you are sharper, yes, that's the purification thing, it just gives; it's so inspiring\u2019 . Negative elements of the outdoors were also referred to as a source of anxiety including litter, dog fouling, rats and chewing gum on the pavements.3.1.5Fear and safety issues were raised in a number of different contexts. Practical safety issues were a concern for some. Personal safety, traffic, lack of footpaths or fear of falling and injury was also a concern, \u2018I get anxious over my physical issues because I'm scared of falling\u2019 . Antisocial behaviour, such as alcohol and drug use in public spaces, was also considered a barrier.Fear relating to social anxiety and experiencing panic attacks was expressed by a number of respondents trying to manage their negative feelings.3.1.6The opportunity that public spaces provide to promote social cohesion also resonated with half of the respondents talking about the social aspect of spending time outdoors, whether this was \u2018talking and walking\u2019  or having the opportunity to \u2018get to meet people\u2019  and \u2018say hello, there is a kind of little community, social thing\u2019 .3.1.7Many of those interviewed were vulnerable and became increasingly isolated during the lockdown. Some people relied on others to spend time outdoors, whether this was a family member or friend or the support services that Praxis Care offered. When these services were withdrawn, this impacted considerably their capacity to leave their home. \u2018Now, we can't meet people. People are important to other people. They help each other\u2019 .3.1.8A number of barriers to spending time in green and blue spaces were discussed. Lack of transport was an issue, especially for those living in rural areas. Physical health and tiredness could be a limiting factor, but problems associated with social isolation impacted being outdoors, \u2018It's just not a place I would go to on my own, you know, I wouldn't ever think of going to a park\u2019 because you would need company to go there . Mental health difficulties also could restrict attempts to socialise or leave the home, \u2018I went and nobody talked to me and then I got really down so that's why I don't go out much\u2019 .3.1.9All of the interviewers were connected to Praxis Care, either as service users or employees, and reference was made to the support the organisation offered people, from providing social contact over the telephone, \u2018you know when you get the phone call, it makes you feel better, makes you think that someone is thinking about you \u201ccause you get lonely on your own\u201d\u2019  to calling to the house and encouraging them to get some exercise and fresh air, \u2018Praxis Care, the wee girls came with me for a walk and it makes me feel good\u2026we go for a walk round the park and we talk, and then come back to the house, it makes me feel out of breath\u2019 . Other factors that contributed to spending time outdoors were associated with promoting mental wellbeing, undertaking functional household\u2010related activities  and the opportunity for social contact.3.23.2.1N\u2009=\u20091791 people participated in the online survey. The demographics of the sample are summarised in Table\u00a0Between September and November 2021, a total of 3.2.2\u03c72\u2009=\u200923.57, p\u2009=\u2009.001) or current wellbeing using the PHQ\u20108 cutoff score of 10 or more \u2009=\u200961.25, p\u2009<\u2009.000). Respondents scoring \u2018high\u2019 on the SWEMWBS scale were also more likely than those rated \u2018low\u2019 or \u2018medium\u2019 to spend time outdoors at least once or more than once a day.The majority of participants had access to green (90.8%) and blue spaces (69.6%) Table\u00a0. People Asked to consider their activity over the past month, respondents endorsed the different reasons why they had spent time outdoors Table\u00a0. Again, \u03c72\u2009=\u20095.36, p\u2009=\u2009.021) or night time \u03c72\u2009=\u200914.06, p\u2009<\u2009.000). Night\u2010time preference was also observed in the PHQ\u20108\u2009>\u200910 samples \u2009=\u20094.18, p\u2009=\u2009.041). A significant relationship was observed between respondents scoring \u2018high\u2019 levels of wellbeing on the SWEMWBS and spending time outdoors in the early morning \u2009=\u200918.98, p\u2009<\u2009.000).Participants were also asked if there was a particular time of day that they preferred to go outdoors, multiple items could be endorsed Table\u00a0. Again s3.2.3\u03c72\u2009=\u20095.85, p\u2009=\u2009.016) \u2009=\u200918.91, p\u2009<\u2009.000). N\u2009=\u2009125 respondents provided an answer to the \u2018other\u2019 open\u2010ended question and exercise (n\u2009=\u200933) was the most popular reason for spending time outdoors, spending time in nature (n\u2009=\u200916) and gardening (n\u2009=\u200911).What