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+{"text": "The ECOG Scale of Performance Status (PS) is widely used to quantify the functional status of cancer patients, and is an important factor determining prognosis in a number of malignant conditions. The PS describes the status of symptoms and functions with respect to ambulatory status and need for care. PS 0 means normal activity, PS 1 means some symptoms, but still near fully ambulatory, PS 2 means less than 50%, and PS 3 means more than 50% of daytime in bed, while PS 4 means completely bedridden. An inter-observer variability study of PS assessment has been carried out to evaluate the non-chance agreement among three oncologists rating 100 consecutive cancer patients. Total unanimity was observed in 40 cases, unanimity between two observers in 53 cases, and total disagreement in seven cases. Kappa statistics reveal the ability of the observers compared to change alone and were used to evaluate non-chance agreement. Overall Kappa was 0.44, (95% confidence limits 0.38-0.51). The Kappa for PS 0 was 0.55 (0.44-0.67), while those for PS 1, 2, 3 and four were 0.48 (0.37-0.60), 0.31 (0.19-0.42), 0.43 (0.32-0.55), and 0.33 (0.33-0.45), respectively. If one observer allocated patients to PS 0-2, then another randomly selected observed placed the patients in the same category with a probability of 0.92. For patients with PS 3-4 the probability that the same category would be chosen was 0.82. Overall, the non-chance agreement between observers was only moderate, when all ECOG Performance Status groups were considered. However, agreement with regard to allocation of patients to PS 0-2 versus 3-4 was high. This is of interest because this cut-off is often used in clinical studies."}
+{"text": "Eighty-eight patients presenting with operable breast cancer of 4 cm or greater in diameter  have received primary systemic therapy. Response was assessed following 12 weeks of systemic therapy by linear regression analysis of changes in tumour volume. Definitive locoregional surgery  was performed on completion of systemic therapy (3-6 months). Response was observed in 24 (39%) of the 61 patients who received endocrine therapy; all 24 had tumours with an oestrogen receptor (ER) concentration of greater than or equal to 20 fmol mb-1 cytosol protein. Cytotoxic therapy was reserved for patients with tumours of ER concentration less than 20 fmol mg-1 cytosol protein (n = 27) or when endocrine therapy had failed (n = 20). Response was observed in 34 patients (72%). The overall survival rate at 3 years was 86%, with 81% remaining free from local relapse. We propose that the treatment policy outlined in this paper should now be tested against orthodox management by controlled randomised trial."}
+{"text": "Recurrence and survival rates were studied in 175 women with breast cancer who, until the development of recurrent disease, received no treatment other than a modified radical (Patey) mastectomy, and in whom the oestrogen (REc) and progesterone (RPc) receptor content of the primary tumour was measured. At the time of first relapse most patients received endocrine therapy. At a minimum follow-up of 58 months post menopausal patients who possessed REc had an increased relapse-free survival (RFS) (P = 0.02). When examined by node status patients with 1-3 axillary nodes containing tumour also had an improvement in RFS (P = 0.02). There was no benefit for node-negative or premenopausal patients. In 163 patients in whom RPc was measured, RFS was unaffected by the possession of this receptor regardless of the degree of node involvement or menopausal status. Patients with REc had a significantly longer survival following mastectomy than patients without it (P = 0.006). This was most marked in post-menopausal (P = 0.003) and node-positive (P = 0.03) patients. Survival following mastectomy was also increased in patients possessing RPc (P = 0.04) and again was most marked for post-menopausal patients (P = 0.01), although no difference could be identified within node subgroups. There were significant differences in the post-relapse survival of REc and RPc positive and negative patients . Patients with both receptors survived approximately 37 months longer than their receptor-negative counterparts. This study failed to confirm that the measurement of REc and RPc can reliably predict early relapse in breast cancer. The greater overall survival of receptor-positive patients is mainly due to an increase in survival following relapse. This may reflect the response of receptor-positive tumours to endocrine therapy given for recurrent disease."}
+{"text": "Tumours from 336 breast cancer patients under the age of 50 were analysed for hormone receptor content and by DNA flow cytometry. Sixty-six percent of the tumours were positive for estrogen receptors (ER), 60% were progesterone receptor (PR) positive and 42% showed DNA diploid profiles. DNA hypodiploid tumours were relatively frequent (7%), especially in patients aged 40 years or less (11%). S-phase fraction (SPF), with a mean of 10%, correlated significantly with receptor status, DNA ploidy, lymph node status, tumour size and age. With a median follow-up period of 34 months, the distant recurrence-free interval was independently predicted by lymph node status, tumour size, SPF and PR content. Amongst the 212 patients who had not received adjuvant systemic treatment, receptor status was, in addition to lymph node status and SPF, independently related to distant recurrence rate. A high SPF identified a subgroup with high recurrence rate, comprising approximately one third of the node-negative patients. Similarly, the one third of node-positive patients who had PR-positive tumours with a low S-phase fraction formed a subgroup with low recurrence rate. We conclude that hormone receptor assays and DNA flow cytometry should be useful tools in the management of breast cancer patients less than 50 years of age."}
+{"text": "Nodal involvement is accepted as the best single marker of prognosis in breast cancer. However, there is little information on the sub-division of node-positive patients according to the oestrogen receptor status of the nodal tissue. We have previously reported  that, in almost all cases, involved nodes are only oestrogen receptor positive (ER+) in patients whose primary tumours are uniformly ER+. This paper presents clinical follow-up on a larger group of patients with node positive breast cancer. For each patient, both soluble and nuclear receptor concentrations were determined in three separate parts of the primary tumour and in at least one involved node . Median follow-up time was 71.5 months. As expected, patients whose tumours were HS++ had a significant (P less than 0.008) survival advantage. More importantly, patients with ER in both the soluble and nuclear fractions of their involved nodes survived significantly (P less than 0.003) longer than those with ER- nodes. Thus, full oestrogen receptor status of involved nodes will give sufficient prognostic information when adequate primary tissue is not available."}
+{"text": "The long term prognostic significance of oestrogen receptors was assessed in a prospective study of 767 patients presenting between the years 1975 and 1981 with stage 1 and 2 breast cancer treated by mastectomy with either full axillary dissection or nodal sampling. Oestrogen receptor binding was determined by a dextran coated charcoal method and median follow up was 11 years. Oestrogen receptors were present in 396 (54%) of tumours. Absence of oestrogen receptors was associated with tumours of high histological grade, but there was no relationship between nodal status or tumour size. Oestrogen receptor status did not predict survival for the group as a whole or when stratified by nodal status. In multivariate analysis both nodal status and tumour size were powerful independent prognostic factors, but oestrogen receptors failed to achieve statistical significance."}
+{"text": "The month of initial detection of tumour was recorded in 2,245 patients with breast cancer and correlated with survival over a follow-up period of 1.5-10 years. Women who initially detected their breast cancer in spring/summer had a significantly longer survival than those detecting their tumour at other times of the year. Overall, this relationship was independent of nodal status, tumour size and hormone receptor status. However, when patients were divided into groups the survival advantage was significantly associated with receptor status and age. Women aged greater than or equal to 50 years with ER-positive and PR-positive tumours who discovered their initial tumour in spring/summer had significantly better survival than those detecting their tumours at other times of the year. Survival was also longer in women aged less than 50 years with receptor-negative tumours who initially found their tumours in spring/summer compared with the rest of the year. This study suggests that the season of first detection of a breast cancer relates significantly to the later behaviour of the tumour, and may reflect seasonal changes in hormone dependent growth."}
+{"text": "More accurate criteria are required for the selection of patients with node-negative breast cancer for systemic adjuvant therapy. Expression of epidermal growth factor receptor (EGFr) has been shown previously to be inversely related to oestrogen receptor (ER) in patients with operable breast cancer and to be associated with a poorer prognosis. Analysis of EGFr and ER was performed on tumour samples from 231 patients with operable breast cancer followed for up to 6 years after surgery. The median duration of follow-up in patients still alive at the time of analysis was 45 months. Thirty-five percent of patients (82) had tumours with greater than 10 fmol mg-1 I125-EGF binding (EGFr+) and 47% (109) and cystolic ER concentrations greater than 5 fmol mg-1 (ER+), with a marked inverse relationship between EGFr and ER (P less than 0.00001). In a univariate analysis EGFr was second only to axillary node status as a prognostic marker for all patients both in terms of relapse-free and overall survival . For patients with histologically negative axillary nodes EGFr was superior to ER in predicting relapse and survival . In a multivariate (Cox model) analysis only EGFr, out of EGFr, ER, size and grade, was predictive for either relapse-free or overall survival for patients with node-negative disease (P = 0.05 and P = 0.026 respectively). EGFr has been shown to be a marker of poor prognosis for patients with node-negative breast cancer. Since patients with EGFr+ tumours are unlikely to respond to hormone therapy it may be possible to select them for trials of systemic adjuvant chemotherapy."}
+{"text": "In a group of 74 patients with advanced metastatic breast cancer, 57% of those with cytoplasmic oestrogen receptor activity in their tumours (REC+) showed a clinical response to endocrine therapy. Of 51 patients whose tumour was assayed for both REC and cytoplasmic progesterone (RPC) activity, 9/12 patients with REC+ RPC+ tumours responded to hormone treatment, whereas only 3/30 patients with REC-RPC-tumours had a clinical response. In a group of 19 patients in whom nuclear oestrogen receptor (REN) was also estimated in the pellets from tumour-tissue homogenates, 5/6 with tumours positive for all 3 receptors showed a clinical response. None of the 9 patients with triply negative tumours responded. Addition of the REN assay appears to reinforce the greater precision of prediction when RPC as well as REC are estimated in breast tumours."}
+{"text": "Sequential biopsies of breast cancer tissue were obtained from a total of 210 women in order to assess any change in oestrogen receptor (ER) status arising spontaneously or as a result of intervening therapy. A combined assay measuring both cytosol and nuclear oestrogen receptors was used for all samples. One hundred and fifty-five patients had biopsies of their primary tumour and of a later loco-regional recurrence; 26 had biopsies of their primary tumour and a recurrence or new primary in the opposite breast; and 29 had sequential biopsies of recurrent disease only. Overall only 61.2% of the primary tumours retained their original status with respect to both cytosol and nuclear oestrogen receptors on recurrence. These results were influenced by intervening therapy, however, and if only untreated patients are considered, over 70% of their recurrences contain the same combination of cytosol and nuclear receptors as found in the primary tumours. For tumours 'recurring' in the opposite breast, the pattern was similar with 69.2% retaining the same status as the first primary. The agent found most likely to alter I:R status was tamoxifen and in the samples taken from patients undergoing treatment with this drug, no tumour was found to contain measurable receptor."}
+{"text": "In the present study, we determined the frequency and intensity of MRP protein expression by monoclonal antibody immunohistochemistry in a series of 259 resected invasive primary breast carcinomas, and we evaluated MRP immunoreactivity in relation to patient and tumour characteristics, relapse-free (RFS) and overall survival (OS). The immunostaining was graded on a semiquantitative scale that ranged from (-) to . Overall, 34% of the tumours were positive for anti-MRP antibody: 19% showed weak cytoplasmic staining (+), 14% had clear cytoplasmic staining (++) and only 1% of the tumours had a strong cytoplasmic as well as membranous staining . MRP expression was not related to patient's age, menopausal status, tumour size, differentiation grade, oestrogen and progesterone receptor level or lymph node involvement. In an exploratory univariate analysis of all patients, only primary tumour size and number of lymph nodes involved were significantly associated with shortened RFS (P < 0.001 and P < 0.001 respectively) and OS (P = 0.02 and P < 0.001 respectively). In Cox univariate analysis for RFS in subgroups of patients stratified by menopausal status, tumour size, nodal status, adjuvant systemic therapy and oestrogen and progesterone receptor status, MRP expression was associated with increased risk for failure in patients with small tumours (T1), in node-negative patients and in node-positive patients who received adjuvant systemic chemotherapy with cyclophosphamide, methotrexate and 5-fluorouracil (CMF); the relative hazard rate (RHR) for relapse was increased in the presence of MRP, with RHR values with 95% confidence limits (CL) of 2.8 (1.2-6.9), 2.1 (1.0-4.2) and 2.8 (0.8-9.9) respectively. In analysis for OS, expression of MRP was also associated with increased risk for failure in patients with small tumours (T1) [RHR (95% CL) 2.3 (0.9-6.0)] and in node-positive patients who received adjuvant systemic chemotherapy with CMF [RHR (95% CL) 3.7 (0.8-17.1)] but not in node-negative patients [RHR (95% CL) 1.1 (0.4-2.6)]. In conclusion, our results show that MRP is frequently overexpressed in primary breast cancer and suggest that MRP expression might be of prognostic significance in the subgroups of patients with the more favourable prognosis, i.e. patients with small tumours and node-negative patients, as well as in the setting of adjuvant systemic chemotherapy. In primary breast cancer, MRP might be related to altered cell biological behaviour, including a more aggressive phenotype, and resistance to adjuvant systemic chemotherapy."}
+{"text": "Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1\u03b1, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy . The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients.Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1 Current prognostic and predictive markers of invasive breast cancer rely largely on histological indicators such as tumour size, grade, nodal status and receptor status, which define broad patient groups for different treatment regimens. However, it is recognised that there are many patients within such groups who are overtreated or undertreated with conventional therapies, strongly suggesting that current stratification does not fully account for the molecular heterogeneity and diverse biological behaviour of breast cancer. Recently cDNA microarray studies have classified breast cancers broadly into five subtypes, based on their different expression profiles: normal breast-like, luminal A (oestrogen receptor (ER) positive), luminal B (ER positive and proliferative), HER2 overexpressing and basal , the latBasal-like breast cancers derive their name from their characteristic expression of basal and myoepithelial markers such as cytokeratins (CK) 5, 14 and 17  and carbonic anhydrase (CA) IX, a downstream target of HIF-1\u03b1 in a series of breast cancers, and compared the expression of each marker by correlating it with various standard clinicopathological parameters.In addition to being mostly high-grade tumours, a so-called triple negative phenotype  and HER2 negative), basal-type breast cancers may also have a characteristic central acellular or necrotic zone , and is Tumour tissue microarray cores (1\u2009mm cores) were collected from 621 invasive breast carcinomas collected from patients who had undergone surgery at the John Radcliffe Hospital, Oxford, UK and from the Garvan Institute, Sydney, Australia. This study has Ethical Committee approvals . All patients had operable breast carcinomas and were not diagnosed with metastatic disease at the time of presentation. Information regarding patient characteristics, including age, tumour size, grade, histology, nodal status, ER and HER2 status, were collected from the clinical and pathological records. The median age of patients included in this study was 55 years (range 24\u201387 years). Eighty-eight percent of tumours were invasive ductal of no specific type, 3% were invasive lobular carcinoma and 3% were of other histological classifications (data was unavailable for the remaining 6%). Median tumour size was 20\u2009mm and the median tumour grade was two. Forty-four percent of patients had nodal disease. Sixty-eight percent of tumours were ER positive and 16% were HER2 positive. Patients less than 50 years of age with node-positive, ER-negative tumours or tumours larger than 3\u2009cm received adjuvant chemotherapy  or adriamycin and cyclophosphamide (AC). Patients with hormone-responsive tumours who were more than 50 years of age received 5 years of endocrine therapy. Patients were followed up for a median period of 131.9 months. During this time, 137 patients developed recurrence (30.0%) and 99 deaths (21.7%) were considered as breast-cancer related.\u03b1 were used as positive controls. In each run a tumour is known to be positive for CAIX was included.Formalin-fixed paraffin-embedded tissue sections were stained for the various hypoxic markers . Substitet al. basal group , luminal group , HER2 group (HER2 positive) and a negative group   and cancer-related death as the endpoints and compared using a log-rank test. Binary logistic regression was used for multivariate analyses and the Cox proportional hazard regression model was used to identify independent prognostic factors for disease-free and overall survival. The analyses were carried out with SPSS 16.0 . A two-tailed P-value test was used in all analyses and a P-value of <0.05 was considered statistically significant.Correlations were evaluated using either the one-way ANOVA or \u03c7Out of 456 tumours 310 (68%) were ER positive; of these, 289 tumours were also HER2 negative and were therefore considered to be of the luminal group . Seventy\u03b1 (Ninety-four out of 188 tumours (50%) stained positive for HIF-1\u03b1  (Supplem\u03b1 . Of thes\u03b1 . None of\u03b1 .P=0.005), high tumour grade (P<0.001) and ER negativity (P<0.001) (P>0.05) (\u03b1 expression (CAIX was positive in 59 out of 407 tumours (14%) . The majP<0.001) . There w(P>0.05) . CAIX expression .P<0.001, 95% CI 3.86\u201320.29) than luminal tumours (\u03b1 or its regulatory enzymes (P>0.05).Basal-like tumours were 8.9 times more likely to express CAIX ( tumours . This asP<0.001), but were not correlated with patient age, tumour size or nodal status (P>0.05). Basal-like tumours and HER2 tumours shared a similar poor disease-free survival outcome  .Basal-like tumours were associated with high tumour grade ( outcome  and wereP<0.001) (P<0.001) (P=0.03) than treated patients with CAIX-negative basal-like breast cancers (P=0.07), or when basal-like carcinomas were stratified according to CAIX expression (P=0.27). There was no significant difference in overall survival in patients not treated with chemotherapy with any type of breast carcinoma (P=0.07) or basal-like carcinoma stratified by CAIX (P=0.27). In a multivariate Cox regression model patients who had tumours that were CAIX positive and who were treated with chemotherapy had a significantly shorter overall survival (P<0.001), whereas CAIX had no effect on survival in patients not treated with chemotherapy (P=0.48) (P=0.04)  . There wP<0.001) . Patient cancers . In the (P=0.48)  and B. T(P=0.04) .\u03b1 is also associated with a poor prognosis and resistance to chemotherapy , where there is significant upregulation of not only HIF-1\u03b1 and CAIX but also other HIF-1\u03b1-regulated genes such as vascular endothelial growth factor-A, BNIP3 and Glut-1. Moreover, patients with basal-like tumours who have not received chemotherapy have a similar prognosis irrespective of CAIX expression, whereas patients with basal-like tumours that are CAIX positive and who have received chemotherapy have a significantly shorter overall survival than patients with basal-like tumours that are CAIX negative. This suggests that CAIX-positive basal-like tumours are particularly resistant to chemotherapy, in contrast to CAIX-negative basal-like tumours, which are able to respond to chemotherapy. A potential mechanism for this action is through trapping of chemotherapeutic agents outside the cell when the extracellular environment is acidic (CAIX expression, like a basal-like phenotype, is associated with an increased risk of recurrence and poorer overall survival (The resistance effect of CAIX/hypoxia seems to hold irrespective of tumour subtype as, unlike patients not treated with chemotherapy, treated patients with any tumour phenotype positive for CAIX also had a significantly worse survival than patients with tumours negative for CAIX in the multivariate analyses. This is in keeping with our studies in the neoadjuvant context, in which CAIX was associated with a shorter relapse-free survival in patients treated with CMF , and in Molecular genetic studies of basal-like breast carcinomas have shown that tumours with a basal-like phenotype have a higher number of unbalanced chromosomal changes and rate of loss of heterozygosity than luminal-type breast tumours  and addiIn summary, the more aggressive phenotype seen in basal-like tumours may be at least partly due to the intrinsically enhanced hypoxic properties of basal-like breast cancers. It suggests that as this tumour type is particularly difficult to treat, because they are both hormonal non-responsive and resistant to chemotherapy, targeting HIF with small molecule inhibitors, HRE-regulated gene therapies, carbonic anhydrase inhibitors or the use of bio-reductive drugs may be of particular use in treating this aggressive cancer ."}