encourages you to spend time outdoors?: Facilitators for those with experience of any mental health problems included \u2018my mood\u2019, \u2018the right clothing/shoes\u2019 and \u2018to get a break from other people\u2019. For those without self\u2010reported mental health problems were more likely to be encouraged by the weather to go outdoors than those with experience of problems , and insects/bugs (n\u2009=\u200910).Not everyone enjoys spending time outdoors, are there times when you don't like going outside?: Participants with experience of past or current problems were statistically significantly more likely to identify barriers such as \u2018feeling unsafe\u2019, \u2018how I look/feel about myself\u2019 and \u2018makes me feel anxious\u2019 than those without mental health problems Table\u00a0. They we4Spending time outdoors, in green and/or blue spaces was recognised as contributing to people's wellbeing, and offered mental health service users opportunities to feel restored, enjoy nature, be active and have social contact. It was mostly structured around errands or specific tasks and was considered to be an important part of a regular routine to help tackle negative aspects of their day. Spending time outdoors had the potential to elevate mood, \u2018lift your spirits, lifts your mind\u2019, helped to reduce unhelpful behaviours\u2014whether these related to challenging feelings of fear, social anxiety or avoiding unhealthy eating habits. A clear link between exercise and wellbeing was understood and the concept that nature was restorative, soothing and clean and required protection and preservation was depicted by those interviewed. However, a range of practical and psychological barriers prevented access was evident. Lack of transport, physical health problems and feeling tired restricted some people's activities but having some social support  to encourage them to leave the house was a valuable resource. Reflecting on the quantitative results, people without experience of mental health problems were more likely to have a higher frequency and dose of green and blue spaces compared to those with mental health problems. Unpicking why these differences were found is complicated however some light may be shed on the variation in motivations to spend time outdoors between the two groups. There were clear differences in how and why people accessed green and blue spaces, with the general sample more likely to endorse positively framed reasons such as getting exercise, enjoy the weather or gardening. Those without mental health problems endorsed items that focused on self\u2010improvement such as increasing physical activity and physical health, enjoying nature and the fresh air and investing time in themselves. Green and blue spaces were considered a way to improve both physical and mental wellbeing. Those with experience of mental health problems portrayed a more negative personal perspective of the barriers they faced. These included issues around self\u2010image, managing anxiety, feeling guilt or dealing with stress. Relying on external motivators was more likely to be endorsed such as walking a pet and having someone to encourage them to get outdoors. There were no differences between the two groups in a number of aspects including taking part in planned outdoor activities such as walking groups and outdoor gyms. Spending time outdoors to improve concentration, and motivation or to work through a problem was also similar. Meeting up with friends and family was equally important to both groups.Going out at night was preferred by those with mental health problems by either measure. Further research could help explore the reasons why this might be the case and hypotheses could include the role of social anxiety, personal safety and disrupted circadian rhythms. Feeling safe at night is a particular issue for many women, creating safer spaces and greater awareness of the vulnerabilities women experience on a regular basis, better lighting and more effective outdoor security measures may help increase feelings of safety for everyone.Like the participants in our study, the general public value the importance and benefits of green and blue spaces for wellbeing.Messaging about the benefits of green and blue spaces is equally important for people with mental health problems and must tackle the anxieties and concerns people raised, people should feel safe, secure and supported to access outdoors and be aware of the mental health benefits that may be available. Although our data did not highlight demographic differences in people's experiences of feeling unsafe, it is important to acknowledge, explore and consider that people may feel unsafe for a wide range of intersecting reasons, for example, in relation to gender, age, sexual orientation, physical appearance and being from visible minorities. Improving access to green and blue spaces has the potential to help address these inequities and the wider social determinants of health, but these complexities need to be considered in the development and promotion of safe, open and accessible places for everyone. How to create and promote accessible spaces for all should be a priority for planners and policymakers, and involving service users and carers in these processes will help to ensure all the relevant and complex issues are considered.4.1It is clear that there are different barriers and facilitators for people who are experiencing mental health problems. The emerging evidence on the contribution that green and social prescribing can make to reduce mental health inequalities is encouraging. Mental health providers, GPs, social workers, education and workplace settings should be encouraged to respond to and incorporate the research learning to establish opportunities and experiences to engage with nature in recognition of its therapeutic benefits. The development of policy guidance would be a welcome starting point and would acknowledge the need to adopt and embed sustainable practices. Inequalities extend to access to green and blue spaces and planning policies already address the need for improving access but this does not tackle existing estate and the fact that social housing stock is often located in areas of social deprivation. Interventions that promote feelings of safety, and reduce anxiety via buddy systems/could promote the social gateway to green and blue spaces. Promoting the benefits of being outdoors should use appropriate and relevant language and identify goals for those experiencing mental health problems such as improving self\u2010image, reducing anxiety, increasing social contact and enhancing mood. Improving access to green and blue spaces is a joint responsibility across government departments and policy needs to reflect this. The longer\u2010term impact of COVID\u201019 on mental health is also an area of developing concern and we need to understand whether the impact of isolation has reinforced anxieties about being in now less familiar, crowded public spaces.4.2We were unable to access an SMI sample because of Covid restrictions. To account for this deficit, we contacted service users living in supported living settings despite our directed efforts to recruit an SMI population, analyses found that almost half of the online participants reported mental health problems, including SMI. The majority of participants in the qualitative study were older adults therefore we cannot generalise these findings to younger people. Using MTurk to access a general population that included a large number of US participants and people were paid a small amount to participate, it is unlikely that participants were a random sample and therefore we are unable to generalise any findings to the United Kingdom.5Promoting green and blue spaces as a mental health prevention, early intervention or treatment option could be a valuable public mental health approach, but it is important that access to safe spaces is improved through public planning and policy. Ways of providing access, on whatever scale, within the existing estate of supported housing for people with mental health problems should be considered. The importance of appropriate messaging could help promote the appeal of green and blue spaces to different users. More research is required into the routine use of green and blue spaces and the potential positive impact on wellbeing.Claire McCartan and Gavin Davidson led all aspects of the project. Lee Knifton, Paul Webb and Chris White contributed to the study design and, with Liam Bradley and Katherine Greer, contributed to writing the research proposal and applying for and securing project funding. Liam Bradley, Katherine Greer and Paul Webb developed the qualitative interview schedule and Paul Webb facilitated data collection. Katherine Greer and Aod\u00e1n Mulholland conducted the qualitative interviews. All authors contributed to the analysis and study write\u2010up. Each author certifies that they have made a direct and substantial contribution to the conception, design and delivery of the study. We are grateful to the service users who participated in the interviews and thanks to Emily Peckham, Piran White, Simon Gilbody and the Closing the Gap team for their support and guidance delivering the project.Gavin Davidson's post at Queen's University Belfast is partly funded by Praxis Care which was one of the partners in this project. The remaining authors declare no conflict of interest.Ethical approval for the qualitative stage was granted by the Research Ethics Committee of the School of Social Sciences, Education and Social Work at Queen's University Belfast in April 2019. The quantitative stage was approved by the NHS West Midlands\u2014Coventry and Warwickshire Research Ethics Committee in July 2020 (REC reference 20/WM/0172). All participants provided written informed consent before taking part."}