+{"text": "A sensitive single-stranded hybridisation probe for the oestrogen receptor mRNA was synthesised using T3 polymerase from oestrogen receptor cDNA cloned in the Bluescript vector. This probe was used to measure oestrogen receptor mRNA in total RNA extracted from breast tumours. Oestrogen receptor mRNA was detected in 41 of 47 (87%) tumours whereas cytosolic oestrogen receptor protein was detected in only 18 out of 39 (46%). There was a significant correlation between the levels of the oestrogen receptor, as measured by 3H-oestradiol binding, and the oestrogen receptor mRNA. Oestrogen receptor mRNA was detected in 15 of the 21 tumours that did not contain detectable oestrogen receptor protein. This suggests that detection of the mRNA is a more sensitive means for establishing oestrogen receptor status than the radioligand oestrogen receptor assay. Oestrogen receptor mRNA was found in all histological tumour types examined. Its level was related to tumour differentiation. All the tumours that did not contain oestrogen receptor mRNA and most of the tumours that contained only low levels of oestrogen receptor mRNA were classified as grade III according to Bloom and Richardson: the median of the oestrogen receptor mRNA levels was significantly lower in this group."}
+{"text": "The relationship to survival after first recurrence of oestrogen receptor (ER), nodal status and TNM stage at diagnosis, and treatment for advanced disease was studied in 457 females whose primary breast cancer was diagnosed in 1975 to 1981. Receptor concentration was the most important predictor of post-recurrence survival, with some additional information conveyed by nodal status. ER predicted survival after recurrence independently of nodal status, clinical stage or mode of therapy. Response to endocrine therapy is only a facet of the generally favourable prognosis of ER positive patients, rather than the sole explanation."}
+{"text": "The oestrogen receptor (RE) status of the primary tumour has been assessed in 466 of a consecutive series of 550 patients with primary operable breast cancer. All patients were followed up (without treatment) until the development of recurrence or metastases. Distant metastases have so far occurred in 124 patients and 82 have had symptomatic local or regional recurrence. A significant correlation exists between the RE status of the primary tumour and subsequent patterns of metastasis. Symptomatic metastases to regional lymph nodes are more common with RE- cancers. There is no significant difference in either time of onset or total incidence of distant metastases between patients with RE+ and RE- tumours. Distribution of distant metastases is influenced by RE status: RE+ tumours tend to recur in bone, RE- tumours show affinity for viscera."}
+{"text": "Fine needle aspirates from 82 patients with breast carcinoma were fixed in methacarn, double embedded in agar or gelatin, and then in paraffin wax. Sequential sections were stained with monoclonal antibodies to the oestrogen receptor-related protein P29 (antibody D5), carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and cytokeratin (CAM 5.2). Sixty-one of 82 (74%) aspirates provided sections suitable for immunostaining. Twenty-six (43%) were D5 positive, 23 (38%) CEA positive, 59 (97%) EMA positive, and 54 (89%) CAM 5.2 positive. Twenty-six of these patients were treated with some form of endocrine therapy. Twelve (46%) showed positive staining for D5. Eleven (92%) of the 12 D5-positive patients responded or had static disease, and 8% progressed. Of the 14 D5-negative tumours 43% responded or remained static, and 57% progressed. The difference in response between the D5-positive and the D5-negative tumours was significant . There was no correlation between staining for CEA, EMA or cytokeratin and response to endocrine therapy."}
+{"text": "Soluble and nuclear oestrogen receptor status was determined in both the central and peripheral portions of tumour for 37 cases of adenocarcinoma of the endometrium. Of these, 29 had functional receptor in the peripheral biopsy, but only 19 retained functional receptor in the centre. Six of the 10 patients whose tumours showed this difference came from the group of 12 patients who were immediately post-menopausal . Receptor status was not related to tumour classification into histological grades I and II. However, receptor-negative central biopsies were significantly more likely (P less than 0.05) to be Grade III. Early relapse was also related to a receptor-negative central biopsy."}
+{"text": "P = 0.008), essentially due to a link with the progesterone receptor status (P = 0.01). By contrast there was an inverse relationship between hSI expression and the oestrogen receptor and/or progesterone receptor positivity (P = 0.098). A significant relationship was shown between both the sigma-1 receptor, hSI expressions and Bcl2 expression, with P = 0.017 and 0.035 respectively. We also assessed whether the expression of the sigma-1 receptor or hSI might be linked with disease-free survival (DFS) and found that the presence of hSI and the absence of sigma-1 receptor expression were associated with a poorer disease-free survival (P = 0.007). Altogether these results suggest that in primary breast carcinomas in association with the evaluation of the steroid receptor status, the sigma-1 receptor and hSI may be interesting new markers useful to identify those patients who might be able to benefit from an adjuvant therapy. \u00a9 2000 Cancer Research CampaignThe purpose of this study was to immunocytochemically investigate two new markers, the sigma-1 receptor and the human sterol isomerase (hSI), in comparison with a series of clinicopathological and immunocytochemical prognostic factors in a trial including 95 patients with operable primary breast cancers. Our results showed no statistically significant relationship between these two markers and the age of the patients, their menopausal status, the tumour size and its histological grade, the nodal status and the expression of the Ki-67 proliferative marker. However, we evidenced a close correlation between the sigma-1 receptor expression and the hormonal receptor positivity ("}
+{"text": "Comparisons between oestrogen-receptor (RE)-positive or negative patients were made on a continuous series of 735 patients with primary breast tumours seen at the major treatment centre in British Columbia between 1975 and 1978. RE positivity was commoner in older patients, and was not associated with menopausal status independently of age. The concentration of receptor protein also increased with increasing age, but was not affected by menopausal status. Neither RE status nor quantity was associated with any of the epidemiological risk factors studied, which included parity, age at first birth, weight, family history and exposure to oestrogenic drugs and oral contraceptives. Patients with RE- tumours were more likely to present with symptoms other than a breast lump, pain or nipple inversion, and had less-differentiated tumours; they did not differ from RE+ patients in terms of stage, size of tumour, or interval from first symptom. These findings are discussed in terms of the biological origin and determinants of oestrogen receptors."}
+{"text": "The c-erbB-2 gene codes for a putative transmembrane protein, similar in structure to the epidermal growth factor (EGF) receptor. Amplification of the gene has been described in a variety of human adenocarcinomas and is particularly well documented in breast carcinoma. It has been suggested that amplification is indicative of poor prognosis and, as such, is comparable with lymph node status as a predictor of clinical outcome. This study examines the suggestion indirectly by an immunohistochemical technique. Archival tissue from 195 patients with primary breast carcinoma was stained with the polyclonal antibody 21N, raised to amino acids 1243-1255, the C-terminus of the predicted amino acid sequence of the c-erbB-2 protein. Up to 10 year verified follow-up data were available on all patients. Staining compatible with significant amplification was observed in 17 patients. Using the chi-squared test for trend a significant correlation was found between staining and grade (P = 0.04) but not with either node or receptor status. No significant association was found between staining and clinical outcome although there was a tendency for patients with stained tumours to have a worse prognosis. A Cox regression analysis was used to adjust for node status and grade and still no correlation was revealed between staining and prognosis. However a study of this size in which only a small number of patients have been found to have stained tumours does have wide confidence limits. Comparable staining observed in in situ and infiltrating components of tumours suggests that amplification is an early event in carcinogenesis. Similar staining in primary and subsequent metastatic lesions was also noted. It is considered that further studies at both the DNA/mRNA and protein levels are required to confirm the significance of c-erbB-2 amplification in human breast carcinoma."}
+{"text": "The soluble oestrogen-receptor content of common breast tumours has been measured by 5 different laboratories, each using their own assay procedure. Good agreement was achieved on whether a sample was positive or negative for oestrogen receptor. Qualitative differences between laboratories could be explained by differences in thiol-reagent content of assay medium and by the method of homogenization. Recommendations are made on some of the factors involved in the routine assay of receptors in breast tumours."}
+{"text": "The clinical course of breast cancer was related to degree of anaplasia (DA) and steroid receptor (SR) content of primary tumours in 743 patients (pts) with clinical recurrence, initially enrolled in the DBCG-77 protocols. The oestrogen receptor (ER) and the progesterone receptor (PgR) content was known in 110 and 67 pts. The recurrence-free interval, survival after recurrence, and the overall survival were all prolonged in patients with well differentiated tumours or with high SR content. The tumour growth rates were estimated as clinical rates of progression . The progression rate was prolonged in relatively well differentiated as well as in receptor rich tumours. The extent of dissemination, as indicated by the number of metastatic sites, was not associated with either DA or SR content. However, the anatomical distribution of metastases varied with both DA and SR content: signs of poor prognosis (high DA or low SR content) were associated with occurrence of visceral metastases. In contrast, SR rich tumours had a propensity for recurrence in bone. The results suggest that the impact on prognosis of the features examined here includes both variations in growth rate and metastatic pattern."}
+{"text": "A total of 98 breast aspirates from patients with breast cancer have been fixed and stained for oestrogen receptors using the Abbott ERICA kit. In a preliminary series of 41 aspirates, cytochemical staining index (% cells staining x mean intensity) related to the receptor concentration determined biochemically on a subsequent biopsy with a correlation coefficient of +0.65. In a second series of 56 aspirates examined after lysis and cytocentrifugation, the correlation coefficient was +0.73. For 14 patients, the response of the primary tumour to endocrine therapy was assessed objectively by serial clinical and mammographic measurements  and was found to relate strongly to the cytochemical staining of the initial aspirate. The potential and limitations of this technique are discussed."}
+{"text": "Oestrogen (ER) and progestin receptors (PR) were measured in cytosols from histologically normal mammary tissues (n = 30), and in benign (n = 59) and malignant mammary lesions (n = 49) from female dogs. Receptor levels greater than or equal to 5 fmol mg-1 protein were considered positive. The presence of histologically normal mammary epithelium within specimens of primary tumours was noticed as a factor that may cause false-positive receptor results. Receptor levels in non-malignant tissues, and the receptor status of primary cancers did not vary significantly with regard to the phase of oestrous cycle (anoestrus/metoestrus) or the influence of exogenous progestins. ER- or PR-positivity was more frequent and levels of both receptors were higher in 'normal' tissues and in benign lesions than in primary cancers (P less than 0.001). ER and PR levels were higher in benign lesions of dogs also developing malignant mammary tumours than in benign lesions of dogs that did not . Regional and distant cancer metastases were frequently receptor-negative. In some dogs heterogeneity of receptor status was found between different sites of the same cancer. These findings indicate that in non-malignant mammary tissues of adult female dogs expression of the genes encoding ER and PR is common. In malignant tumours this property may become lost, in particular in advanced states of disease."}
+{"text": "The proportion of positive results and the range of concentrations were found to be somewhat higher in postmenopausal than in premenopausal patients. Despite detailed examination, no histological feature was found which could explain the variation in receptor concentration; neither could it be accounted for by differences in the cellularity of the biopsies. Of the 41 benign breast biopsies examined only 3 contained any high-affinity oestradiol receptor and in these the concentration was very low, ranging from 0\u00b73-0\u00b76 \u00d7 10-15 moles/mg. tissue. The receptor has not been detected in normal breast tissue. The relationship between the presence of oestrogen receptors and hormone responsiveness in tumours is discussed.An assay is described for measuring the concentration of specific, high-affinity oestradiol receptors in the cell supernatant fraction of breast tumour biopsies. The method has been applied to biopsies from 94 patients with malignant and benign diseases of the breast. Of the 53 biopsies classified as carcinomas, 37 contained high-affinity oestradiol receptors in concentrations ranging from 0\u00b73-22\u00b76 \u00d7 10"}
+{"text": "Expression of the bcl-2 proto-oncogene was studied immunohistochemically in 251 invasive ductal breast carcinomas  and the results were correlated with clinicopathological data and prognostic variables. Sixty-three (25%) tumours were scored bcl-2 negative and 188 (75%) tumours were bcl-2 positive. No relationship could be observed between bcl-2 status and tumour grade, pTNM staging or menopausal status. A strong positive relationship was demonstrated between bcl-2 immunoreactivity and oestrogen receptor status (P < 0.001) and progesterone receptor status (P < 0.001). No prognostic value was demonstrated for bcl-2 expression on disease-free survival and overall survival in axillary node-negative breast cancer patients. However, in axillary node-positive breast cancer patients multivariate analysis demonstrated absence of bcl-2 expression to be independently related to shortened disease-free survival (P = 0.003) and shortened overall survival (P < 0.001). Our results suggest a potential important role for bcl-2 expression as a modulator of response to adjuvant therapy in breast cancer."}
+{"text": "A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation  with the dye during the stimulation of the nerve . This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles . After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis , process that can be followed by monitoring the fluorescence intensity decrease (destaining).The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy  The Drosophila larvae is pined dorsal side-up in a Sylgard dish; the dorsal side is sectioned longitudinal, and the internal organs are removed. The preparation is then stretched and pined. Several ventral muscles can be then used.It is advisable to do all the following steps in low light conditions, in order to protect FM dyes from bleaching.For chemical stimulation of the nerves, high potassium buffer is used. Prepare Drosophila saline (see 1.1) containing 90 mM of KCl and 10 \u03bcM of FM1-43 dye. Keep the solution protected from light.Bath the preparation with the previously prepared buffer for 1 minute at room temperature. Wash with standard Drosophila saline (1.1) to remove extracellular FM1-43 dye. Proceed rapidly to fixation.Bath the preparation with 2.5% glutaraldehyde in phosphate-buffered saline (PBS) for 45 minutes at room temperature. For good preservation of morphological features, glutaraldehyde is preferred to paraformaldehyde.4Cl. This step is done in order to quench the free aldehyde groups of the remaining glutaraldehyde fixative. Wash once with PBS and then leave the preparation submerged for 15 minutes in 100 mM of NH4Cl solution out with normal PBS.Wash the NHIncubate the NMJ preparation for 30 minutes at 4\u00b0C in PBS containing 1.5 mg ml-1 of diaminobenzidine (DAB). Position the sample under the fluorescent microscope. Find and focus the fluorescent signal using a relatively low magnification water immersion objective (20x 0.5 NA).Illuminate the sample with the maximum lamp intensity until the FM dye is completely bleached . To control if photoconversion has occurred, is advisable to check at the sample under transmission light before and after the FM dye-bleaching step. If photoconversion takes place, a dark brown precipitate is expected . The illumination time is variable, depending upon the illumination strength and also upon the DAB penetration into the preparation. For most preparations, we found illumination times of ~30-45 minutes to be optimal when using a 20x objective. Shorter illumination periods are used for higher magnification objectives (60x).For illumination we prefer to use a mercury lamp, with a lamp housing containing a back mirror, which collects the back-scattered light beams, and therefore increases total intensity.Prepare a solution with one volume of osmium tetroxide in tree volumes of water (approx. 300 \u03bcl per sample). Work using osmium tetroxide must be done under the hood, using gloves and aye protection equipment.Incubate the preparation in the osmium tetroxide solution for 1 hour at room temperature (Under the hood).Wash extensively with PBS (4-5 times 5 minutes) and transfer each samples to a clean glass flask.Osmication Prepare separate solutions containing 30, 50, 70 90, 95 and 100% of ethanol.Add 1 ml of 30% ethanol to each sample for 5 minutes.Add 1 ml of 50% ethanol to each sample for 5 minutes.Add 1 ml of 70% ethanol to each sample for 5 minutes.Add 1 ml of 90% ethanol to each sample for 5 minutes.Add 1 ml of 95% ethanol to each sample for 5 minutes (repeat 3x).Add 1 ml of 100% ethanol to each sample for 5 minutes (repeat 3x).Dehydration Mix 1 volume of Epon resin with one volume of ethanol. Add it to the preparation under continuous rotation (2-4 hours).Place the preparation in 100% Epon in open flasks, allow the remaining ethanol to evaporate (4-6 hours).Place the preparation in moulds at 60\u00b0C for 36 hours.Electron microscopy processing. Process the preparation in 50-80 nm thin sections, using standard ultramicrotomy procedures.Embedding and further processing The preparation is imaged using conventional EM procedures.Electron microscopy imaging The expected results are outlined in Figures 1C and 2. The illumination procedure results in the formation of brown DAB precipitate, which will be visible in both fluorescence and transmission imaging. The first occurrence during illumination is the disappearance of fluorescence associated with the FM dye staining. The background fluorescence induced by the aldehyde fixative, in contrast, will be visible throughout the experiment. The bleaching takes place typically in ~10-20 minutes after the start of illumination. Typically no photoconversion product can be observed at this time point.iv) It is unclear why DAB precipitation and FM dye bleaching do not take place simultaneously; it is possible that a relatively large amount of oxidized DAB needs to accumulate before aggregation and precipitation, and that therefore this reaction is slower than the bleaching process. At this stage, however, the preparation is not ready for electron microscopy processing, as the DAB precipitation is likely incomplete. We prefer to wait 5-10 minutes longer, during which the preparation  assumes a dark-brown (or black) color, indicative of complete conversion. A slight conversion of the general surface of the preparation  can be observed at this stage. It is most likely related to a conversion of DAB induced by autofluorescence (and/or fixative fluorescence), and it is not detrimental to the procedure.Illumination should be continued, and after ~10 minutes the preparations will turn to a brown shade due to DAB accumulation . To increase the chance of distinguishing well the different types of vesicles, no contrast-enhancing post staining of the sections should be performed (no uranyl acetate or lead citrate staining); the osmium staining is sufficient to observe most cellular elements, and is not as dark as DAB precipitate.The preparation is then processed for electron microscopy, and should be checked for the presence of dark (labeled) vesicles. As we have demonstrated before           Figure 1: FM dyes and it use for photoconversion. (A) Represent the steps of a classical experiment for loading and distaining vesicles using FM dye under stimulation. (i) Incubate the sample in FM dye containing buffer. (ii) Stimulate  to induce vesicle fusion in presence of FM dye. (iii) The subsequence endocytosis after stimulation will load some vesicles with FM dye still present in the extracellular buffer. (iv) Replace the extracellular FM dye by washing with buffer. (v) A new stimulation will induce loaded vesicles to release their FM dye upon exocytosis. (B) Schematic showing the head, bridge and tail of the FM1-43 dye. (C) Example of a photoconversion experiment in Drosophila NMJ. (i) After loading the NMJ with FM1-43, washing external dye molecules and fixing, the fluorescence of a nerve terminal can be observed with a conventional epifluorescence microscope (63x). (ii-iii) Under continuous illumination the dye is completely bleached. (iv) When illumination continues, a black color appears due to diaminobenzidine precipitation. Scale bar represents 10 \u03bcm.                    Figure 2: EM examples of photoconverted samples. (A) The image shows a nerve terminal containing synaptic vesicles, but not of them are photoconverted; this might be caused by not having enough illumination or poor diaminobenzidine penetration in the tissue. (B) Synaptic bouton with several dark (filled) vesicles that went through FM photoconversion. (C) Excess of illumination results in an overall dark terminal where no vesicles or organelles are distinguishable. Scale bars represent 200 nm.          A few critical steps should be taken into account:The DAB incubation should be performed only after thorough washing and quenching of the preparations. Otherwise the non-reacted glutaraldehyde will interact with DAB and cause its precipitation . Preparations where this precipitation takes place abundantly are rarely usable for electron microscopy.Illumination times should be optimized, by testing several identical preparations with different illumination times. In our experience, optimal conversion  is achieved in a time window of about 5 minutes . Insufficient illumination  is characterized by a lack of labeled organelles, and/or partially labeled organelles . Long illumination, in contrast, results in over-conversion   the DAB conversion product accumulates, distorts the organelles and escapes into the cytosol . The preparations appear as black structures in electron microscopy, with little observable morphology.Caenorhabditis elegans, Drosophila melanogaster, zebrafish (Danio rerio) and frog (Rana pipiens and Rana esculenta). We have also used the technique in cultured cells, including cultured neurons and neuroendocrine cells. It can also be used successfully in purified synapses from rat brain (synaptosomes). Therefore, we expect the technique to be usable in most preparations which use vesicular membrane uptake.  The technique it is the most sensitive technique to date in determining the positioning and morphology of endocytosed organelles. It was used to determine the exact location of recently endocytosed organelles, including specific physiological pools of vesicles 5,6,7,8. It has also been used in determining the number and morphology of the organelles in the recycling pathway see for example 9,10. We have used the technique in several preparations, including neuromuscular junctions from"}
+{"text": "CNTNAP2) gene for single-nucleotide polymorphisms (SNPs) in Japanese patients with the exfoliation syndrome (XFS).To investigate the contactin-associated protein-like 2 , directly sequenced, and genotyped.=5.43XE-4, OR=1.86, 95% CI; 1.31\u20132.65) were statistically significantly different between XFS and controls. In addition, there were significant differences in these genotype frequencies (p=0.0197 and 1.75XE-3). The allele and the genotype frequencies of rs2107856 and rs2141388, which were statistically significant SNPs in an earlier study, were not significantly different.The allele frequencies of rs1404699 =1.59, 95% confidential intervals (CI); 1,12\u20132.24) and rs7803992 (pCNTNAP2 should be associated with XFS in the Japanese population.The variants, rs1404699 and rs7803992, of  This association was replicated in the United States of America [Thorleifsson et al.  found a  America -13 and a America -23.LOXL1 is a member of the lysyl oxidase family of proteins that catalyzes the oxidative deamination of lysine residues of tropoelastin [LOXL1 plays an important role in elastogenesis. Thus, it is quite possible that defects in LOXL1 can cause features of XFS that result from an aberrant production of elastin and accumulation of fibrillar materials in the anterior segment of the eye.oelastin . The homoelastin , and LOXCNTNAP2) locus had significant associations between XFS and exfoliation glaucoma and two SNPs (rs2107856 and rs2141388). These findings were confirmed in an independent German cohort but not in an Italian cohort [CNTNAP2 is a large gene spanning 2.3\u00a0mb of DNA on chromosome 7 and has 24 exons, and codes for the contactin-associated protein-like 2 . CNTNAP2 is member of the neurexin superfamily [A GWAS was recently performed using a DNA-pooling approach, and a single genotype at the contactin-associated protein-like 2  and 199 controls  were studied. The controls were matched by age and gender. The XFS group included 85 exfoliation glaucoma (XFG) patients. They were examined at the ophthalmic clinic of the Tohoku University Hospital, Sendai, Japan, and the Ehime University Hospital, Ehime, Japan. The purpose and procedures were explained to all patients, and an informed consent was obtained. This study was approved by the Institutional Review Boards of the Tohoku University and Ehime University, and the procedures used conformed to the tenets of the Declaration of Helsinki.Routine ophthalmic examinations were performed on all patients. The criteria used to classify a patient as having XFS was an open anterior chamber angle with accumulation of abnormal fibrillar material in the anterior segment of the eye. In addition, three other criteria for XFG had to be met: 1) applanation intraocular pressure (IOP) >22\u00a0mmHg in each eye; 2) glaucomatous cupping in each eye including a cup-to-disc ratio >0.7; and 3) visual field defects determined by Goldmann perimetry and/or Humphrey field analyzer consistent with the glaucomatous cupping in at least one eye. The control subjects had the following characteristics: 1) IOP less than 22\u00a0mmHg; 2) normal optic discs; and 3) no family history of glaucoma.rs1404699, rs700308, rs4725736, rs2107856, rs2141388, and rs6970064, were chosen from the earlier studies. Two newly identified SNPs, rs826802 and rs7803992, were designed around intron 9 of the gene. The CNTNAP2 gene structure with the location of the 8 SNPs is shown in TM Terminator v3.1 Cycle Sequencing Kit  by an automated DNA sequencer . The allele frequencies, genotypes, and haplotypes of the CNTNAP2 SNPs were determined.Genomic DNA was extracted from the leukocytes of peripheral blood and purified with the Qiagen QIAamp Blood Kit . Genomic DNA was extracted from the leukocytes of the peripheral blood, and the 6 SNPs, CNTNAP2 allele frequency and the phenotype. For each odds ratio, the 95% confidence intervals were calculated. The inferred haplotypes, quantified between all pairs of biallelic loci, were estimated using the SNPAlyze program version 7.0 . Additionally, a permutation test was performed to test the deviations of allelic frequencies of the SNPs and haplotypes. The Hardy\u2013Weinberg equilibrium was analyzed using gene frequencies obtained by simple gene counting and the chi-square test with Yates' correction for comparing observed and expected values.The significance of associations between the phenotype and SNPs were determined by contingency table analysis using chi-square or Fisher's exact test. The odds ratios, approximating to relative risks, were calculated as a measure of the association between the CNTNAP2 SNPs, rs826802, rs1404699, rs7803992, rs700308, rs4725736, rs2107856, rs2141388, and rs6970064, were determined in the XFS patients.The allele frequencies and genotypes of the 8 rs1404699 =1.59, 95% confidential intervals (CI); 1,12\u20132.24) and rs7803992  were statistically significant between the XFS group and the control group , but the allele frequencies were not significantly different (p=0.553 and 0.0524) between the XFS group and the control group. All SNPs adhered to the Hardy\u2013Weinberg expectations (p>0.05).The genotype frequencies of rs1404699 and rs7803992 were estimated  in Japan, instead of G-G in Europeans. We investigated how the variants in LOXL1 gene were related to CNTNAP2. We sorted our subjects for carriers and non-carriers of the risk haplotype T-G (CNTNAP2 SNPs with the LOXL1 non-risk haplotype (A strong correlation between variants in reported , LOXL1 ctype T-G . The numCNTNAP2 were strongly associated with XFS. In an earlier study [rs2107856 and rs2141388 SNPs in CNTNAP2 were confirmed in an independent German cohort but not in the Italian cohort. Although neither the rs2107856 or rs2141388 SNPs was significant in our study, rs1404699 and nearby rs7803992 were statistically significant between the XFS group and the control group. Thus, it is possible that CNTNAP2 could be associated with XFS. Like other susceptible variants of a complex disease, the OR in the earlier study was modest at about 1.4. In our study, the highest OR was 1.86 for rs7803992. This difference can be explained by racial differences and heterogeneities. Because the number of XFG patients was small, it seemed that the statistical power was weak.We compared the findings of Krumbiegel and colleagues  to that er study , the freLOXL1 in XFS has been reported [CNTNAP2 locus based on the presence of the identified Japanese LOXL1 common risk haplotype T-G. We found no significant association to allele T of the rs1404699 and rs7803992 SNPs of CNTNAP2 in carriers of LOXL1 the risk T-G haplotype (CNTNAP2 and LOXL1 in the Japanese. This would then mean that a LOXL1-independent mechanism is involved in CNTNAP2 function.Because a strong association of variants in reported , we compaplotype , and alsLOXL1 gene strongly influence the phenotypic expression of the XFS.In a molecular genetic study, the most promising loci at 18q12.1\u201321.33 and 2q, 17p, and 19q have been proposed to be the susceptible loci in a Finnish population in an autosomal dominant mode of inheritance . In a mirs1404699 and rs7803992 SNPs are located in intron 9 of the CNTNAP2 gene. Exon 9, nearby to intron 9, codes for the laminin globular domain, which contains proteins that play a wide variety of roles in cell adhesion, signaling, migration, assembly, and differentiation of cells. We suggest that alterations in membrane stabilization may contribute to the abnormal exfoliation matrix processes, which are associated with cell-surface irregularities, basement membrane destruction and degenerative alterations.CNTNAP2 is a single-pass transmembrane protein with multiple protein-interaction motifs typical of the neurexins, e.g., epidermal growth factor repeats, laminin globular domains, and F5/8-type C domain, and a putative PDZ-binding site. Poliak et al.  reportedCNTNAP2rs1404699 and rs7803992 are significantly associated with XFS in the Japanese population. More studies of the functions and genotype-phenotype correlation of CNTNAP2 are required to determine the pathophysiology of XFS. In addition, further studies searching for secondary genetic and environmental factors that contribute to XFS is required to gain better understanding of the complex etiology of XFS.Identification of XFS-associated SNPs that will allow early detection of an increase in the IOP, or even before an elevation of IOP, would be desirable. Our findings showed that variants of"}
+{"text": "Background. Data is limited on the burden of common comorbidities, such as cardiovascular disease (CVD), respiratory disease and diabetes, or comorbidities related to cancer and its treatment, such as anemia and depression, in patients with soft tissue sarcoma (STS). Patients and Methods. From the Dutch Pathology Registry linked to the PHARMO database , 533 patients with STS were selected during 2000\u20132007 and matched 1\u2009:\u200910 to cancer-free controls. The occurrences of comorbidities were assessed in the 12 months before and after STS diagnosis. Results. STS patients were 2\u20134 times more likely to have comorbidities at diagnosis compared with cancer-free controls. The incidence of CVD, anemia, and depression after STS diagnosis differed significantly from cancer-free controls and decreased during followup from 40\u2013124 per 1,000 person-years (py) during the first six months to 11\u201338 per 1,000\u2009py more than 12 months after diagnosis. The incidence of respiratory disease and diabetes among STS patients remained stable during followup  and did not differ significantly from cancer-free controls. Conclusions. STS patients were more likely to have comorbidities before cancer diagnosis and to develop CVD, anemia, and depression after diagnosis compared to cancer-free controls. Soft tissue sarcomas (STSs) are a rare and heterogeneous group of tumors arising from mesenchymal tissue . They acLittle epidemiologic data on STS is available due to large variations in tumor biology, histology, location, stage at presentation, and the relative rarity of these tumors . The devPrevious studies using administrative healthcare databases showed that cardiovascular disease (CVD), respiratory disease, and diabetes are the most common concomitant illnesses in cancer patients , 15. CurThis retrospective cohort study describes the prevalence and incidence of comorbidities including cardiovascular disease, respiratory disease, diabetes, anemia, and depression among a population-based cohort of STS patients. These rates were compared to those observed in a matched noncancer control population. In addition, risk factors for developing these morbidities after STS diagnosis were determined.Data from patients with a STS pathology specimen were obtained from PALGA, the Dutch Nationwide Network and Registry of Histo- and Cytopathology. PALGA contains data of all histological, cytological, and autopsy examinations in The Netherlands . CurrentKIT, with the protein tyrosine kinase inhibitor imatinib and sunitinib as specific treatments for this tumor [Patients with a record of a STS pathology specimen in the period January 1, 2000 to December 31, 2007 were selected from the PALGA database. We excluded Kaposi's sarcomas . Date oThe selection of the control cohort was stepwise. First, each STS patient was matched 1\u2009:\u200920 by age and gender with a patient from the PHARMO RLS. Each of these potential controls was assigned the cohort entry date of the STS patient case match. Potential cases could not be selected as controls. Second, we eliminated from the pool of 20 possible controls, any control that did not have 12 months of history before cohort entry date in the PHARMO RLS and/or who had a hospitalization with a primary diagnosis for any cancer (ICD-9-CM codes: 140\u2013239) in the period of ten years before the cohort entry date until December 31, 2008. Finally, out of the remaining controls that fulfilled the above criteria, ten were randomly selected and included in the control cohort.We determined gender, age, and duration of followup of the patients included in our study cohort. Topography of the tumor was obtained from PALGA and classified as follows: extremities, head and neck, thorax, abdomen or (retro)peritoneum, and overlapping sites or sites not otherwise specified. Histologic type of STS was also obtained from PALGA and determined by using morphology ICD-O-3 codes  \u201326. In aComorbidities were defined via hospital admissions (ICD-9-CM codes) and/or drug dispensings (ATC codes) captured in the PHARMO RLS. Patients were considered to have a comorbidity if they either had been hospitalized for the condition or had been dispensed a medication used to treat the condition twice in a 365-day period. For the calculation of incidence rates, the event date of the first of each comorbidity was defined as the date of the hospitalization or the date of the first dispensing, whichever came first. The following comorbidities were considered: cardiovascular disease, including heart disease, thrombosis, and hypertension , respiratory disease, including asthma, and chronic obstructive pulmonary disease , diabetes (ICD-9-CM code: 250 or ATC code: A10), anemia , or depression . Comorbidities were assessed in 12 months, of history and during followup .Prevalence proportions in the 12 months before cohort entry were assessed for each comorbidity and expressed with accompanying 95% confidence intervals (CI). Conditional logistic regression was used to compare the prevalence of the comorbidities between the STS patients and the cancer-free control group. For incidence calculations, patients with a prevalent comorbidity  were removed from the at-risk population for that specific event. Incidence rates per 1,000 person-years with accompanying 95% CI based on Byar's approximation  were calN = 173, 32%). The most common known histological tumor type was leiomyosarcoma (23% (84/360)), followed by liposarcoma (21% (77/360)). Treatment in the first six months after STS diagnosis was mainly surgery (82%). Chemotherapy was administered to 10% of the patients included in our study cohort.A total of 533 patients with a STS resection specimen between 1 January 2000 and 31 December 2007 were selected from the PALGA database and linked to the PHARMO RLS . Mean  at time of diagnosis, and 10% had evidence of respiratory disease. Diabetes, anemia, and depression occurred in 6-7% of the patients at time of diagnosis. In a previously performed study  in The NIn the current study, STS patients were twice as likely to have any of the comorbidities prior to diagnosis compared with the cancer-free control group. This can likely be attributed to several risk factors that are both related to the development of the comorbidity and STS such as immunodeficiency, environmental factors, lifestyle factors, and genetic factors , 9.Cardiovascular diseases, anemia, and depression were common incident comorbidities, especially in the first six months after STS diagnosis when rates were 124, 85, and 40 per 1,000 py, respectively, and 3\u20137 times higher than among cancer-free controls. These incidence rates decreased during followup, which suggests that cardiovascular diseases, anemia and depression are comorbidities related to the cancer itself or its treatment. This was investigated in our risk factor analysis. We found few significant independent risk factors, which might be explained by the relatively small patient cohort as STS is a rare tumor. We did find that male gender and STS located in the abdomen or (retro)peritoneum significantly increased the risk of developing cardiovascular disease after correction for other risk factors. A possible explanation for this finding is that abdominal or (retro)peritoneal STS tumors are mostly larger and of higher stage at presentation than those located elsewhere . This maIn addition to risk factors for cardiovascular disease, a clear association was demonstrated between chemotherapy in the first six months after STS diagnosis and the risk of anemia during followup. It is well recognized that anemia might arise during or shortly after myelotoxic chemotherapy , 30. NexNext to missing data on radiotherapy, another study limitation concerns the way the comorbidities were captured. In this study we did not use data from general practitioners or outpatient medical visits. The presence of comorbidities was based on hospitalizations and/or drug dispensings and consequently comorbid cases with a mild course were not included. Furthermore, identifying only the medically treated depression, limits the ability to directly compare these results with studies using structured clinical interviews or screening questionnaires to define depressive disorders. Regarding cardiovascular comorbidity, the definition used is very broad. However, low numbers of the patients included in this study did not allow sub classification of the results. Finally, as STSs are a heterogeneous group of tumors and there is a considerable variation in incidence patterns of sarcomas by histological type and anatomic site , future This study quantifies the significant burden of prevalent comorbidity in patients diagnosed with STS and presents the incidence of comorbidity after STS diagnosis and treatment. Prevalent comorbidity at STS diagnosis implies that more personalized supportive care is required for these patients. Incident comorbidity after diagnosis and treatment provides insight into the pharmacovigilance of STS treatment which can be used to improve the outcome of these patients."}