{"text": "Prior studies suggested that antidepressant use is associated with an increased risk of dementia compared to no use, which is subject to confounding by indication. We aimed to compare the dementia risk among older adults with depression receiving first-line antidepressants  versus psychotherapy, which is also considered the first-line therapy for depression.This retrospective cohort study was conducted using the US Medical Expenditure Panel Survey from 2010 to 2019. We included adults aged\u2009\u2265\u200950 years diagnosed with depression who initiated SSRI/SNRI or psychotherapy. We excluded patients with a dementia diagnosis before the first record of SSRI/SNRI use or psychotherapy. The exposure was the patient\u2019s receipt of SSRI/SNRI (identified from self-report questionnaires) or psychotherapy . The outcome was a new diagnosis of dementia within 2 years  identified using ICD-9/ICD-10 codes from the Medical Conditions file. Using a multivariable logistic regression model, we reported adjusted odds ratios (aORs) with 95% confidence intervals (CIs). We also conducted subgroup analyses by patient sex, age group, race/ethnicity, severity of depression, combined use of other non-SSRI/SNRI antidepressants, and presence of underlying cognitive impairment.Among 2,710 eligible patients , 89% used SSRIs/SNRIs, and 11% received psychotherapy. The SSRI/SNRI users had a higher crude incidence of dementia than the psychotherapy group (16.4% vs. 11.8%), with an aOR of 1.36 (95% CI\u2009=\u20091.06\u20131.74). Subgroup analyses yielded similar findings as the main analyses, except no significant association for patients who were aged\u2009<\u200965 years , male , Black , had a higher PHQ-2 , and had underlying cognitive impairment .Our findings suggested that older adults with depression receiving SSRIs/SNRIs were associated with an increased dementia risk compared to those receiving psychotherapy.The online version contains supplementary material available at 10.1186/s12877-023-04475-z. \u2022\u00a0Our findings suggested that SSRI/SNRI use was associated with an increased dementia risk compared to psychotherapy among older adults with depression.\u2022\u00a0Subgroup analyses yielded similar findings as the main analyses, except no significant association for patients who were aged\u2009<\u200965 years, male, Black, had a higher PHQ-2 score, and had underlying cognitive impairment.Why does this matter?Clinicians should be aware of SSRI/SNRI-associated dementia risk when considering the first-line treatment for older adults with depression. Patient characteristics may also play a critical role in such an association.The online version contains supplementary material available at 10.1186/s12877-023-04475-z. We certify that this work is novel. This work is the first to compare first-line antidepressants  to an active comparator  to reduce confounding by indication instead of comparing antidepressant use to no use in prior studies.One out of ten older adults aged\u2009\u2265\u200965 years suffers from dementia in the United States (US), and the prevalence dramatically increases with age . The ecoPsychotherapy and antidepressants are considered the mainstay treatments for depression . PsychotAlthough antidepressants are beneficial for managing depressive symptoms, some studies reported the association between antidepressants and risk of dementia. For example, a meta-analysis found an increased long-term risk (follow-up >\u20091 year) of dementia associated with antidepressant use compared to no use among older adults . Kodesh Prior studies are limited by only comparing antidepressant exposure with no exposure, which may be subject to confounding by indication and severity . That isThis study used the 2010\u20132019 US Medical Expenditure Panel Survey (MEPS) data, a longitudinal, large-scale survey of noninstitutionalized adults in the US . Each pahttps://meps.ahrq.gov/mepsweb/ ). Due to the nature of de-identification and public availability of the MEPS data, the University of Florida IRB determined the study exempt and did not require informed consent to participate.MEPS data is reviewed and approved by the Westat Institutional Review Board (IRB) annually and is established under a multi-project assurance (MPA M-1531) granted by the Office for Protection from Research Risks. After carefully removing individual\u2019s identifiable information, an annual series of Public Use Files of de-identified MEPS data are made publicly available to researchers  codes  . We furtOur exposure of interest was the patient\u2019s receipt of SSRIs/SNRIs