+{"text": "CXCR4, CXCR7), matrix remodeling (COL4A3), angiogenesis and tumor growth . EV-stimulated MSCs exhibited in vitro an enhancement of migration toward the tumor conditioned medium. Moreover, EV-stimulated MSCs enhanced migration of renal tumor cells and induced vessel-like formation. In vivo, EV-stimulated MSCs supported tumor development and vascularization, when co-injected with renal tumor cells. In conclusion, CSC-derived EVs induced phenotypical changes in MSCs that are associated with tumor growth.Renal carcinomas have been shown to contain a population of cancer stem cells (CSCs) that present self-renewing capacity and support tumor growth and metastasis. CSCs were shown to secrete large amount of extracellular vesicles (EVs) that can transfer several molecules  and induce epigenetic changes in target cells. Mesenchymal Stromal Cells (MSCs) are susceptible to tumor signalling and can be recruited to tumor regions. The precise role of MSCs in tumor development is still under debate since both pro- and anti-tumorigenic effects have been reported. In this study we analysed the participation of renal CSC-derived EVs in the interaction between tumor and MSCs. We found that CSC-derived EVs promoted persistent phenotypical changes in MSCs characterized by an increased expression of genes associated with cell migration ( The tumor is composed by different cell populations. A subset of tumor cells, defined as cancer stem cells (CSCs), are characterized by self-renewal and continuous proliferation capacity, providing the ability to initiate tumor and to generate other heterogeneous cell populations . Tumor set al. showed that bone marrow progenitor cells can be \u201ceducated\u201d by exosomes secreted by melanoma to increase tumor metastasis [Bone marrow-derived mesenchymal stromal cells (MSCs) are known by their migratory capacity to injury and tumor sites. These cells are susceptible to signaling molecules secreted by the tumor and tumor-related stromal cells, promoting their recruitment from bone marrow into the circulation and subsequent engraftment within tumor microenvironment . The roltastasis .Our group previously demonstrated the presence of a CSC population in renal carcinoma that releases EVs that favor tumor growth by increasing vascularization and development of a pre-metastatic niche , 24. In CSC-EV uptake by MSCs was assessed by maintaining cells incubated with stained CSC-EVs for different periods of time. As shown in Fig. When unstimulated MSCs were plated in the upper chamber of transwell, no direct chemoattraction was observed by CSC-EVs (EVs) Fig.  or CSC cMMP1, MMP2 and MMP3), collagens (COL3A1 and COL4A3) and CXCR4 and CXCR7. The analyses were performed in MSC stimulated with CSC-EVs for 72 hours or 2 weeks  when compared with unstimulated control MSCs (white bars). After 2 week-stimulation, a significant increase of MMP1, MMP3, CXCR4, MMP2, COL4A3 and CXCR7 was observed .To evaluate whether MSCs stimulated for 2 weeks with CSC-EVs promoted angiogenesis, MSCs were plated in the upper transwell chamber and human umbilical vein endothelial cells (HUVEC) in the lower. As shown in Fig. To evaluate whether MSCs stimulated for 2 weeks with CSC-EVs promoted renal carcinoma (K1) cell migration, MSCs were plated in the lower transwell chamber and K1 cells in the upper. CSC-EV-stimulated MSCs significantly increased the migration of tumor cells in respect to control unstimulated MSCs (TUM + MSC STI) Fig. .The specificity of MSC changes promoted by CSC-EVs were evaluated by comparing the effects of EVs derived from renal carcinoma K1 cells and non-tumorigenic cells renal proximal tubular epithelial cells (PTEC) on MSC using a 2 week-stimulus protocol. MSCs stimulated by PTEC-EVs did not induce angiogenesis (HUVEC+MSC STI PTEC-EVs) or tumor migration (TUM+MSC STI PTEC-EVs) Fig. . EVs derTo evaluate which EV fraction induced MSC angiogenic and migratory properties, we isolated by differential ultracentrifugations, 10K and 100K EV fractions from CSC-EVs. When the two fractions were compared, we found that the MSCs stimulated with 10K fraction was more effective in inducing angiogenesis (HUVEC+MSC STI 10K CSC-EVs), whereas MSCs stimulated with 100K fraction mostly stimulated tumor migration (TUM+MSC STI 100K CSC-EVs) Fig. .Cytokine array analysis was performed in cell free supernatants of unstimulated and 2 week-stimulated MSCs to observe variation in the secreted molecules Fig. . From thThe size of tumors formed by subcutaneous injection in SCID mice of K1 cells within Matrigel was significantly increased in the presence of 2 week CSC-EV-stimulated MSCs (TUM MSC STI) in respect to tumor alone (TUM) or tumor co-injected with unstimulated MSCs (TUM MSC CTR) Fig. . Such stPCNA staining by immunohistochemistry showed a higher proliferative rate in tumors co-injected with CSC-EV-stimulated MSCs (4.7 fold increase) than in tumor containing unstimulated MSCs Fig. . In addiTumor development has been associated with the interactions between tumor cells and the surrounding stromal cells. This interaction has been, at least in part, ascribed to EV secretion -9. MSCs CXCR4 expression, which was increased after CSC-EV stimulation, is known to be involved in MSC migration through a SDF-1 concentration gradient secreted by tumor cells [CXCR7 expression, a SDF-1 receptor associated with survival and paracrine actions of MSCs [To reach and engraft the tumor, MSCs must be capable to sense and respond to tumor secretory stimuli , 26. Wheor cells , 28. Sti of MSCs , 30. In  of MSCs , 32.MMP1, 2 and 3 expression observed after stimulation with CSC-EVs suggested the capacity of MSCs to modulate matrix remodeling within tumor microenvironment. MMPs are proteolytic enzymes that are increased in the majority of human cancers and are associated with invasion, metastasis processes and with several other steps of tumor development like growth and angiogenesis [COL4A3 gene. This gene has been involved in regulation of cell adhesion, migration and metastasis in different tumor types [MMP1, MMP2, MMP3, CXCR4/7, and COL4A3) was maintained for at least 2 weeks after CSC-EV stimulation, suggesting persistent epigenetic alterations. Furthermore, one of the characteristics of MSCs stimulated with CSC-EVs, but not with EVs released from a total tumor cell population or from non-tumor cells, was the capacity to attract tumor cells and to promote angiogenesis, suggesting a role of MSCs in tumor spread. This observation suggests that EVs released from CSC rather than total tumor cell population play a central role in MSC pro-tumorigenic changes.The enhanced ogenesis . The paror types -36. The + that promotes a S-nitrosylation of caspase-3, inhibiting its activity [Communication between CSCs and MSCs was not unidirectional. In fact, MSCs, after CSC-EVs incorporation, changed their secretory profile increasing IL-8, osteopontin (OPN) and myeloperoxidase (MPO) production. MPO is known to be involved in oxidative stress response in tumors . MPO hasactivity . This alactivity . IL-8 caactivity . In addiactivity , 42. OPNactivity . The rolactivity . OPN alsactivity .in vivo tumor growth by increasing proliferation and vascularization. In contrast, unstimulated MSCs were unable to promote tumor growth suggesting the need of MSC pre-conditioning by tumor microenvironment. In fact, several studies reported the role tumor-EVs in the communication with surrounding cells creating an environment favorable to tumor growth [In our experimental condition, we observed that MSCs stimulated by CSC-EVs were capable to support r growth -10. Exper growth . CSCs mar growth -17, MSCsr growth , 47.In conclusion, the results of the present study suggest the importance of EVs in tumor-MSC interaction. In particular, CSCs can alter MSC phenotype through secretion of EVs. Altered MSCs became more responsive to tumor chemoattractive stimuli and supportive to tumor cell migration and proliferation, and may favor tumor vascularization. The phenotypic changes were maintained even after removal of stimulation, suggesting a persistent change in MSC phenotype.+) clones previously isolated and characterized were used for the experiments. The CD105+ cells were isolated from human renal carcinomas and presented clonogenic ability, stem cells markers, ability to differentiate and can be cultured in suspension. In addition, CSC clones (1\u00d7105 cells) were able to generate heterogenic tumors [K1 cell lines were previously isolated from renal carcinoma cells and characterized. These cells are tumorigenic cells positive for CD40 and CD154 markers . K1 cellc tumors . CSCs wec tumors .in vitro was performed with HUVECs that were isolated and characterized as previously described [Angiogenesis escribed .Human MSCs were obtained from Lonza , cultured in the Mesenchymal Stem Cells Basal Medium  and characterized as previously described . All theo C. In selected experiments CSC-EVs were separated in two fractions by a centrifugation at 10,000 g (10K) for 1 hour at 4o C and followed by ultracentrifugation at 100,000 g (100K) for 1 hour at 4o C of supernatants deprived of the 10K fraction. Vesicles were studied as previously reported [\u00ae Dil (Molecular Probes) [4 vesicles/stimulated cell for different periods of time . MSCs were stained in green with Syto\u00ae RNA select (Molecular Probes). The incorporation of CSC-EVs by MSCs was analyzed by confocal microscopy.The CSCs, K1 and PTEC were cultured overnight in RPMI medium without phenol red (Life Technologies). The supernatants were centrifuged at 300 g for 20 min to remove debris and subsequently centrifuged at 100,000 g (Beckman Coulter Optima L-90K ultracentrifuge) for 1 hour at 4reported  by cytof Probes)  to evalu4 vesicles/stimulated cell) for 72 hours (short stimulation); or MSCs were repeatedly stimulated every 5 days with CSC-EVs (5\u00d7104 vesicles/stimulated cell) for 2 weeks (long stimulation), performing a total of 3 stimuli.Functional experiments were performed under two protocols of stimulation: MSCs were incubated with CSC-EVs (5\u00d710The migration assay was performed using a transwell system (Becton Dickinson) with 8 \u03bcm pore size. To observe the changes in the migratory capacity, unstimulated or CSC-EV-stimulated MSCs were plated in the upper compartment of a transwell. Based on experimental conditions, the inferior compartment was fulfilled with CSC-EVs (to determine vesicles chemoattraction) or EV-deprived CSC conditioned medium  or medium alone. After 24 hours incubation, non-migrated cells were removed from the membrane upper surface with cotton swabs. Migrated cells were then fixed with absolute methanol and stained with 0.2% crystal violet solution. MSC migration rate was determined by counting the number of cells contained in the photos of 5 different fields at original magnification \u00d7 10.4 cells) were co-cultured for 24 hours with unstimulated or with CSC-EV-stimulated MSCs (ratio 1:1). The analyses of tumor migration were performed as previously described [To analyze the capacity of stimulated MSCs to induce tumor migration, K1 cells (2\u00d710escribed .4 cells) were plated into 24-well plates coated with growth-factor depleted Matrigel\u00ae (Corning) and co-cultured with unstimulated or 2 week stimulated MSCs (2\u00d7104 cells) in a transwell system of 0.4 \u03bcm pore size (Becton Dickinson) for 24 h. The MSC angiogenic potential was determined by the number of vascular-like structures formed by endothelial cells.HUVECs (2\u00d710\u00ae Reagent (Life Technologies) from unstimulated, 72 h-stimulated, 2 week-stimulated MSCs or 2 week-stimulated MSCs maintained after washing for 2 additional weeks in culture without CSC-EVs. The obtained RNA was quantified by spectrophotometry (Nanodrop ND-1000) and 200 ng total RNA of each sample condition was reversely transcribed to cDNA using High Capacity cDNA Reversion Transcription kit (Life Technologies). The mRNA expression in MSCs was assessed by qRT-PCR using Power SYBR\u00ae Green PCR Master Mix (Life Technologies), performed in a 96-well StepOne Real-Time System (Applied Biosystems). The sequence-specific oligonucleotide primers were all obtained from MWGBiotech AG, Ebersberg, Germany (www.mwg-biotech.com). The relative gene expression was measured using GAPDH as housekeeping gene. List of primers used is reported in RNA was isolated with TrizolTo identify changes in the pattern of molecules secreted by MSCs, the supernatants of unstimulated and 2 week-stimulated MSCs cultured in RPMI overnight were collected and centrifuged to remove cellular debris. Experiments were then performed using a Human XL Cytokine Array Kit (Proteome Profiler\u2122 Array - R&D Systems) according to the manufactory protocol. Quantification was performed by ChemiDoc\u2122 XRS+ System and data analyses were performed using Quantity One\u00ae Analysis Software (Bio-Rad).in vivo effects on tumor growth of MSCs stimulated with CSC-EVs for 2 weeks, SCID mice were divided in 3 groups (n=6 for each group): Group 1 was subcutaneously injected with renal carcinoma (K1) cells (1\u00d7106 cells); Group 2 was subcutaneously injected with K1 cells (1\u00d7106 cells) mixed with unstimulated MSCs (0.5\u00d7106 cells); Group 3 was subcutaneously injected with K1 cells (1\u00d7106 cells) mixed with MSCs (0.5\u00d7106 cells) stimulated for 2 weeks with CSC-EVs. Prior to the injection harvested cells were counted and mixed in 250 \u03bcL of cold DMEM. The cells were then added to 250 \u03bcL of growth-factor depleted Matrigel\u00ae at 4\u00b0C and subcutaneously injected using a 1-ml syringe with 26-gauge needle. Tumor volume was measured with a caliper and calculated using the formula: v= L \u00d7 l2 \u00d7 0.5 where \u201cL\u201d indicates the large diameter and \u201cl\u201d indicates the small diameter as previously described [\u00ae goat anti-mouse peroxidase conjugate secondary antibody . The substrate 3,3\u2032 \u2013diaminobenzidine (DakoCytomatation) was used for color development. The sections were then counterstained with hematoxylin. PCNA positive cells were counted in 10 random fields for each section at \u00d7200 magnification. Tumor vascularization was measured by counting the number of erythrocyte-containing vessels in 10 random fields of trichrome stained tumor sections at original magnification \u00d7 20 per each sample.All animal experiments have the approval of the Ethics Committee of Turin University. To evaluate the escribed . After 4Statistical analyses were performed using t-test or ANOVA followed by the Newman-Keuls multicomparison test when appropriate. Statistical significance was set at P < 0.05. Data were analyzed using the GraphPad Prism 5.0 Demo program."}
+{"text": "At present, the percutaneous nephrolithotripsy (PCNL) is performed both in supine and in prone position. The aim of this paper is to describe an innovative position during PCNL.  We describe a supine position. The patient's legs are slightly abducted at the hips. The thorax is laterally tilted (inclination 30\u00b0\u201335\u00b0) and kept in the right position by one or two gel pads placed between the scapula and the vertebrae. External genitalia can be accessed at any time, so that it is always possible to use flexible instruments in the upper urinary tract. We used this position for a period of 12 months to treat with PCNL 45 patients with renal lithiasis.  All the procedures were successfully completed without complications, using the position we are describing. The following are some of its benefits: an easier positioning of the patient; a better exposure of the flank for an easier access to the posterior renal calyces of the kidney; a lower risk of pressure injuries compared to positions foreseeing the use of knee crutches; the possibility of combined procedures (ECIRS) through the use of flexible instruments; and a good fluoroscopic visualization of the kidney not overlapped by the vertebrae.  This position is effective, safe, easy, and quick to prepare and allows for combined anterograde/retrograde operations. Percutaneous access to the kidney was first used in 1954 when radiologists dared to puncture the pelvis of hydronephrotic kidneys to perform anterograde pyelography .in vivo experience with 557 patients [The traditional prone position has in fact been developed by radiologists, who used to puncture the pelvis directly, and not through the renal parenchyma. In 1987-88, Valdivia et al.  reportedpatients . The supThe advantages of percutaneous nephrolithotripsy (PCNL) performed in the supine position are both of an anesthesiological and urological kind. The surgeon does not experience the cardiovascular , ventilaAuthors have suggested many different versions of the supine and prone positions to optimize the technique: the lateral , lateralIn our department, we perform an average of 79 percutaneous nephrolithotripsy per year. The need for an easy and quick-to-prepare position, which could at the same time allow for a safe and effective execution of the procedure, led us to develop the supine double-S (Slightly tilted thorax-Slightly split legs) position, which will be described in the following lines.This is a preliminary study, carried out by the Department of Urology at the University of Cagliari  with the purpose to first describe the innovative position we developed.The objective of the study was explained to all enrolled patients. Informed consents to participate and use personal data and images were signed by all enrolled patients.For every case study, we recorded the patient's BMI, the dimension and the position of the stones, the operating time, and the complications occurred (using the Clavien-Dindo classification).The patient is placed on the operating table with the glutei at the lower end of the bed and the external genitalia exposed for an easy access.It is important not to place the patient too close to the metallic side of the table, which is radiopaque and may project on the fluoroscopic field, obscuring the images. The patient's legs are secured independently and slightly abducted at the hips without being flexed . This poThe renal puncture and the remaining phases of the surgery do not differ from the traditional procedure in the Valdivia position .Between March 1, 2016, and March 1, 2017, the double-S position was used to treat 45 patients.Patients presented an average body mass index (BMI) of 27.4.In the CT (computed tomography) study, the mean stone size was 2.2\u2009cm. The stones were positioned in the renal pelvis (48.8%), in the inferior calyx (22.2%), and in the medium calyx (8.8%). No staghorn stones were documented. In 9 cases, we treated multicalyceal stones (20%). In these 9 cases, we preferred not to perform a secondary access but a combined anterograde-retrograde procedure using a flexible ureteroscope. When possible, the stone was relocated in a place where lithotripsy could be performed through the percutaneous access; when this maneuver was not feasible, the stone was shattered using laser energy.Patients were treated with an antibiotic prophylaxis before any procedure.The effectiveness of all the operations was evaluated on the basis of the stone-free status after one month from the procedure, ascertained with CT abdomen examination without medical contrast medium (stone-free status\u2009<\u20093\u2009mm).All the procedures performed in the described position were successfully completed. The mean operating time was 43.5 minutes.At the end of each operation, a flexible pyelocalyceal-scopy and an anterograde pyelogram were performed, and in all cases the patients resulted 100% stone-free. With a CT abdomen without medical contrast medium after one month, we had the confirmation of the stone-free status for all operations.There were neither high (III\u2013V) nor low level (I-II) complications according to Clavien-Dindo.A smaller number of nurses in the operating room, who have an easier position to prepare and lighter loads to shift.A better exposure of the flank and an increased distance between the last rib and the iliac crest (as reported by Desoky), providing a wider space for puncture, dilatation, and maneuverability of the nephroscope. This is feasible thanks to the fact that a single support is placed under the shoulder, and not under the lumbar region as in the Valdivia position .A lower risk of pressure injuries to vascular and nervous structures compared to positions foreseeing the use of knee crutches .The absence of flank support, which prevents the cephalad sliding of the kidney, making upper-pole puncture more feasible.A lower degree of thorax rotation, allowing for a better fluoroscopic view of the kidney, which is not overlapped by the vertebrae, as it may happen in semi-supine (more tilted) positions.The possibility to always have a retrograde access to the high urinary tract.Besides the well-known anesthesiological advantages of the supine position, the double-S position presents other benefits. It indeed combines all the advantages of the several variations of the supine position already described in the scientific literature \u201317:A smaSomeone could state that the lack of space between the legs, as they are only slightly abducted, could represent an issue for the assistant carrying out the retrograde intrarenal procedure. We, however, did not experience such a drawback. For every retrograde procedure, we chose the use of a ureteral sheath that allowed for an easy access and the possibility, when necessary, of multiple entries and exits. Sometimes, with female patients, we had to ask for the assistance of the scrub nurse to find the urethral meatus. Even in those cases, however, after the positioning of the sheath, the access was always easy.Arab Journal of Urology in 2012 [A similar supine position has been reported by Desoky et al. and published in the  in 2012 . Desoky'Another similar position is the Barts \u201cflank-free\u201d modified supine one , which dThe supine double-S position we describe turned out to be efficient, safe, easy, and quick to prepare. It allows for an easy access to the upper urinary tract and therefore for the performance of combined anterograde/retrograde operations. In our clinical practice, it has become the standard position for PCNL procedures in a supine position. In the age of flexible endoscopy, we believe that this position could be a new valid option and we recommend it both to those with a good experience in percutaneous kidney operations as well as to those who are approaching this technique."}
+{"text": "Single and multi\u2010component hydrogels of l\u2010phenylalanine are used herein as model materials to develop an NMR\u2010based analytical approach to gain insight into the mechanisms of supramolecular gelation. Structure and composition of the gel fibres were probed using PXRD, solid\u2010state NMR experiments and microscopic techniques. Solution\u2010state NMR studies probed the properties of free gelator molecules in an equilibrium with bound molecules. The dynamics of exchange at the gel/solution interfaces was investigated further using high\u2010resolution magic angle spinning (HR\u2010MAS) and saturation transfer difference (STD) NMR experiments. This approach allowed the identification of which additive molecules contributed in modifying the material properties.Supramolecular hydrogels are composed of self\u2010assembled solid networks that restrict the flow of water.  Smart Materials.Hydrogels are semi\u2010solid colloidal materials which contain a three\u2010dimensional 3D) network with a fine structure that can often be defined by non\u2010covalent interactions.D networkLow molecular weight gelators (LMWG), with a molecular mass normally under 1000\u2005Da, are molecules capable of self\u2010assembling into organised gel networks.l\u2010phenylalanine (Phe) is the smallest molecule known to form supramolecular hydrogels.To date, Despite the wide range of existing and potential applications of hydrogels,m. Throughout the gel forming concentrations, 303\u2005mm Phe represented the rheologically strongest material.Solutions of Phe have shown thermoreversible formation of hydrogels that sustain their own weight under inversion in the range of concentrations from 212 to 605\u2005ml\u2010leucine (Leu), l\u2010serine (Ser), l\u2010tryptophan (Trp) and l\u2010tyrosine (Tyr) formed clear solutions when heated. When subjected to the same cooling conditions, all formed crystalline precipitates in the range of 5 to 500\u2005mm. Suspensions of Phe mixed with these non\u2010gelating additives  were heated and quenched, resulting in white opaque hydrogels obtained from the mixtures Phe/Leu (5:1), Phe/Ser (5:1), Phe/Trp (5:1) and Phe/Tyr (5:1). The pH values for the pure sample and mixtures were not significantly different (Table\u2005S1 in Supporting Information).The hydrogelation ability of a range of structurally diverse amino acids with different hydrophilicities Figure\u2005 was alsoTgel). Tgel values for hydrogels of Phe were not affected upon the introduction of these additive molecules. These temperatures were in the range of 322 to 327\u2005K for all the hydrogels (Table\u2005S2 in Supporting Information).The temperature above which supramolecular arrangements were broken  was defined as the gel\u2010to\u2010solution transition , can range from 0\u00b0, for ideal solids, to 90\u00b0, for Newtonian liquids.G\u2032) reflects the elasticity of a material, while the loss modulus (G\u2032\u2032) shows the amount of energy lost from the system by non\u2010elastic behaviour.G\u2032 values for the majority of these hydrogels were in the order of 105\u2005Pa (G\u2032\u22482.0\u00d7105\u2005Pa) and were typically two to five times greater than the G\u2032\u2032 values (G\u2032\u2032\u22484.5\u00d7104\u2005Pa) , indicating that the presence of Tyr or Leu did not modify significantly the response of the hydrogel fibres to stress. However, significant differences between these systems and the hydrogels of Phe/Trp (G\u2032=3.5\u00d7105\u2005Pa) and Phe/Ser (G\u2032=7.5\u00d7104\u2005Pa) were identified. The addition of Trp increased the storage modulus, indicative of a rheologically stronger network. In contrast to Trp, the addition of Ser severely decreased the resistance of the hydrogel to deformation.Similar \u03b8, assigned to water molecules.The crystalline nature of the hydrogel fibres of Phe was revealed by the presence of diffraction peaks in PXRD patterns of single and multi\u2010component hydrogels Figure\u2005. All patm.\u22121 at 298\u2005K),Hydrogels of Phe can self\u2010organise into the monohydrate phase of Phe in the range of 212 to 303\u2005m1H\u201013C cross\u2010polarisation magic angle spinning (CP/MAS) solid\u2010state NMR experiments of hydrogels provided information on the rigid components, which are characterised by strong heteronuclear dipolar couplings that efficiently transfer magnetisation between 1H and 13C spins.1H\u201013C CP/MAS NMR spectrum of the hydrogel of Phe displayed peak splitting for each carbon site, due to the presence of two magnetically inequivalent molecules in the asymmetric unit of the monohydrate phase of Phe  NMR spectra can be used to probe semi\u2010solid components at the interfaces between the hydrogel fibres and the pools of water. According to Iqbal et\u2005al., the application of a diffusion filter in pulsed\u2010field gradient (PFG) experiments eliminates the contribution of fast moving molecules, hence enabling us to filter out free gelator molecules from D) were determined .The dynamic behaviour of valine\u2010based organogels has been described by Escuder et\u2005al. The authors discussed various equilibria between free gelator molecules and oligomeric aggregates in solution, in the light of different time scales of several NMR experiments.d Figure\u2005. These DD values for Leu and Trp were in the same range as Phe (D\u22487\u00d710\u221210\u2005m2\u2009s\u22121). On the contrary, a higher apparent self\u2010diffusion coefficient was determined for Ser, in the hydrogels of Phe/Ser. This indicated that Ser exists mainly as a free mobile molecule in the pools of solvent. Tyr presented poor signal\u2010to\u2010noise (due to significant overlap with Phe aromatic peaks and poor water solubility of Tyr), preventing accurate determination of its apparent self\u2010diffusion coefficient.The values of apparent self\u2010diffusion coefficients for water and Phe were very similar for all hydrogels Figure\u2005 and are 1H solution\u2010state NMR spectra at room temperature immediately after cooling down a hot solution of Phe. Very sharp and intense peaks of Phe were recorded three minutes after quenching . 24\u2005h ageing of these hydrogels led to reduced peak intensities for Trp and Tyr. However, no significant peak variations were identified for either Leu or Ser . This is characteristic of large molecules which transfer magnetisation efficiently through dipolar interactions.The intermolecular interactions responsible for formation of the hydrogel network were probed using nuclear Overhauser effect (nOe) spectroscopy (NOESY). Negative nOe enhancements were detected in 1H solution\u2010state NMR longitudinal relaxation times (T1) measured during the gel\u2010to\u2010solution transitions of single and multi\u2010component hydrogels , which correspond to averaged values resultant from exchange phenomena of the gelator molecules between solution and gel states. Bouguet\u2010Bonnet et\u2005al. reported a similar observation for single component organogels of a derivative of phenylalanine and naphthalimide.T1 times typical of Phe solutions was observed, reflecting the disassembly of the network and the presence of more Phe molecules in the solution state. The process of the molecular gel\u2010to\u2010solution transition, Tgel*,Tgel (here at 313\u2005K). As temperature was raised further, gradual dissolution of the fibres led to progressive destruction of the hydrogel, which finally resulted in a loss of structural integrity (reflected by Tgel). This temperature marked the beginning of stage III, after which the distribution of T1 times increased further and resulted in an overall increase of T1 times for Phe Arom and Phe C\u03b1H . Sharpening of the peaks was also consistent with faster molecular motions occurring at higher temperatures. However, 1H T1 times for Phe C\u03b2H2 followed a different trend, with rapid decay throughout the range of temperatures and a minimum at 326\u2005K (T1C\u03b2H2=1.76\u2005s). This minimum possibly reflected the point after which 1H T1 values corresponded mainly to fast tumbling molecules dissolved in solution.Further insight into gelation mechanisms and molecular motional states was obtained by  Figures\u2005 and S\u2009101H T1 times of Phe determined at 298\u2005K increased according to the following trend: Phe/Trp < Phe/Tyr \u2264 Phe \u2264 Phe/Leu < Phe/Ser (Table\u2005S8 in Supporting Information). The strongest hydrogel, composed of Phe and Trp, presented the fastest relaxation rates for Phe Arom protons. The weakest material, formed by Phe and Ser, contains the highest concentrations of dissolved Phe, the slower relaxation profiles of which contribute to a higher averaged 1H T1 value. 1H longitudinal relaxation times may therefore mirror the strength of the hydrogel fibres, although such a comparison should only be performed when discussing systems with similar composition.For multi\u2010component hydrogels, 1H sites displayed an evolution of T1 times with temperature similar to the single component system. Furthermore, 1H T1 times for Trp and Tyr presented similar temperature dependence to those in hydrogels of Phe/Trp and Phe/Tyr, respectively. At low temperatures, 1H T1 times were similar for different 1H sites followed by a gradual dispersion of values as temperature was increased .For all hydrogels, Phe  Figures\u2005 and S10.1H T1 times of Leu and Ser was observed throughout the range of temperatures with a distribution of values similar to solutions  NMR experiments.STD NMR spectroscopy is applied frequently to identify the functional groups of a ligand responsible for binding to its receptor .\u03b7STD, with saturation time (build\u2010up curves) supported the presence of fast exchanging processes  of Phe between free and bound states in the hydrogel of Phe \u2005the increase in the binding kinetics with temperature, reducing the fraction of bound species, and, more importantly, 2)\u2005the gradual dissolution of the supramolecular network, reaching full dissolution at 338\u2005K. This temperature was higher than the macroscopically determined Tgel, since the former reflects the dissolution of the supramolecular network (that acts as reservoir of magnetisation), whereas the latter describes the loss of structural integrity. Relaxation studies corroborated these findings, since 338\u2005K was the temperature above which the distribution of 1H T1 values resembling that in solutions of Phe was observed  and current investigations are underway in our research group.The ability of STD NMR to detect the exchange of gelator molecules between the network and the solution at temperatures above \u03b7STD  in multi\u2010component systems. \u03b7STD values of Phe protons decreased in all multi\u2010component hydrogels in comparison with the hydrogel of Phe. In contrast to classical exchange processes in protein\u2010ligand interactions (in which there is a determined number of binding sites decorating the surface of the protein), in a supramolecular system such a decrease in saturation transfer may be attributed to several phenomena, which are discussed below.The effect of introducing an additive was assessed through the evaluation of  Figures\u2005 and S\u200911\u03b7STD might result from: 1)\u2005decreased binding strength of Phe, due to perturbations of the network structure from the binding of the additive, or 2)\u2005from competition of the additive with Phe for the interaction sites, assuming the network interaction sites remain the same in the presence and in the absence of the additive. In both cases, there will be an increased amount of Phe in solution, which reduces the fraction of bound molecules, and hence the STD (Equation S1 in Supporting Information). On the other hand, slow exchange on the NMR relaxation time scale, associated with strong binding, would result in longer residence time in the network\u2010bound state, lower accumulation of saturation in solution and, therefore, lower STD signals.Lower values of \u03b7STD values in the same range as protons of Phe  and the enhanced resistance to deformation found for the hydrogel of Phe/Trp  NMR experiments in understanding exchange dynamics and binding processes in multi\u2010component supramolecular hydrogels. The application of the presented approach enables better understanding of the role of guest molecules, such as drugs, co\u2010gelators or non\u2010gelating additives, in the self\u2010assembly processes of supramolecular soft materials, which is of paramount importance in knowledge based design of new functional materials.l\u2010leucine, l\u2010phenylalanine, l\u2010serine, l\u2010tryptophan, l\u2010tyrosine, and hexamethylbenzene (HMB) were purchased from Sigma\u2013Aldrich. Deuterium oxide, 4,4\u2010dimethyl\u20104\u2010silapentane\u20101\u2010sulfonic acid (DSS), and tetramethylsilane (TMS) were purchased from Goss Scientific. Milli\u2010Q water was obtained with a Thermo Scientific Barnstead NANOpure purification system coupled to a Barnstead hollow fibre filter.Reagent grade (>98\u2009%) 2O) or heavy (D2O) water in a glass vial (2\u2005cm diameter) promoting dissolution with a vortex mixer for 30\u2005s. These samples were heated up to 363\u2005K with a hot plate and immediately quenched in a water bath at 273\u2005K. The hydrogel samples were left resting overnight at room temperature and analysed 24 hours after preparation. Hydrogelation was assessed through the vial inversion test. Temperature of gelation was determined by heating up the hydrogel samples with a hot plate at a heating rate of 1\u2005K\u2009min\u22121 while performing vial inversion tests. The resulting hydrogels were dried under vacuum.Hydrogels were prepared by dispersing amino acids in 1\u2005mL of light (HMorphology of the hydrogel fibres was determined using scanning electron microscopy (SEM). SEM experiments were carried out using a Jeol JSM\u20105900 LV Oxford instrument with an accelerating voltage of 2.1\u2005kV. Hydrogels were mounted on aluminium stubs with double sided carbon adhesive and allowed to dry before analysis. Dried samples were then gold\u2010coated using a Quorum Technologies Polaron SC7640 gold sputter coater.\u22121, operated with intermittent contact for imaging. Hot solutions (ca. 10\u2005\u03bcL) were pipetted onto a sample holder and allowed to dry before analysis.Nanoscale morphology of the hydrogel fibres was determined using atomic force microscopy (AFM). AFM experiments were conducted using a JPK NanoWizard instrument with a silicon cantilever at a nominal spring constant of 40\u2005N\u2009mnano Rotonetic 2 drive, equipped with a Julabo F12 water cooler and circulator controlling the temperature of the bottom Peltier plate, and an aluminium cone and plate geometry system . Hot solutions (ca. 1.5\u2005mL) were pipetted into a 300\u2005\u03bcm gap, with the temperature of the plate maintained at 323\u2005K for sample preparation. The temperature was then lowered to 293\u2005K, covered with a solvent trap to prevent solvent evaporation, and the hydrogels were left stabilizing for 1\u2005h. Phase angle, storage and loss moduli were monitored and recorded as a function of frequency and stress. All samples were subjected to frequency sweeps in the range of 0.1 to 100\u2005Hz and applied stress of 700\u2005Pa, as well as stress amplitude sweeps in the range of 500 to 10\u2009000\u2005Pa.Resistance of fibres to mechanical stress was investigated using rheology. Measurements were performed using a Bohlin Gemini HRK\u03b1 radiation (\u03bb=1.54\u2005\u00c5). Samples were analysed in the 2\u03b8 range of 5 to 36\u00b0, with a step size of 0.01\u00b0 and a scan time of 6\u2005s. Hydrogels (ca. 1\u2005mL) and dried samples were placed onto stainless steel sample holders and analysed immediately to prevent dehydration.Long\u2010range ordering of the obtained hydrogels was investigated using powder X\u2010ray diffraction (PXRD) with a Thermo Scientific ARL XTRA powder diffractometer under Cu1H) and 100.64\u2005MHz (13C). Hydrogels were prepared by pipetting 40\u2005\u03bcL of hot solutions into Kel\u2010F plastic inserts and allowing them to cool down and gelate inside the insert. 1H\u201013C cross\u2010polarisation magic angle spinning (CP/MAS) NMR experiments of reference solid powders were acquired using 128 scans at an MAS rate of 10.5\u2005kHz, a recycle delay of 30\u2005s and contact time of 2\u2005ms. 1H\u201013C CP/MAS NMR spectra of hydrogels were acquired using 8192 scans and an MAS rate of 8.5\u2005kHz with a recycle delay of 20\u2005s and contact time of 2\u2005ms. The Hartmann\u2013Hahn matching condition was established with hexamethylbenzene (HMB). All spectra were referenced using tetramethylsilane (TMS) as a standard.Structure and dynamics of hydrogels were characterised using nuclear magnetic resonance spectroscopy. Solid\u2010state NMR experiments were performed using a Bruker Avance III spectrometer equipped with a 4\u2005mm triple resonance probe operating at frequencies of 400.23\u2005MHz  NMR experiments were carried out using the stimulated echo and longitudinal eddy current delay diffusion\u2010filtered pulse sequence (ledgpgp2s), with 5 to 95\u2009% of the maximum gradient intensity. A smoothed\u2010square shaped gradient (SMSQ10.100) was used. A diffusion delay (\u0394) of 70\u2005ms, a diffusion gradient length (\u03b4) of 1\u2005ms, a recycle delay of 2\u2005s and MAS rates of 1\u2005kHz were used in each experiment. Attenuated peak intensities were plotted against the b factor, b=\u2212\u03b32g2\u03b42(\u0394\u2212\u03b43).D) were obtained from the mathematical fitting of the resulting curves to the mono\u2010exponential function [Eq.\u2005High\u2010resolution magic angle spinning (HR\u2010MAS) NMR experiments were performed using a Bruker Avance III spectrometer operating at a tion Eq.\u2005:(1)I=I0eI is the observed intensity, I0 is the unattenuated signal intensity and \u03b3 is the gyromagnetic ratio of the observed nucleus.where 1H frequency of 499.69\u2005MHz equipped with a 5\u2005mm probe. Hydrogels were prepared by pipetting 600\u2005\u03bcL hot solutions into NMR tubes and allowing them to cool down and gelate inside the tube. All experiments were carried out with hydrogels prepared in D2O. Variable temperature (VT) experiments were carried out in the range of temperature from 298 to 353\u2005K, allowing thermal stabilisation of the sample for 15\u2005min prior to spectra acquisition. 1H\u2005NMR spectra were acquired using excitation sculpting for water suppression (zgespg) with a recycle delay of 10\u2005s. DSS was used as an internal standard inside a coaxial insert.Solution\u2010state NMR experiments were performed using a Bruker Avance I spectrometer operating at a 1H longitudinal relaxation times (T1) were measured using a standard inversion recovery pulse sequence with a recycle delay of 10\u2005s. 16 points were recorded at variable time delays ranging from 0.1 to 20\u2005s. The evolution of intensities was fitted mathematically to the mono\u2010exponential function [Eq.\u2005tion Eq.\u2005:(2)Mz(\u03c4)Mz is the z\u2010component of magnetisation, M0 is the equilibrium magnetisation and \u03c4 is the time delay.where 1H frequency by a train of 40 Gaussian pulses with the duration of 50\u2005ms each (stddiffgp19.2), acquired with a constant total experiment duration of 6\u2005s. STD spectra were created by the subtraction of an on\u2010resonance spectrum (STDon), in which a spectral region was selectively saturated, from an off\u2010resonance spectrum (STDoff), acquired with no selective saturation. STDon spectra were acquired at a saturation frequency of 0\u2005ppm (where only resonances of the network can be encountered), whereas the STDoff saturation frequency was set for 40\u2005ppm. Interleaved acquisition of STDon and STDoff spectra was performed as a pseudo\u20102D experiment to minimise artefacts caused by variations throughout the experiment. Each pair of experiments was acquired at variable saturation times ranging from 0.25 to 6\u2005s. Signal intensity in the difference spectrum relative to the signal intensity in the STDoff spectrum was used to determine the fractional STD response, \u03b7STD [Eq.\u2005Saturation transfer difference (STD) NMR experiments were performed with selective saturation of a given STD Eq.\u2005:(3)\u03b7STD=I0 is the signal intensity from the STDoff spectrum, ISAT is the signal intensity from the STDon spectrum and ISTD is the signal intensity from the difference spectrum.(tsat)=STDmax(1\u2212e(-\u03basat\u00b7tsat)), where STDmax is the maximum possible STD at very long saturation times, \u03basat is the saturation rate constant and tsat is the saturation time. Initial slope values of the build\u2010up curves, STD0, were obtained from the product STDmax\u00d7\u03basat.where The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re\u2010organized for online delivery, but are not copy\u2010edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file."}