versus psychotherapy. We identified SSRI/SNRI use from the questionnaires in the prescribed medicines files using therapeutic classification variables from Cerner Multum, Inc.  and ICD-10 codes  from the Medical Conditions file  . Given tTo evaluate the heterogeneity in the drug effect among different patient subgroups, we grouped patients by sex , age group , race/ethnicity , severity of depression , concomitant use of non-SSRI/SNRI antidepressants , and underlying cognitive impairment . Then, we repeated all the steps in the main analysis in each subgroup.To test the robustness of our findings, we performed two sensitivity analyses. First, we used a 1:1 greedy nearest neighbor PS matching approach to match patients using SSRI/SNRI to those receiving psychotherapy. Similar to the main analysis, PS matching with the PROC SURVEYLOGISTIC procedure allows us to estimate the population ATT. Second, we used a subset of covariates  to estimate the PS and repeated the sIPTW approach.As shown in Fig. In Table Most subgroup analyses reported consistent results with the main analysis, except for patients who were aged\u2009<\u200965 years, male, Black, had a PHQ-2 score of 3\u20136, and had underlying cognitive impairment, for whom the adjusted ORs (95% CI) were 1.23 (0.93\u20131.62), 1.34 (0.95\u20131.90), 0.76 (0.48\u20131.19), 1.39 (0.90\u20132.15), and 1.06 (0.80\u20131.42), respectively. The sensitivity analyses yielded similar findings as the main analysis. The adjusted ORs (95% CI) were 1.27 (1.18\u20131.37) using the 1:1 PS matching and 1.12 (1.05\u20131.20) when removing comedications from the PS calculation.In this retrospective cohort study using nationally representative survey data in the US, we found that older adults with depression receiving SSRIs/SNRIs were associated with a 31% increased risk of dementia within two years compared to those receiving psychotherapy after adjusting for patient characteristics such as age, sex, race/ethnicity, depression severity, underlying cognitive impairment, comorbidities, and concomitant drugs. Most subgroup analyses yielded similar results, except for patients who were aged\u2009<\u200965 years, male, Black, had a PHQ-2 score of 3\u20136, and had underlying cognitive impairment, which did not show significant differences in the risk of dementia between SSRI/SNRI use and psychotherapy.Unlike previous studies largely comparing antidepressant users with nonusers, our study adopted the active comparison group approach to reduce confounding by indication and severity in older adults with depression. Our findings were consistent with some of the prior studies. For example, a meta-analysis including observational studies with at least a 1-year follow-up period showed that SSRI use was associated with an increased risk of dementia compared to no SSRI use, with a pooled risk ratio (RR) of 1.75 (95% CI: 1.03\u20132.96). However, the heterogeneity in the meta-analysis was extremely high, and one out of five included studies suggested that SSRI use was associated with a lower risk of dementia  . AnotherIn the subgroup analyses, we found that the association between SSRI/SNRI use and dementia risk did not exist in patients with a higher PHQ-2 score. Patients with a higher PHQ-2 score might reflect those with more severe depression or uncontrolled depression, which may confound the drug effect . That isThere are some limitations in our study. First, we used MEPS data, which only follow a patient for at most 2 years, which may not be long enough for dementia to occur and underestimate the risk of dementia . HoweverOur findings provide valuable insight into the complex association among depression, antidepressants, and risk of dementia, providing additional evidence for clinicians while prescribing antidepressants for patients with depression. Future longitudinal studies are warranted to allow the identification of new users of antidepressants and the evaluation of long-term dementia risk.Additional file 1: Appendix Figure 1. Directed acyclic graph illustrating the relationships among exposure, outcome, and confounders. Appendix Figure 2. Detailed steps of the statistical analysis. Appendix Table 1. Variables included in our study."}