+{"text": "Bace1 (encoding \u03b2-secretase BACE1 involved in A\u03b2 production), Mme, Ide, Ece1, Ace2 (encoding enzymes involved in A\u03b2 degradation), Epo  in the frontal cortex, hippocampus, striatum, hypothalamus, and amygdala of OXYS and Wistar (control strain) male rats. Starting from the age of 14\u00a0weeks, animals received CEF  or saline. mRNA levels were evaluated with RT-qPCR method. Biochemical parameters of plasma were measured for control of system effects of the treatment. The dominant hypothesis about the pathogenesis of Alzheimer\u2019s disease (AD) is the \u201camyloid cascade\u201d concept and modulating the expression of proteins involved in the metabolism of amyloid-beta (A\u03b2) is proposed as an effective strategy for the prevention and therapy of AD. Recently, we found that an antibiotic ceftriaxone (CEF), which possesses neuroprotective activity, reduced cognitive deficits and neurodegenerative changes in OXYS rats, a model of sporadic AD. The molecular mechanisms of this effect are not completely clear, we suggested that the drug might serve as the regulator of the expression of the genes involved in the metabolism of A\u03b2 and the pathogenesis of AD. The study was aimed to determine the effects of CEF on mRNA levels of Ace2 in the frontal cortex and hypothalamus, and of Actb in the amygdala of untreated OXYS rats. Analysis of potential effects of CEF revealed its novel targets. In the compound-treated OXYS cohort, CEF diminished mRNA levels of Bace1 and Ace2 in the hypothalamus, and Aktb in the frontal cortex. Furthermore, CEF augmented Mme, Ide, and Epo mRNA levels in the amygdala as well as the levels of Ece1 and Aktb in the striatum. Finally, CEF also attenuated the activity of ALT and AST in plasma of OXYS rats.To better understand strain variations studied here, we compared the gene expression between untreated OXYS and Wistar rats. This comparison showed a significant decrease in mRNA levels of Those findings disclosed novel targets for CEF action that might be involved into neuroprotective mechanisms at early, pre-plaque stages of AD-like pathology development. Alzheimer\u2019s disease (AD) is one of the most common neurodegenerative disorders of the central nervous system. Loss of neurons in multiple brain regions  leads to cognitive dysfunctions including memory impairment as well as to behavioral and physiological changes. The dominant hypothesis of the pathogenesis of AD is the \u201camyloid cascade\u201d concept. The excessive accumulation and aggregation of amyloid-beta (A\u03b2) in AD is assigned as a key role in triggering the chain of pathological neurodegenerative processes , 2. MajoThe A\u03b2 levels in the brain depend on its de novo formation by amyloidogenic APP processing with \u03b2-secretase BACE1 and also on its elimination via different mechanisms including its proteolytic degradation, transport processes, cell mediated clearance, and its deposition into insoluble aggregates , 6. It id-galactose. The OXYS rats are known for shortened lifespan and an early development of age-related pathological phenotypes  and no significant effect of the \u201cGenotype\u201d factor  or of the interaction between the factors  on the mRNA levels of Ace2 in the amygdala of the rats.According to two-way ANOVA, there was a tendency in influence of the \u201cCeftriaxone\u201d factor  on mRNA levels of Bace1.Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d [F\u2009<\u20091] or \u201cCeftriaxone\u201d [F\u2009<\u20091] factor as well as of the interaction between the factors  or \u201cCeftriaxone\u201d [F\u2009<\u20091] factor as well as of the interaction between the factors [F\u2009<\u20091] on the mRNA levels of P\u2009<\u20090.05] and of the interaction between the factors  but no significant effect of the \u201cCeftriaxone\u201d factor  on the mRNA levels of Epo in the amygdala of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in Wistar rats of \u201cWistar\u2009+\u2009saline\u201d (P\u2009<\u20090.05) and \u201cWistar\u2009+\u2009CEF\u201d (P\u2009<\u20090.01) or \u201cOXYS\u2009+\u2009saline\u201d (P\u2009<\u20090.05) group.According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor , a tendency for the interaction between the factors  and no significant effect for the \u201cCeftriaxone\u201d factor [F\u2009<\u20091] on the mRNA levels of Ide in the amygdala of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in Wistar rats of \u201cWistar\u2009+\u2009saline\u201d (P\u2009<\u20090.05) or \u201cWistar\u2009+\u2009CEF\u201d (P\u2009<\u20090.01) group.According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor , and no significant effect for the \u201cCeftriaxone\u201d factor [F\u2009<\u20091] or for the interaction between the factors  on the mRNA levels of Mme in the amygdala of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in Wistar rats of \u201cWistar\u2009+\u2009saline\u201d (P\u2009<\u20090.05) or \u201cWistar\u2009+\u2009CEF\u201d (P\u2009<\u20090.01) group.According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor , and no significant effect for the \u201cCeftriaxone\u201d factor [F\u2009<\u20091] or for the interaction between the factors [F\u2009<\u20091] on the mRNA levels of Actb in the amygdala of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cWistar\u2009+\u2009CEF\u201d group versus that in OXYS rats of \u201cOXYS\u2009+\u2009saline\u201d (P\u2009<\u20090.05) or \u201cOXYS\u2009+\u2009CEF\u201d (P\u2009<\u20090.05) group.According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor  and of the \u201cCeftriaxone\u201d factor  but no significant effect of the interaction between the factors  on the mRNA levels of Ace2 in the hypothalamus of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cWistar\u2009+\u2009saline\u201d group versus that in rats of \u201cWistar\u2009+\u2009CEF\u201d (P\u2009<\u20090.05) and \u201cOXYS\u2009+\u2009saline\u201d (P\u2009<\u20090.01) or \u201cOXYS\u2009+\u2009CEF\u201d (P\u2009<\u20090.001) group.According to two-way ANOVA, there were a significant influence of the \u201cGenotype\u201d factor  and no significant effects of the \u201cGenotype\u201d [F\u2009<\u20091] or of the \u201cCeftriaxone\u201d  factors on the mRNA levels of Bace1 in the hypothalamus of the rats. LSD post hoc test revealed that the parameter was significantly lower in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in rats of \u201cOXYS\u2009+\u2009saline\u201d (P\u2009<\u20090.05) group.According to two-way ANOVA, there was a significant influence of the interaction between the factors , Epo , and Ide . For Mme, Kruskal\u2013Wallis ANOVA by Ranks showed a significant difference between the groups: . However, multiple comparisons of mean ranks for all groups demonstrated no significant intergroup differences.Due to the lack of normal distribution of the data in the studied groups, P\u2009>\u20090.05] factor as well as of the interaction between the factors [F\u2009<\u20091] on the mRNA levels of Actb.Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d [F\u2009<\u20091] or \u201cCeftriaxone\u201d , Bace1 , Epo , and Actb .Due to the lack of normal distribution of the data in the studied groups, P\u2009<\u20090.05], a tendency for the interaction between the factors  and no significant effect of the \u201cCeftriaxone\u201d factor [F\u2009<\u20091] on the mRNA levels of Ece1 in the hippocampus of the rats. LSD post hoc test revealed that the parameter was significantly lower in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in rats of \u201cWistar\u2009+\u2009CEF\u201d group (P\u2009<\u20090.01).According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor  factor as well as of the interaction between the factors .Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d [F\u2009<\u20091] or \u201cCeftriaxone\u201d  and no significant effect of the \u201cGenotype\u201d factor  or of the \u201cCeftriaxone\u201d factor [F\u2009<\u20091] on the mRNA levels of Mme in the hippocampus of the rats.According to two-way ANOVA, there was a tendency for influence of the interaction between the factors , Bace1 , Epo , and Mme . For Ece1, Kruskal\u2013Wallis ANOVA by Ranks showed a significant difference between the groups . Multiple comparisons of mean ranks for all groups revealed that the parameter was significantly higher in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in rats of \u201cWistar\u2009+\u2009saline\u201d group (P\u2009<\u20090.05).Due to the lack of normal distribution of the data in the studied groups, P\u2009>\u20090.05] or \u201cCeftriaxone\u201d [F\u2009<\u20091] factor as well as of the interaction between the factors [F\u2009<\u20091].Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d , and no significant effects of the \u201cCeftriaxone\u201d factor  and of the interaction between the factors [F\u2009<\u20091] on the mRNA levels of Actb in the striatum of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in Wistar rats of \u201cWistar\u2009+\u2009saline\u201d (P\u2009<\u20090.05) or \u201cWistar\u2009+\u2009CEF\u201d (P\u2009<\u20090.05) group.According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor , and no significant effect of the \u201cCeftriaxone\u201d factor [F\u2009<\u20091] or of the interaction between the factors [F\u2009<\u20091] on the mRNA levels of Ace2 in the frontal cortex of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cWistar\u2009+\u2009saline\u201d group versus that in rats of \u201cOXYS\u2009+\u2009saline\u201d (P\u2009<\u20090.05) or \u201cOXYS\u2009+\u2009CEF\u201d (P\u2009<\u20090.05) group.According to two-way ANOVA, there was a significant influence of the \u201cGenotype\u201d factor .Due to the lack of normal distribution of the data in the studied groups, P\u2009>\u20090.05] factor as well as of the interaction between the factors [F\u2009<\u20091].Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d [F\u2009<\u20091] or \u201cCeftriaxone\u201d  and no significant effects of the \u201cGenotype\u201d [F\u2009<\u20091] and \u201cCeftriaxone\u201d [F\u2009<\u20091] factors on the mRNA levels of Epo in the frontal cortex of the rats.According to two-way ANOVA, there was a tendency for influence of the interaction between the factors  factor as well as of the interaction between the factors [F\u2009<\u20091] on the mRNA levels of Ide in the frontal cortex of the rats.Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d [F\u2009<\u20091] or \u201cCeftriaxone\u201d  or \u201cCeftriaxone\u201d  factor as well as of the interaction between the factors [F\u2009<\u20091] on the mRNA levels of Mme in the frontal cortex of the rats.Two-way ANOVA did not show a significant effect of the \u201cGenotype\u201d , and no significant effect of the \u201cGenotype\u201d factor [F\u2009<\u20091] or of the interaction between the factors  on the mRNA levels of Actb in the frontal cortex of the rats. LSD post hoc test revealed that the parameter was significantly higher in the rats of \u201cOXYS\u2009+\u2009saline\u201d group versus that in Wistar rats of \u201cWistar\u2009+\u2009CEF\u201d (P\u2009<\u20090.01) or \u201cOXYS\u2009+\u2009CEF\u201d (P\u2009<\u20090.05) group.According to two-way ANOVA, there was a significant influence of the \u201cCeftriaxone\u201d factor  and the interaction of the factors  on AST activity, and there was a tendency for the \u201cCeftriaxone\u201d factor . CEF treatment significantly reduced the augmented AST activity in OXYS rats. Similarly, chronic treatment with CEF attenuated the activity of ALT in OXYS rats. Two-way ANOVA revealed significant effect of the \u201cCeftriaxone\u201d factor  and a tendency for the interaction of the factors  but not of \u201cGenotype\u201d factor [F\u2009<\u20091] on ALT activity. For the levels of Ca2+, two-way ANOVA showed significant influence of the \u201cGenotype\u201d  and \u201cCeftriaxone\u201d  factors while the interaction of the factors was insignificant [F\u2009<\u20091]. LSD post hoc test revealed that the parameter was significantly lower in the rats of \u201cOXYS\u2009+\u2009CEF\u201d group versus that in Wistar rats of \u201cWistar\u2009+\u2009saline\u201d (P\u2009<\u20090.01) group.Two-way ANOVA showed significant influence of the \u201cGenotype\u201d factor , triglycerides , LDL-C , and HDL  while the effects of \u201cCeftriaxone\u201d factor or the interaction of the factors were insignificant.Significant influence of the \u201cGenotype\u201d factor was found on the levels of total cholesterol  and the levels of total bilirubin were measured using clinical chemistry analyzer Konelab 30i and Konelab kits  according to the manufacturer\u2019s instructions .P\u2009<\u20090.05. STATISTICA 10.0 software was used to perform all the statistical analyses.All results were presented as mean\u2009\u00b1\u2009SEM and compared using two-way ANOVA followed by post hoc Fisher LSD test or nonparametric Kruskal\u2013Wallis ANOVA followed by multiple comparisons of mean ranks for all groups . The independent variables for two-way ANOVA were Genotype (Wistar or OXYS strain) and Treatment . The level of significance was defined as"}
+{"text": "PT-symmetry) phononic crystals (PCs) with balanced gain and loss medium. Using the super cell method of rectangular lattice, we exhibit the thresholdless spontaneous PT-symmetry breaking in the band structure. The numerical results show that the asymmetric scattering properties obviously occur in a non-Hermitian system. At two specific incident frequencies, unidirectional reflectionless and perfect transmission behaviors exist individually in opposite directions, which are accompanied by a phase transition of \u03c0. Based on the generalized Snell\u2019s law, combining such a PT-symmetric medium, we design a novel metamaterial crystal for PT-symmetric acoustic flat focusing. Its focus frequency can also be modulated by the gain/loss parameter. The novel flat focusing based on the PT-symmetry that we propose opens a new door for high-dimensional applications of non-Hermitian metamaterials in acoustic wave manipulation.We present a two-dimensional (2D) parity-time-symmetric ( In particular, in flat focusing, PCs are implemented in many interesting manners, including negative refraction6 and metasurfaces9. However, most studies focused on Hermitian system, except a few works related to the complex domain17. Since the concept of PT-symmetry was introduced in Bender\u2019s work20, it began to arouse much interest on research of non-Hermitian systems, which remain invariant under the combination of parity (P) and time reversal (T) operations. Operator P is defined as: T is: 32, and has introduced many interesting features.Over the past two decades, there have been many studies on acoustic wave manipulations. The periodically structured PCs, metamaterials and metasurfaces are well-designed for applications such as acoustic waveguide33 and bound states of the defect mode34 have been systematically investigated. These acoustic studies are only studied in 1D space. Very recently, Zhu et al. designed a curved PT-symmetric metamaterials crystal in 2D space, to achieve the unidirectional sound focusing effect in simulation and experiment35. However, until now, there has been no comprehensive analysis about the effect on the dispersion relation caused by PT-symmetry breaking in 2D acoustic systems. In addition, not confined to a curved structure, achieving the optional geometric manipulation of PT-symmetric metamaterials is a challenging problem.Recently, non-Hermitian acoustics have been introduced to the 1D structure. The properties of one-way propagationPT-symmetric PC with complex parameters. A complex bulk modulus in active practical systems can be uesd36, and non-Hermitian acoustic systems are also proven to be feasible in practice40. We analyze the degenerated band structure which experiences thresholdless PT-symmetry breaking. Moreover, we study the asymmetric scattering properties caused by PT-symmetry breaking. The unique unidirectional reflectionless and perfect transmission behaviors with a phase transition of \u03c0 are shown in the following sections. Based on the phase characteristic, which is associated with the generalized Snell\u2019s law41, we further design a PT-symmetric planar metamaterials lens for acoustic focusing, which opens a new degree of freedom of geometric shapes for acoustic wavefront engineering in non-Hermitian systems.In this letter, we introduce a 2D PT-symmetric PCs, which is presented in Fig.\u00a0\u03b1 to denote the imaginary strength of the gain/loss materials. The distribution in the x direction with balanced gain and loss satisfies the PT-symmetry condition that the real part of the modulus is an even function, while the imaginary part is odd. Figure\u00a0a and a rod radius of 0.3a. The band structures of the underlying 2D Hermitian system and non-Hermitian system are calculated, respectively. Figure\u00a0\u03b1\u2009=\u20090). The wave vectors sweep along the boundary of the irreducible Brillouin zone for rectangular lattice. Although the 2\u2009\u00d7\u20091 super cell method was used, the calculation result is identical to that of the unit cell, except a band corresponding to the unit cell is folded into two bands in the x direction of the super cell. Some bands also cross and degenerate in the Brillouin zone. As shown in Fig.\u00a0PT-symmetry breaking spontaneously occurs when the imaginary component (\u03b1\u2009=\u20090.25) is added to form a balanced complex elastic modulus. Many degenerated bands appear along the boundary, especially in the \u0393-X and Y-M directions. Similar to the case for 1D structures, some bands merge at the high symmetry points (labeled as dashed box A). However, in dashed box B, the originally degenerated bands split and merge again with other multiple bands, which does not occur in low-dimensional situations. The compression of merged bands also causes the appearance of directional bandgaps.We propose a structure of 2D \u03b1\u2009=\u20090) is shown in Fig.\u00a0kx\u2009=\u20090.5\u03c0/a in the band structure because of the fold features in the super cell system. When the gain and loss are added to form a non-Hermitian system with a coefficient \u03b1\u2009=\u20090.25, Fig.\u00a0PT-symmetry breaking. A particularly interesting phenomenon occurs, where the folded bands merge together outwards from the primary degenerate contour, and a new contour appears at the boundary between the merged and the independent regions. Thus, degeneracy in 3D k-space is exhibited as bands merging in a continuous area, which includes high-symmetry points and arbitrary low-symmetry points. Due to the extra spatial degree of freedom, the degenerate points in the 1D structure evolve into an irregular contour in the 2D system. A comparison of data values also shows that the bands nearly flatten in the x direction perpendicular to the degenerate contour after they merge. The imaginary part, as presented in Fig.\u00a0To provide a vivid illustration of the degenerate phenomenon, the 3D dispersion surface for the first and second bands are plotted. The band structure for the super cell of the underlying Hermitian acoustic system  with obvious unidirectional behavior in the frequency range of our study. As shown in Fig.\u00a0T\u2009=\u20091. In addition, the relation between reflection and transmission in our 2D structure excellently satisfies the generalized unitarity relation. As illustrated in Fig.\u00a0\u03c91, while the left reflected waves experience the same transition at frequency \u03c92. The phase between the left and right reflections is \u03c0 for the case of T\u2009>\u20091 but is invariably equal for T\u2009<\u20091. One-way transport without reflection occurs when the PT-symmetric phase changes into the broken phase mainly because the reflected waves are completely absorbed by the loss part. However, the unitary behavior of transmitted acoustic waves is only contributed by the gain part in the lossy system.In our 2D \u03c91\u2009=\u20090.475, but the right-incident waves are reflectionless. Conversely, Fig.\u00a0\u03c92\u2009=\u20090.555. Based on the property of EPs, it can be designed into a novel acoustic switch device using the unidirectional reflectionless behavior, which is also applied to other multifunctional devices in acoustic wave manipulations.The simulated scattering pressure fields in Fig.\u00a0PT-symmetric metamaterials. Due to different arrays of gain and loss mediums, the reflected waves of two units correspond to the left and right reflected waves, respectively. Here, the lattice length a is 1\u2009cm. To clearly exhibit the discrete phase shifts, as plotted in Fig.\u00a0a. At this wavelength, the reflection amplitudes in two units are nearly equal, which are approximately 3/5 of the amplitude of the incident wave. The pressure strips are obviously shown, and there is a shift of \u03bb/2, i.e., the \u03c0 phase between adjacent peaks of two reflected waves, so that two such unit structures can shift up to provide a discrete phase gradient for the entire 2\u03c0 range.There is always a phase difference \u03c0 between the left- and right-reflected waves when the transmittance is greater than 1. It can be utilized to frame two units of metamaterials, which can realize the discrete phase gradient of \u03c0 that covers the full range of 2\u03c0. Using the above structure and parameters, Fig.\u00a0\u03b8r(\u03b8i) is the angle of reflection (incidence), ni is the refractive indices of the media, and d\u03d5/dy is the phase shift per unit distance along the y direction. When the plane wave is normally incident on the metamaterials, Eq.  in the y direction must satisfy the equation:Based on this property, we design a non-Hermitian acoustic flat lens to focus on the acoustic plane wave. Figure\u00a0f\u2009=\u20095\u03bb as the focal length. For the sake of clarity, there is a rule to convert a continuous phase profile to a discrete phase profile. Because the \u03c0 phase difference of two structural arrangements, the units providing \u03c0 phase shift is utilized to displace the continuous phase range \u03c0\u2009\u00b1\u2009\u03c0/2 at the specified position in the y direction. From Eq.  are easier to find around the bandgap. On both sides of the EPs, there will always be a case of T\u2009>\u20091, which determines the frequencies of the phase difference of \u03c0. Thus, the imaginary parameter \u03b1 has a great effect on the frequencies of the phase transition. We can control the frequency, which has a phase difference of \u03c0, by changing the value of the imaginary part. Theoretically, a high focusing efficiency of reflected waves can also be acquired by modulating the gain/loss strength \u03b1. Therefore, both the frequency and the amplitude of acoustic flat focusing can be manipulated.We have demonstrated the effect of imaginary parameter \u03b1 in Fig.\u00a0PT phononic structure from opposite directions, there are significant unidirectional behaviors and phase transitions in the scattering phenomenon. The right-reflected waves vanish at We have designed a novel 2D non-Hermitian PC for acoustic flat focusing. It is composed of soft rubber rods with a complex conjugate elastic modulus, which are periodically embedded in water. When plane waves propagate through the PT-symmetric PC considered in this paper is composed of gain/loss materials in water  based on the software COMSOL Multiphysics 5.3a is employed for the simulations. The calculations of the band structure are performed with the super cell method. For Fig.\u00a0The 2D 39. More specifically, the proposed structures can be realized by using the sensors and pre-amplifiers. The sensed signal is amplified in a pre-amplifier of appropriate gain progression. In addition, an equivalent acoustic gain system can be constructed by combining auxiliary devices such as microphones and loudspeakers.The structures consist of the loss and gain medium with a positive and negative imaginary part respectively. Generally, the acoustic loss materials are ubiquitous in nature because acoustic loss can be easily caused by viscous elasticity. It should be noted that a natural acoustic gain media has not yet been found. However, it can be effectively realized by well-designed feedback systems"}
+{"text": "In order to efficiently replicate, viruses require precise interactions with host components and often hijack the host cellular machinery for their own benefit. Several mechanisms involved in protein synthesis and processing are strongly affected and manipulated by viral infections. A better understanding of the interplay between viruses and their host-cell machinery will likely contribute to the development of novel antiviral strategies. Here, we discuss the current knowledge on the interactions between influenza A virus (IAV), the causative agent for most of the annual respiratory epidemics in humans, and the host cellular proteostasis machinery during infection. We focus on the manipulative capacity of this virus to usurp the cellular protein processing mechanisms and further review the protein quality control mechanisms in the cytosol and in the endoplasmic reticulum that are affected by this virus. In mammalian cells, protein homeostasis or proteostasis maintenance is assured through an integrated network that ensures efficient biogenesis, folding and assembling of proteins, as well as the degradation of abnormal conformers. Cells are frequently exposed to external stimuli that can disrupt proteostasis leading to the accumulation of misfolded proteins and, under unmitigated chronic stress conditions, to the formation of potentially pathogenic cytotoxic aggregates ,2. To coAs opportunistic infectious agents, viruses employ several strategies to hijack and control cellular activities, including protein production and processing, in order to efficiently replicate. Multiple viruses specifically alter organelle morphology and dynamics as part of their replication cycle ,9, as weIn this review, we summarize and discuss the induced disruption of the cellular machinery, with focus on proteostasis imbalance, throughout the course of influenza A virus (IAV) life-cycle and explore its significance for infection efficiency. Although information on the interplay between influenza B virus (IBV) and host-cell proteostasis is scarce, we have established a parallel, where appropriate, between the two viruses.IAV has been the causative agent for most of the annual respiratory epidemics in humans as well as the for the major influenza pandemics in the last century, associated with high morbidity and mortality especially in the elderly . ImunoseCurrently, the permanent risk of influenza epidemics and pandemics is due to the continuous viral antigenic evolution, linked to the accumulation of point mutations within the viral genome or the genetic reassortments of viral genome segments from different viruses or virus strains . For thiThe IAV genome consists of eight single-stranded negative-sense linear RNA segments (ssRNA), encoding for a different number of proteins depending on the virus strain . The eigTo successfully establish infection, viruses can manipulate the host cell to favor the synthesis of viral proteins, at the expense of host proteins, involving changes in translation and RNA synthesis and stability ,22 Figu. IAV useThe viral non-structural protein 1 (NS1) can block the processing of cellular pre-mRNA  and targNS1 has also been described to enhance the translation initiation rate of viral mRNA , by alloNS1 was reported to prevent the activation of the double stranded DNA (dsDNA)-dependent protein kinase R (PKR), both upon IAV and IBV infection . This inOther mechanisms have been proposed to explain host shutoff by IAV such as cap snatching, in which the virus uses short oligomers from host pre-mRNA to prime its own mRNA synthesis . This prHence, the dynamic virus\u2013host interactions can elicit alterations in the host proteome to benefit viral replication. Several groups have studied the establishment of temporally and spatially regulated virus\u2013host protein\u2013protein interactions throughout the course of infection, essentially to identify unanticipated cellular factors required for influenza replication and pathogenicity, or related to host antiviral responses, and targetable host factors to guide antiviral drug development ,46,47,48Furthermore, due to the importance of the transfer RNA (tRNA) pool in protein synthesis, viruses have evolved sophisticated strategies to manipulate tRNA populations and optimize tRNA usage to enhance the efficiency of viral translation (reviewed in ) Figure. IAV, asIn addition, the IAV has evolved to regulate host cellular microRNAs (miRNAs) to subvert antiviral immunity and escape host-mediated inhibition of viral replication ,53,54,55Eukaryotic cells have evolved several cytosolic protein quality control mechanisms that ensure high fidelity on protein synthesis and processing. Various chaperones, mainly the heat shock proteins (HSPs), have evolved to assist the efficient folding and assembly of newly synthesized and stress-damaged proteins, in order to prevent potentially pathogenic protein aggregation . The expThe Hsp40/DnaJB1, involved in protein translation, folding, and degradation, has shown to be required for an efficient IAV replication as it interacts at early stages of infection with the viral NP, which encapsidates the viral RNA, and facilitates the nuclear import of incoming viral ribonucleoproteins (RNPs)  Figure . The Hsp40/DnaJA1 protein can enhance viral RNA synthesis, and ultimately IAV replication, by interacting with the viral RNA polymerase, through its polymerase basic protein 2 (PB2) and PA subunits  Figure . SimilarIPK, a cellular inhibitor of PKR, consequently enhancing PKR activation [IPK and Hsp40 to mitigate PKR-mediated antiviral response against IAV infection and ease viral replication [IPK was shown to regulate IAV mRNA translation and promote viral protein synthesis through PKR [IPK interaction.At late stages of infection, matrix protein 2 (M2) IAV protein is suggested to form a stable complex with Hsp40 and p58tivation . Moreovelication . Consideough PKR , these rAt later times of infection, the heat shock cognate protein 70 (Hsc70), a member of Hsp70 family, was demonstrated to interact with viral matrix protein 1 (M1) in infected cells, showing to be required for the nuclear export of vRNP-M1 complex [Interestingly, IAV can modulate apoptosis through the action of both Hsp70 and Hsp90 proteins. In fact, M1 was shown to bind to Hsp70, which results in the disruption of the Hsp70-Apaf-1 complex, consequently leading to apoptotic caspase-9 recruitment, cell lysis, and virus release . LikewisOverall, during the course of viral infection, IAV seems to take advantage of several cellular chaperones in different crucial steps of the life cycle, including nuclear import, viral genome replication, nuclear export, and further virion lytic release from the host cell to spread infection. Alternatively, when proteins cannot be refolded and aggregation cannot be prevented, molecular chaperones may contribute, along with the ubiquitination machinery, to target the misfolded proteins to degradation by the ubiquitin-proteasome system (UPS) or the autophagosome\u2013lysosome pathway . A functional UPS is important for the replication of influenza viruses, in order to maintain proper function and the level of viral proteins, although it can act as a host antiviral strategy to mark viral components for degradation ,74. StudHowever, influenza proteins can be targeted for ubiquitin-mediated degradation as an antiviral mechanism employed by the cells. The E3 ubiquitin ligase TRIM32 is known to interact and directly ubiquitinate the PB1 viral protein, leading to its degradation and the subsequent reduction of its polymerase activity . BesidesOn the other hand, IAV has evolved to modulate ubiquitination events to evade immune responses, by degrading or inactivating cellular proteins that limit viral growth and support virus replication. For example, NS1 interferes with the domain of TRIM25 required for RIG-I ubiquitination to circumvent downstream antiviral IFN production ,81,82, bYet, the ubiquitination of viral proteins has been shown to be essential for its correct biological activity. Concretely, the ubiquitination of M2 protein has shown to be crucial for the efficient packaging of the viral genome into infectious particles . SimilarThe autophagosome\u2013lysosome pathway is similarly manipulated upon infection with IAV, in a way that the formation of autophagosomes is triggered, but the fusion with lysosomes is inhibited by the virus  Figure . AutophaThis might imply that the virus benefits from autophagy to accumulate viral elements during replication, without affecting progeny virus production. In fact, it was recently suggested that autophagy plays a role in supporting IAV-induced apoptosis and replication . InteresWhen the amount of misfolded proteins in cells exceeds the refolding or degradative capacity of the quality-control machinery, or when this machinery is disrupted, the misfolded or aggregated proteins may be sequestered and compartmentalized into stress foci to minimize their toxic effects and interference with the normal cellular functions . Upon viIn response to immune activation, rather than impaired proteolysis, the formation of aggresome-like induced structures (ALIS) in the cytosol is also induced as an early event in the cellular adjustment to proteostasis disruption, consisting in the temporary aggregation of ubiquitinated proteins . In IAV-On the other hand, the IAV PB1-F2 protein was shown to form highly cytotoxic amyloid-like fibers in IAV-infected cells. The accumulation of these fibers can be a crucial virulence factor and presumably one of the causes for the immunopathological disorders observed during IAV infections . During Thus, IAV seems to induce the formation of inclusion bodies to escape the host cellular antiviral response and induce pathogenicity. In specific cases, the presence of RNA sensors, viral RNA, and MAVS in infected cells within cytoplasmic inclusion bodies correlates with the inhibition of an antiviral IFN-mediated response . A mechaUpon viral infection, the deregulation of proteostasis may lead to the accumulation of insoluble misfolded proteins that, along with chaperones and proteasome components, are often targeted to juxtanuclear structures termed aggresomes. These proteins are actively transported in a retrograde dynein-based manner along the microtubule cytoskeleton to the perinuclear site at the microtubule-organizing center, where they are sequestered into aggresomes . It invoWhether IAV leads to the accumulation of misfolded proteins is still not known. Yet, it is known that IAV usurps the HDAC6 ubiquitin-binding function, taking advantage of the aggresome processing machinery for capsid disassembly and uncoating during entry in cell . The fusAlternatively, misfolded proteins strongly tend to accumulate in stress granules (SGs), stress-induced cytosolic ribonucleoprotein complexes that assemblies when translation is inhibited. By sequestering polyribosome-associated components, SGs can reprioritize translation and rout the stalled components for re-initiation or degradation. The continuing accumulation of misfolded proteins can affect SGs dynamics, promoting its conversion into an aberrant aggresome-like structure that can be eventually transported to aggresomes and further degraded. The presence of cellular chaperones, specifically HSP27 and HSP70, has shown to prevent the formation of aberrant SGs and promote SGs disassembly by helping to maintain its normal composition and dynamism  Figure .It was already reported that the formation of SGs was not observed upon infection with IAV, suggesting that this virus can efficiently prevent global translation arrest . HoweverIPK activation and subsequent inhibition of PKR-mediated host response during IAV infection by exploiting Hsp40 [Besides NS1, NP and PA-X contribute to block the SG assembly ,112. In ng Hsp40 . RemarkaIn eukaryotic cells, the ER is the major site for protein folding and trafficking. Therefore, ER proteostasis perturbations, including those caused by viral infection, may lead to accumulation of misfolded proteins and their aggregation within the organellar lumen, which induces stress in the ER . Its abiOnce a threshold of misfolded protein accumulation in the ER has been reached, it activates the unfolded protein response (UPR) (reviewed in ). As an Upon infection, the UPR might either be mobilized by the host in an attempt to restrict viral propagation or be manipulated by the virus to benefit its replication. For instance, the innate sensing of viruses by ER stress was found to be responsible for the pathogenicity of pandemic IAV . On the The role of the UPR during IAV infection is unclear, since the results on the subject are somewhat controversial. Hassan et al.  reportedUpon IAV infection, several processes within the cytosolic and the ER protein quality control mechanisms, namely protein folding, degradation and sequestration, have shown to contribute either as part of an effective replication cycle or as part of an effective cellular antiviral response to the virus. IAV seems to take advantage of the protein quality control components to replicate and spread infection, although some of the observed effects may also be part of the establishment of cellular reactions to counteract the virus. Some reports show contradictory findings and further studies are needed in order to obtain more clarity and insight into the control of cellular proteostasis mechanisms by both the virus and the host cell."}
+{"text": "AbstractArulidae found off the coast of Kota Kinabalu, Sabah, East Malaysia; Bungapayunggen. nov. et sp. nov. and Laetawaheedaegen. nov. et sp. nov. As well, the stoloniferan genus Phenganax Alderslade & McFadden, 2011 belonging to the family Clavulariidae is expanded with three new species, P.marumisp. nov., P.subtilissp. nov., and P.stokvisisp. nov., which are all sclerite-free. Additionally, we report a possibly undescribed species, closely related to the clavulariid genera Azoriella Lopez-Gonzalez & Gili, 2001 and Cervera Lopez-Gonzalez et al., 1995. As this and other recent studies have shown, discoveries of small stoloniferous octocorals are helping to fill gaps in our knowledge of the overall systematics of Octocorallia.Sabah, Malaysia, is well known for its extensive and diverse coral reefs. It is located on the northwestern edge of the Coral Triangle, the region with the highest marine biodiversity. Much of the marine fauna here is still unknown, especially inconspicuous animals, such as small stoloniferous octocorals, which are common on coral reefs. Here, we describe two new monospecific genera of the family  Coral reefs fringe one-sixth of the world\u2019s coastlines and constitute the most biologically diverse shallow-water marine ecosystems, supporting thousands of species . The est2 with the majority (>75%) situated in Sabah : Gaya, Manukan, Sapi, Sulug, and Mamutik islands cover an area of approximately 50 km2, including their surrounding reefs and sea  and colonies, and are hard to find, one reason for the scant amount of information available for this group , Tokyo, Japan  and at the Borneo Marine Research Institute (IPMB), Universiti Malaysia Sabah (UMS), Sabah, Malaysia .A total of 25 stoloniferous octocoral specimens Table  was collSEM) operated at high vacuum at 15 kV.Sclerites were isolated by dissolving different parts of the specimens  in 4% hypochlorite (household bleach). Rinsing and visualisation of sclerites followed the same protocol as described in COI), mitochondrial mutS-like protein (mtMutS), and the ND6 subunit (ND6), and one nuclear ribosomal marker, 28S ribosomal DNA (28S rDNA) followed the protocols in COI, mtMutS, and ND6 were checked for introns, exons, and stop-codons in AliView . PCR amplification and sequencing of three mitochondrial markers, cytochrome c oxidase subunit I , including sequences of Cornulariapabloi McFadden & van Ofwegen, 2012 and Cornulariacornucopiae  as outgroup . Alignments of 893 bp for 28S rDNA, 717 bp for COI and 923 bp for mtMutS were separately run in Maximum Likelihood (ML) analyses  were also run in ML analyses to check for congruency .A separate phylogenetic analysis was made using a concatenated four-marker dataset to further investigate the phylogenetic position of collected clavulariid and arulid specimens. The four separate markers  among sequences , 20 March 2018 and collected by YW Lau. Holotype: NSMT-Co 1679, 9 m depth. Paratype: IPMB-C 01.00017, 10 m depth.All specimens are from Sepangar, Sepangar Island, Kota Kinabalu, Sabah, Malaysia . Polyps connected through stolons attached to rock. Stolons are thin and rounded  and polyps are spaced apart irregularly, either adjacent to one another or spaced apart up to ~5 mm. Expanded polyps are ~2.2\u20133.0 mm in width and retract fully into calyces of ~1 mm wide and up to ~2 mm in height. Calyces do not retract into the stolon. The oral disk of the polyps is expanded into a circular membrane by fusion of proximal regions of adjacent tentacles Figure , as is cAnthocodial sclerites are smooth rods, with simple tubercles at distal margin ends, 0.1\u20130.15 mm long Figure . CalycesPolyps are brown coloured in life with a whitish oral disk, but yellowish white when preserved in ethanol. Zooxanthellate.The paratype is a colony consisting of ~10 polyps. Polyps of the paratype colony show variation in colouration; the whitish colour is not restricted to the oral disk but is also seen in the tentacles. This could be due to differences in sclerite density Figure , as descSepangar Island, Kota Kinabalu, Sabah, Malaysia.Bungapayung gen. nov. et sp. nov. The 6-radiate type of sclerite was not observed in this genus but is present in Arula and Hana, the two other genera in the family Arulidae.Anthocodial rods are scarce in payung, which means umbrella; denoting the shape of the oral disc of the polyps, which resemble the shape of an umbrella.From the Malaysian and Indonesian word Taxon classificationAnimaliaAlcyonaceaArulidaeGenus34C1E76EFA0854E1ADF3CA66CF0B00A9http://zoobank.org/02B40F3A-AA78-49A5-ADA8-12F3374CDF21Laetawaheedae sp. nov., by original designation and monotypy.Colony with polyps connected through stolons, which are ribbon-like. Oral disk with eight furrows, running from intertentacular margin to the mouth of the polyp, dividing the membrane into eight shallow lobes. Distal two-thirds of tentacles extend from fused margins of the oral membrane. Sclerites of anthocodiae are rods. Sclerites of calyx are table-radiates and three types of rods; (1) branched rods with high tuberculate processes, (2) club-like rods and (3) table-radiate-like rods. Sclerites of stolon are fused table-radiates, which form a sheet. Sclerites colourless. Zooxanthellate.Laetawaheedae gen. nov. et sp. nov., and species of the genera Arula, Hana, and Bunga gen. nov. is found in the presence of all three types of rods in its calyces. The club-like rod with tubercles at one distal end of the rod is also seen in HanahanatabaLaetawaheedae and not in the calyx as in Hana. Similar to Bunga, Laeta does not have 6-radiates in the calyx. Similar to Hana and Bunga, Laeta has fused table-radiates in the stolon. Outer margins of the oral membrane are much less pronounced than in Arula and Hana, and are more similar to margins of the oral disk in Bunga.The main morphological difference between the type species, laeta, meaning bright, charming, cheerful. Gender: feminine.From Latin Taxon classificationAnimaliaAlcyonaceaArulidae289F7404F31D5F01A1C9F9599E3EF929http://zoobank.org/761E7919-D311-4F0B-8B67-17E8ECDEC370Holotype: NSMT-Co 1680, Udar, east of Udar Island , 10 m depth. Paratypes: IPMB-C 01.00018, Mid Reef, east of Manukan Island, TARP , 8 m depth. IPMB-C 01.00019, Gaya, Gaya Island, TARP , 11 m depth.All specimens are from Kota Kinabalu, Sabah, Malaysia and collected by YW Lau. The colony consists of ~50 polyps, which are connected through ribbon-like stolons (0.5\u20130.6 mm width). Polyps are spaced apart quite regularly (~3 mm). Anthocodiae retract fully into low oval to cylindrical calyces , which do not retract into the stolon. Expanded polyps 3.5\u20134.0 mm in diameter. Oral membrane with eight shallow furrows running from intertentacular margin to mouth of polyp, dividing the membrane into eight shallow lobes. Distal two-thirds of tentacles extend from fused margins of the oral membrane. Tentacles with 6\u20138 pairs of widely spaced pinnules are arranged in a single row on either side of rachis.Sclerites of anthocodiae are small smooth rods, 0.035\u20130.1 mm in length, with little ornamentation Figure . ScleritOral membrane has a blue colour in life (yellowish white when preserved in ethanol). The tentacles are brown in colour, partially with a greenish shimmer. Zooxanthellate.The paratype colony consists of ~30 polyps. Tentacles of paratypes show variation in pinnule pairs, partly having nine pairs of widely spaced pinnules arranged in a single row on either side of rachis.TARP, Kota Kinabalu, Sabah, Malaysia.Udar Island, Kota Kinabalu, Sabah, Malaysia. Gaya Island, Laetawaheedae gen. nov. et sp. nov. have the same blue-purple colour in life as those of Arulapetunia McFadden & Ofwegen, 2012, but there is a difference in the outer margins and lobes of the oral membrane; both are more pronounced in Arulapetunia. The holotype colony was attached to sponge tissue, but this epibiotic relation is not obligate.Polyps of Named after Dr. Zarinah Waheed, for her dedication to coral reef research and her guidance during fieldwork in Sabah.Taxon classificationAnimaliaAlcyonaceaArulidaeGenusAlderslade & McFadden, 20115B94F4202707519BA68EA0EAA5435705Alcyonacea with erect polyps and stolons with encrusting, stoloniferous habit. Sclerites are absent. Zooxanthellate. Distribution tropical, Indo-Pacific. Type species: Phenganaxparrini Alderslade & McFadden, 2011.