{"text": "Actinobacillus pleuropneumoniae (apxIVA) and Pasteurella multocida (kmt1). The methods showed good linearity, precision, and accuracy. In both healthy and diseased pigs, a significant correlation was observed between LC/MS and the microbiological assay  and between LC/MS and the enzyme-linked immunosorbent assay (ELISA) . A strong correlation was observed between the microbiological assay and the ELISA in both healthy and diseased pigs . A Bland-Altman analysis revealed good agreement between the methods, i.e., 95% of the differences were within the limits of agreement. Therefore, the microbiological assay and the ELISA, which demonstrated sufficient precision and accuracy, can be viable alternatives to LC/MS when it is unavailable.Employing affordable and uncomplicated sample preparation techniques to recommend the most efficient antibacterial therapy could help reduce antibiotic-resistant bacteria. This study evaluated the suitability of immunoassays and microbiological assays as alternatives for liquid chromatography/mass spectrometry (LC/MS) in determining plasma tylosin concentrations after intramuscular administration at a dose of 20 mg/kg to both healthy and diseased pigs in clinical veterinary practice. The diseased pigs were confirmed using the target genes  Staphylococcus, Streptococcus, and Corynebacterium [Actinobacillus pleuropneumoniae and Pasteurella multocida in pigs [Tylosin is an antibiotic commonly used in veterinary medicine for the treatment of respiratory infections in pigs . Tylosinacterium . Tylosin in pigs ,5.The prevention and treatment of pathogenic respiratory bacterial infections in pigs can be enhanced by directly measuring plasma antibiotic concentration and conducting antibiotic susceptibility testing, and thereby increasing the effectiveness of antimicrobial therapy . In addiMethods such as liquid chromatography/mass spectrometry (LC/MS), microbiological assay, and enzyme-linked immunosorbent assay (ELISA) can determine plasma concentration ,9. TheseIf we can establish a strong correlation between LC/MS and field-applicable microbiological methods or ELISA in healthy and infected swine plasma following the administration of tylosin to pigs, it will be possible to prescribe optimal antibacterial therapies in the field using simple and low-cost sample preparation technology. This could help reduce the emergence of resistant bacteria. Thus, this study aimed to evaluate the correlation of LC/MS with a microbiological assay and an ELISA when determining the plasma concentration of tylosin in healthy and infected pigs.apxIVA and kmt1 polymerase chain reaction (PCR) target genes for A. pleuropneumoniae and P. multocida with respective fragment sizes of 377 bp , out of which 5 infected pigs exhibited clinical signs such as reduced activity, labored breathing, and coughing. Similarly, the rest of the infected pigs were lethargic and less engaged with their surroundings compared with their healthy counterparts. The diseased pig samples had the f 377 bp a and 460f 377 bp b, confirThe concentration\u2013time profiles of tylosin in the plasma after intramuscular injection in healthy and infected pigs were determined using three methods, namely LC/MS, microbiological assay, and ELISA, and the results are presented in 2), slope, and y-intercept were 0.98, 1.00, and 0.16, respectively. The limit of detection (LOD) was 0.014 \u00b5g/mL and the limit of quantification (LOQ) was 0.042 \u00b5g/mL. Both the intra-assay precision and the inter-assay precision were determined to be <20%, and the accuracy of the assays were 101.34\u2013113.62% and 97.38\u2013108.36% for the intra- and inter-assays, respectively  values from the assays on three different days, and these values ranged from 4.08% to 7.64%. The intra- and inter-assay accuracy rates were 85.95\u2013116.04% and 84.20\u2013102.02%, respectively  and the slope of 0.806 indicate a dose-dependent relationship. Likewise, a correlation was observed in the diseased pigs, with a Pearson correlation coefficient of 0.950 (p < 0.001) and a slope of 1.330  and a slope of 0.694 (p < 0.001) (of 0.694 b. A sign< 0.001) e.To assess the agreement between LC/MS and the ELISA, a Bland\u2013Altman plot was produced, and this displayed the differences between the LC/MS and ELISA datasets versus the mean tylosin concentrations obtained using these two methods. The mean difference in the concentrations obtained using the two methods was \u22120.17 in the healthy pigs, with limits of agreement of \u22122.10 and 1.77 b, whereap < 0.001) and a slope of 0.782 were observed, whereas for the diseased pigs, a Pearson correlation coefficient of 0.910 (p < 0.001) and a slope of 0.881 were observed, indicating a dose-dependent relationship  and LC/MS are the most widely accepted methods of assessing drug plasma concentrations, and they are both highly accurate and capable of analyzing multiple compounds in a single sample . HoweverIn this study, we aimed to develop a low-cost method for determining plasma tylosin concentrations that can be used in laboratories where LC/MS is inaccessible. Alternative methods for measuring drug concentrations, such as immunoassays and microbiological assays, are available ,19. AlthComparing microbiological and instrumental methods with other biological methods is a common practice used to investigate potential variations in the pharmacological activity of drugs and determine drug concentrations in the plasma. It has been found that microbiological assays may have limited sensitivity compared with other analytical methods. The detection limit of the assay may not be sufficient to accurately measure low drug concentrations in plasma samples. This can be a limitation when analyzing drugs that exhibit low plasma concentrations, or when studying drug pharmacokinetics in the body . A previIn this study, we analyzed plasma tylosin concentrations over time in healthy and diseased pigs and found similar concentration changes over time using all three methods. Each method demonstrated satisfactory performance with inter- and intra-assay coefficients of variation within \u00b120.0% for the RSD (%). These results suggest that the microbiological and ELISA methods may be used in place of LC/MS for measuring tylosin, and that they have potential for rapid and on-site application. To confirm this possibility, we statistically analyzed the data to compare the three methods.p < 0.001), between LC/MS and the ELISA , and between the microbiological assay and the ELISA  in the healthy pigs, and by those between LC/MS and the microbiological assay , between LC/MS and the ELISA  and between the microbiological assay and the ELISA  in the diseased group.Bland and Altman developed the Bland\u2013Altman plot to evaluate similarities between two sets of numerical measurements. In this study, the Bland\u2013Altman plot indicated a high level of agreement between the measurement methods, with 95% of the differences between the two methods falling within the appropriate limits. However, the suitability of these limits should be determined based on clinical and biological considerations . To use Although the ELISA is a valuable tool, there are certain limitations and obstacles that may affect the precision and consistency of its results . Its speThis study found good correlation between different analytical methods used to measure tylosin levels in plasma. ELISAs and microbiological assays have several advantages, including simplicity, cost-effectiveness, and the ability to provide important insights into a drug\u2019s effectiveness and potency. However, it is important to acknowledge that these assays may not be suitable for all types of drugs, and their limitations should be taken into account. Certain drugs may not be accurately detected or quantified by these assays due to factors such as interference from other substances, complex drug formulations, or the absence of specific microbial targets. Therefore, while microbiological assays can be valuable in drug testing, it is crucial to supplement them with other analytical methods to ensure a thorough and precise evaluation of a drug\u2019s properties.A. pleuropneumoniae and P. multocida displayed various symptoms, including anorexia, high body temperature, labored breathing, and coughing, all of which are symptoms that are typically observed in diseased pigs [This study involved creating a diseased porcine model through intranasal inoculation. The pigs infected with sed pigs . After tsed pigs . Therefosed pigs . Hence, Standard tylosin (>96%) was purchased from Sigma . The injectable tylosin was obtained from Samyang Anipharm . Nicotinamide adenine dinucleotide (NAD) was obtained from Sigma. All chemicals and reagents used in this study were of HPLC grade.A. pleuropneumoniae (BA2000013) and P. multocida (BA1700127) were provided by the Animal and Plant Quarantine Agency . Before use, the bacterial isolates were streaked on brain heart infusion agar  supplemented with 0.02% NAD and incubated at 37 \u00b0C in 5% CO2. For the microbiological assay, M. luteus KCCM 11236 was obtained from the Korean Culture Center of Microorganisms (KCCM) and cultured on tryptic soy broth  at 37 \u00b0C for 18 h.9 cfu/mL of A. pleuropneumoniae and P. multocida to establish a disease model. During the experiment, clinical respiratory disease score, intestinal function score, appearance/abnormal signs, and clinical signs were recorded [A. pleuropneumoniae and P. multocida infections were monitored by culturing nasal swabs, and infections were confirmed by the presence of the apxIVA gene [kmt1 gene [A. pleuropneumoniae and P. multocida, respectively.The study was carried out on 14 pigs (Duroc \u00d7 Landrace \u00d7 Yorkshire) aged 5\u20136 weeks with an average weight of 9.5 \u00b1 1.1 kg. The animals were acclimatized for 1 week with free access to the water and feed. The animal study was approved by the Animal Ethics Committee of the Petobio Clinical Institute (PTB-2022-IACUC-013-A). After proper adaptation, the pigs were divided into a healthy group and an infected group, with each group consisting of seven animals. The bacterial challenge was performed according to a previously described method, but with some modifications . The 40 recorded ,35. A. pIVA gene  and the mt1 gene , used fog for 10 min at approximately 5 \u00b0C. The plasma from each Vacutainer was divided into aliquots of about 0.6 mL which were deep frozen at approximately \u221275 \u00b0C until analysis.The healthy and diseased pigs received intramuscular tylosin injections at a dose of 20 mg/kg. The selection of a 20 mg/kg drug dose was based on previous studies  that hadg for 10 min at 4 \u00b0C. The 2 mL of supernatant was collected to dryness in a water bath using nitrogen at 50 \u00b0C. The residue was then dissolved in 200 \u03bcL of methanol, agitated for 1 min, and centrifuged at 12,000 rpm for 10 min at 4 \u00b0C. The final 70 \u03bcL of supernatant was analyzed using LC/MS.Preparation for the LC/MS analysis was as follows. The 250 \u03bcL plasma samples were thawed at room temperature and then deproteinized with methanol (2 mL) via vortexing for 15 min and centrifuged at 3300\u00d7 M. luteus KCCM 11236 as the test organism [M. luteus KCCM 11236 bacterial suspension was added to tempered nutrient agar from Becton, Dickinson and Company, Franklin Lakes, NJ, USA, at a concentration of 106 cfu/mL. The agar mixture was immediately poured onto assay plates in 2.2 mm layers. After allowing the samples to solidify for 45 min, wells with 0.5 cm diameters were created and filled with 60 \u03bcL plasma samples or tylosin standards covering a range of concentrations from 1 \u03bcg/mL to 16 \u03bcg/mL. The agar plates were then incubated for 18 h at 37 \u00b0C. The zones of bacterial inhibition were measured using a digital caliper from Mitutoyo, Japan. This method was chosen for its high sensitivity, simplicity, and strong correlation with HPLC measurements [Plasma tylosin concentrations were determined via a microbiological assay using organism . The bacurements . The meturements .0) \u00d7 100%. Here, A = average absorbance of standard or sample and A0 = Average absorbance of 0 ppb standard. For the standard curve, the absorbance percentage of each standard was plotted on the y-axis, and the logarithmic concentration was plotted on the x-axis, resulting in a semi-log plot. The precision of the assays was evaluated by assessing repeatability, which was expressed as the relative standard deviation (RSD). To determine accuracy, a control sample was introduced at the beginning of the procedure, and the measured value was calculated by dividing it by the nominal value and multiplying the result by 100.Plasma tylosin concentrations were assessed using an ELISA kit (E-FS-E058) obtained from Amsbio . A 1 mL serum sample was transferred to a 2 mL e-tube and centrifuged for 5 min at 4000 rpm at room temperature. Following centrifugation, 0.1 mL of the supernatant was collected and mixed with 0.9 mL of the reconstitution buffer provided by the ELISA kit. The mixture was then oscillated for 30 s. Finally, a 50 \u03bcL sample was extracted for detection, following the manufacturer\u2019s instructions. The optical density was determined at 450 nm using an Epoch microplate reader . The percentage of absorbance was determined using the following formula: . The flow rate was 0.4 mL/min and the sample injection volume was 3 \u03bcL. The column temperature was maintained at 40 \u00b0C. The monitored precursor ion for tylosin was 916.3 m/z. The validation of the assay was performed according to a procedure described elsewhere [The LC/MS analysis was performed using a 1200 HPLC system from Agilent Technologies  that was connected to a 6140 single mass spectrometer (Agilent Technologies). The mass spectrometer was set up with an electrospray positive ionization mode using a capillary voltage of 3500 V, and it had optimal ESI-MS parameters, such as a drying gas temperature of 350 \u00b0C, a drying gas flow of 5 L/min, and a nebulizing gas pressure of 45 psi. The separations were accomplished using an Eclipse Plus C18 column  from Agilent Technologies. The mobile phase consisted of a mixture of 0.1% formic acid in water (Eluent A) and 0.1% formic acid in acetonitrile solution (Eluent B), with a ratio of 30:70 . The agreement of the three analytical methods was evaluated using the Pearson correlation coefficient method and the Bland-Altman method ,43.In this study, the results of comparing three different methods for measuring tylosin plasma concentrations were significant, considering the importance of monitoring antibiotic concentrations in animals to prevent the emergence of antibiotic-resistant bacteria, which can be caused by the overuse of antibiotics in farming practices. Thus, the microbiological assay and the ELISA, both of which are cost effective and accessible, could replace LC/MS. Nevertheless, these simpler methods still require improvements in their precision and consistency before they can be extensively used in clinical applications for antibacterial therapy."}