(after Alderslade & McFadden 2011). Taxon classificationAnimaliaAlcyonaceaArulidae27333CB5803E5D2685480AE7926D5905http://zoobank.org/29410835-EB6E-4E9D-9361-CF60FE36ECFDHolotype: NSMT-Co 1683, Manukan, Manukan Island, TARP , 13 m depth. Paratypes: IPMB-C 01.00001: Edgell Patches, west of Sapi Island , 16 m depth. IPMB-C 01.00002: Mid Reef Slope, east of Manukan Island, TARP , 13 m depth. IPMB-C 01.00003: Mid Reef Slope, east of Manukan Island, TARP , 11 m depth. IPMB-C 01.00004: Edgell Patches, west of Sapi Island , 19 m depth. IPMB-C 01.00005: Edgell Patches, west of Sapi Island , 16 m depth. IPMB-C 01.00006: Gaya Clement Reef, west of Gaya Island, TARP , 12 m depth. IPMB-C 01.00007: Gaya Clement Reef, west of Gaya Island, TARP , 11 m depth. IPMB-C 01.00008: Manukan, north of Manukan Island, TARP , 12 m depth. IPMB-C 01.00009: Manukan, Manukan Island, TARP , 12 m depth. IPMB-C 01.00010: Manukan, Manukan Island, TARP , 12 m depth.All specimens are from Kota Kinabalu, Sabah, Malaysia and collected by YW Lau. Colony is attached to rock and sponge and consists of ~15 polyps. Stolons are consistent in width throughout the colony (~0.35 mm). Polyps are spaced apart irregularly (from 0.5 mm up to 6 mm) and retract fully into calyces; calyces do not retract into stolon and are approximately 1 mm in width and 2 mm in height. Expanded polyps 4.0\u20135.5 mm in width. The pinnules are arranged on either side of the rachis in pairs of 12\u201315, sometimes spaced apart irregularly. The pinnules give a swollen appearance and are mostly conical shaped, otherwise diamond-shaped. The colony is sclerite-free. Polyps are brownish yellow in colour, with a bright yellowish white oral disk in life (whitish yellow when preserved in ethanol). Tentacles have a greenish colour interrupted with brown specks. Zooxanthellate.IPMB-C 01.00003 and IPMB-C 01.00007 show variation in the colouration of the polyps, having a paler and pinkish appearance. There is also variation in the number of pinnule pairs (~8 pairs) and the space between the pinnules; pinnules are sometimes so densely packed there is no space between them. Like the holotype, all paratype colonies are small colonies consisting of ~20 polyps.Paratypes TARP, Kota Kinabalu, Sabah, Malaysia.Gaya, Sapi and Manukan Islands, Phenganaxmarumi sp. nov. resembles Phenganaxparrini Alderslade & McFadden, 2011 in polyp morphology, but differs in colour. The numerous and densely packed polyps described for a P.parrini colony  in which it was kept for scientific research purposes , 7 m depth. Paratypes: IPMB-C 01.00011: Sepangar II, west of Sepangar Island , 7 m depth. IPMB-C 01.00012: Manukan, north of Manukan Island, TARP , 12 m depth. IPMB-C 01.00013: Manukan, north of Manukan Island, TARP , 12 m depth.All specimens are from Kota Kinabalu, Sabah, Malaysia and collected by YW Lau. The colony consists of ~50 polyps, which are connected through thin rounded stolons with a width of approximately 0.13\u20130.2 mm, growing over coral rubble. Polyps are partly clustered and spaced apart irregularly (1.0\u20133.0 mm). Polyps retract into calyces (0.65\u20130.77 mm width) that are barrel shaped. Expanded polyps are 3.0\u20133.5 mm in width and have tentacles with pinnules that are widely spaced and arranged in pairs of 12 on either side of the rachis. No sclerites were found in any of the specimens. Polyps are brown coloured in life and whitish yellow when preserved in ethanol. Zooxanthellate.Paratypes show variation in the number of pinnules on either side of the rachis (10\u201313 pairs).TARP, Kota Kinabalu, Sabah, Malaysia.Sepangar Island, Kota Kinabalu, Sabah, Malaysia. Manukan Island, Phenganaxsubtilis sp. nov. differs from P.parrini and P.marumi sp. nov. mainly in the shape of the pinnules. Phenganaxsubtilis does not have the plump, diamond-shaped pinnules as seen in P.marumi and P.parrini. Additionally, the colour of P.subtilis is different from P.parrini and P.marumi; brown instead of the more characteristic white-grey.subtilis, meaning simple, subtle, plain; denoting the subtleness of the polyps blending into the reef background. Gender: masculine.From Latin Taxon classificationAnimaliaAlcyonaceaArulidaeDD32586E0B2E5DD7BE5BF6DB1821F2E1http://zoobank.org/5871284F-05F8-469D-AED1-0BE797C0FEB205\u00b058'35.8\"N, 116\u00b000'52.2\"E) TARP, Kota Kinabalu, Sabah, Malaysia and collected by YW Lau. Holotype: NSMT-Co 1685, 5 m depth. Paratypes: IPMB-C 01.00015: 4 m depth. IPMB-C 01.00014 4 m depth.All specimens are from dive location Mid Reef, east of Manukan Island (The colony consists of ~100 polyps which are densely packed and are connected through flattened ribbon-like stolons with irregular width (~0.21\u20130.55 mm). The colony is fragmented into two pieces with the rock it was attached to. Expanded polyps are approximately 3.0 mm in diameter and have tentacles with pinnules arranged in pairs of ten on either side of the rachis. Polyps are clustered closely together (adjacent to one another) and partly spaced apart (~1.0 mm in between clustered polyps). Polyps retract into calyces (~1.0 mm width) that are barrel shaped. No sclerites were found. Polyps are whitish grey coloured in life (whitish yellow when preserved in ethanol). Zooxanthellate.TARP, Kota Kinabalu, Sabah, Malaysia.Manukan Island, Phenganaxstokvisi sp. nov. shows similarity to the densely packed P.parrini, polyps being in the same size range (2.5\u20133.0 mm width in P.parrini), only showing difference in the number of pinnule pairs and colour of the polyps (possibly due to zooxanthellae).Named after Frank Robert Stokvis, whose passion for octocoral research has never changed since his first study on this topic.Taxon classificationAnimaliaAlcyonaceaArulidaesp.4F253F95E71D5A93952393F39A2E08B4NSMT-Co 1686, Edgell Patches, west of Sapi Island , 18 m depth, coll. YW Lau. IPMB-C 01.00016, Gaya Clement Reef, west of Gaya Island, TARP , 11 m depth, coll. YW Lau.All specimens are from Kota Kinabalu, Sabah, Malaysia. Colonies with 20\u201330 polyps are connected through flattened stolons, which have a varying width of 0.5\u20131 mm. Colonies can be loosely attached to sponge or rocky substrates, such as coral rubble. Polyps are transparent and clustered in groups, connected by stolons with lengths up to 4\u20135 mm. Expanded polyps were ~6.0\u20137.0 mm in width when alive, with the pharynx visible in all polyps. Polyps retract fully into the calyx, which is cylinder-shaped  and do not retract fully into the stolon. The tentacles have approximately 11 pairs of pinnules, which are widely spaced apart. No sclerites were found in the specimens. Polyps are whitish translucent when alive (yellowish white when preserved in ethanol). Azooxanthellate.TARP, Kota Kinabalu, Sabah, Malaysia.West of Sapi and Gaya Islands, Clavulariidae sp., can be identi\ufb01ed to the family level Clavulariidae Hickson, 1894 by its initial morphological resemblance to the type species Azoriellabayeri Lopez-Gonzalez & Gili, 2001 and Cerveraatlantica Lopez-Gonzalez et al., 1995 in having similar whitish translucent polyps, although, the polyps of C.atlantica are translucently orange. However, more diagnostic morphological features and more specimens are necessary before a genus- and species-level distinction can be made.This material, henceforth Clavulariidae sp. and A.bayeri can be found in the absence of sclerites in Clavulariidae sp. Additionally, both type species C.atlantica and A.bayeri have polyps that are smaller than in Clavulariidae sp.; C.atlantica, ~5.1 mm width, A.bayeri, ~3.6 mm width, and Clavulariidae sp., ~6.0\u20137.0 mm width. As well, C.atlantica and A.bayeri have more pinnules on either side of the tentacles than seen in Clavularidae sp.; both C.atlantica and A.bayeri have 12\u201314 pinnules and Clavulariidae sp. has tentacles with 11 pairs.The main difference between ML analyses of the separate markers  formed a completely-supported clade with the arulid genera Arula and Hana; 100%/1.00 for both the three- and four-marker datasets . Nonetheless, all genera in the clade have high support values.Sequences of arulids P, expressed as percentage) between specimens of Bunga gen. nov., Laeta gen. nov., Arula, and Hana  indicated that the levels of pairwise differences were well above those observed among congeneric species , which were only observed in Laeta but not in the other sister genera, and the absence of 6-radiates in Bunga, which were present in all sister genera. There was also a difference in sclerites of the stolon; Arula is the only genus without fused table-radiates, but instead has table-radiates in the calyx. The polyp morphology did not show many obvious differences, although two of them could be observed in the outer margins of the characteristic fused oral membrane; both Bunga and Laeta had polyps with oral discs that were less pronounced at their outer margins than those of other genera, having a smooth transition from oral disc to the connecting tentacles, while the furrows dividing the oral disc into eight lobes were shallower in both of these genera  and Phenganaxparrini Alderslade & McFadden, 2011 in a well-supported clade in both the three-marker and four-marker analyses . However, there was disparity in the relationships within the remaining clade in the two phylogeny reconstructions and the relationships within the remaining clade were not clearly resolved. The four-marker phylogeny reconstruction, with more specimens, gave a higher resolution .Sequences of 18 clavulariid specimens grouped together with n Figure  and herePhenganax were 0.57% for COI and 3.10% for mtMutS, percentages within the range of congeneric species commonly observed in octocorals  in which it was kept for scientific research , but showing differences in the number of pinnule pairs and colour of the polyps (possibly due to the presence of zooxanthellae).Phenganaxparrini; P.marumi sp. nov., P.subtilis sp. nov., and P.stokvisi sp. nov.Differences in morphology combined with genetic distances and high support values in the four-marker phylogeny reconstruction supported the erection of the three new sister species of Clavulariidae sp. had a similar DNA profile to two genera within the Clavulariidae; sequences from the two specimens clustered in a well-supported clade (96%/1.00), together with the genera Azoriella  and Cervera Lopez-Gonzalez et al., 1995, with Cervera as sister to Azoriella and Clavulariidae sp. .d clade 9%/1.00, t2 (TARP + area around Udar and Sepangar Islands), five species and two stoloniferan genera new to science were discovered. These results are not completely unexpected, as TARP is located on the outer edge of the Coral Triangle, where studies on stoloniferous octocorals are in their infancy. Of the seven families that are considered to belong to the subordinal group Stolonifera , three have members that are confirmed to occur in the Indo-Pacific  and by not having table-radiates which are elongated in shape; the other three genera all have stolons with fused table-radiates which form a sheet and all have larger table-radiates up to 0.10\u20130.19 mm length.COI, mtMutS, and ND6 sequences) resulted in similar phylogenetic positions for Bungapayung gen. nov. et sp. nov. and Laetawaheedae gen. nov. et sp. nov. when compared to our analyses of the concatenated three marker dataset . It should be noted that the two analyses including more specimens for Bunga and Laeta placed the four genera in slightly different phylogenetic positions. In the three-marker dataset . This clade was sister to Hana and Arula, which also formed a clade. In the four-marker dataset  next to the conventional DNA markers  commonly used in octocoral phylogenetic analyses , Clavulariacelebensis Hickson, 1894, Clavulariapregnans Thomson & Henderson, 1906.Multiple factors made it initially difficult to establish with certainty if the currently described species were either congeners or possibly already described. The most important factor in this matter is the small number of morphological features that represent the new Phenganax spp. described in this study and the above mentioned Clavularia spp. are the \u201cnumerous densely packed pinnules\u201d, which are sometimes as many as 30 pinnules per row; all Phenganax spp. in the current study have six to 15 pinnules on either side of the tentacle rachis. Not all measurements (for example expanded polyp width) were included in the Clavularia spp.  resulted in obtaining higher phylogenetic resolution and increased branch support values, and therefore should represent a more accurate reconstruction of evolutionary relationships. Future octocoral taxonomy will unquestionably build upon a combination of molecular and more traditional morphological techniques   . Scleriteniidae) . Scleriteniidae) . It couleniidae) , and morTARP area, off the coast of Kota Kinabalu, in Sabah, Malaysia, harbours stoloniferous octocoral diversity that was previously unknown to science, which is obvious since it represents one of the first studies focusing on Stolonifera in this region. However, the newly described sclerite-free Phenganax spp. as well as Clavulariidae sp. are only small pieces of the puzzle in the needed thorough investigation of the polyphyletic family Clavulariidae (Stolonifera and ultimately Octocorallia. Molecular data indicate that the entire Octocorallia classification needs re-structuring, but for now researchers still rely on the traditional morphological classification system (Our results demonstrate that the lariidae , and an n system , as evoln system . Integran system ."}
+{"text": "Quantifying the impact of climate change and human activities on grassland dynamics is an essential step for developing sustainable grassland ecosystem management strategies. However, the direction and magnitude of climate change and human activities in driving alpine grassland dynamic over the Tibetan Plateau remain under debates. Here, we systematically reviewed the relevant studies on the methods, main conclusions, and causes for the inconsistency in distinguishing the respective contribution of climatic and anthropogenic forces to alpine grassland dynamic. Both manipulative experiments and traditional statistical analysis show that climate warming increase biomass in alpine meadows and decrease in alpine steppes, while both alpine steppes and meadows benefit from an increase in precipitation or soil moisture. Overgrazing is a major factor for the degradation of alpine grassland in local areas with high level of human activity intensity. However, across the entire Tibetan Plateau and its subregions, four views characterize the remaining controversies: alpine grassland changes are primarily due to (1) climatic force, (2) nonclimatic force, (3) combination of anthropogenic and climatic force, or (4) alternation of anthropogenic and climatic force. Furthermore, these views also show spatial inconsistencies. Differences on the source and quality of remote sensing products, the structure and parameter of models, and overlooking the spatiotemporal heterogeneity of human activity intensity contribute to current disagreements. In this review, we highlight the necessity for taking the spatiotemporal heterogeneity of human activity intensity into account in the models of attribution assessment, and the importance for accurate validation of climatic and anthropogenic contribution to alpine grassland variation at multiple scales for future studies. Finally, the relative contribution of climatic and anthropogenic factors to alpine grassland changes can be identified by comparing the trends of NPPH and NPPP , and Aqua  climatic force, (2) nonclimatic force, (3) combination of anthropogenic and climatic force, or (4) alternation of anthropogenic and climatic force. These views also show spatial inconsistencies and lack validation. The current challenges in distinguishing climatic and anthropogenic contributions to alpine grassland variation on Tibetan Plateau mainly include the uncertainties in quality of remote sensing products, uncertainties in the accuracy of models, especially for simulating the potential vegetation growth, and overlooking spatiotemporal heterogeneity of human activity intensity. Thus, it is necessary to evaluate the differences among the multiple remote sensing datasets before inputting some of them into the models of attribution assessment, to take the spatiotemporal heterogeneity of human activity intensity into account in the models, and further to improve the accuracy of models. We also proposed a framework for accurate validation of anthropogenic and climatic contribution to alpine grassland variation at multiple scales for future studies.None declared.Zhang Y. and Liu L. designed the study, and Li L. wrote most of the first draft. Wu J., Li S., and Zhang H. contributed to the analysis and interpretation of the results. Zhang B., Ding M., Wang Z., and Paudel B. contributed to revise the manuscript and provided conceptual advice. All authors contributed to final approval for publication.\u00a0Click here for additional data file."}
+{"text": "Achromobacter and four Pseudomonas strains isolated from sediments of the River Elbe which are highly tolerant toward the xenobiotic target compound diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) and emerging contaminant.Here, we report the genome sequences of one  Achromobacter and four Pseudomonas strains isolated from sediments of the River Elbe which are highly tolerant toward the xenobiotic target compound diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) and emerging contaminant.Here, we report the genome sequences of one  Diclofenac, an over-the-counter medication used in many countries, has a yearly production volume of about 90 tons in Germany and 800 tons in India. Insufficient removal by wastewater treatment plants has led to an increasing occurrence of diclofenac, as well as that of other pharmaceuticals and personal care products (PPCPs), in surface waters such as the River Elbe in Germany . MoreoveIsolates were obtained from enrichment cultures of sediment samples from the River Elbe downstream of the Hamburg harbor  with 0.5de novo with Velvet (version 1.2.10) and VelvetOptimiser (version 2.2.5) . The contigs obtained were further annotated with the Rapid Annotations using Subsystems Technology (RAST) server version 2.0 . The quality and quantity of the DNA were assessed with the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). To obtain the draft genome of each isolate, 300-bp paired-end sequencing libraries were prepared using the Illumina Nextera XT DNA library version 3 sample preparation kit, and sequencing was performed with the Illumina MiSeq platform at the Center for Scientific Instrumentation of the University of Granada (Spain). Sequence reads were assessed for quality using FastQC , filtered using Trimmomatic , and assn 2.2.5)  within asion 2.0 , and forsion 2.0 .rpoD, recA, and gyrB genes (Achromobacter strain from 69.2 to 70.7%, which are all well below the 95 to 96% species threshold (Genome sizes vary between 5,862 and 7,346 Mbp and GC contents from 61.8 to 67.6% . TaxonomrB genes  with a BrB genes . As possrB genes  ranged bhreshold .The whole-genome sequences have been deposited in DDBJ/ENA/GenBank under the accession numbers listed in"}
+{"text": "Neisseria meningitidis (N.meningitidis) bacteria belonging to clonal complex 4821 (CC4821) have been mainly reported in China and have been characterized by a high resistance rate to ciprofloxacin (CIP). The aim of this study was to assess the evolution of the DNA gyrase A (gyrA) gene from N.meningitidis CC4821 strains collected in China between 1978 and 2016. The complete sequence of gyrA gene from 77 strains are reported in this study and analyzed in the context of publicly available sequences from N. meningitidis of other CCs as well as other Neisseria species.gyrA gene reveals at least 5 distinct genetic clusters. These clusters are not CC4821-specific showing that gyrA evolution is independent of CC4821 evolution. Some clusters contain sequences from other Neisseria species. Recombination within N.meningitidis strains and between Neisseria species was identified in SimPlot analysis. Finally, amino acid substitutions within GyrA protein were analyzed. Only one position, 91 (83 in E.coli gyrA gene), was linked to CIP resistance. Thirty-one additional putative resistance markers were identified, as amino acid substitutions were only found in resistant strains.The phylogenetic analysis of CC4821 gyrA gene of CC4821 N.meningitidis strains is not dependent on CC4821 evolution or on CIP resistance phenotype. Only amino acid 91 is linked to CIP resistance phenotype. Finally, recombination inter- and intra-species is likely to result in the acquisition of various resistance markers, 31 of them\u00a0being putatively mapped in the present study. Analyzing the evolution of gyrA gene within CC4821 strains is critical to monitor the CIP resistance phenotype and the acquisition of new resistance markers. Such studies are necessary for the control of the meningococcal disease and the development of new drugs targeting DNA gyrase.The evolution of  This disease can have multiple causes like bacteria or virus but also fungus, parasite or even non-infectious agent like lupus. Bacterial meningitis can be caused by several types of bacteria including Streptococcus pneumonia or Neisseria meningitidis (N.meningitidis). In addition to N.meningitidis, the genus Neisseria contains at least 30 distinct species colonizing humans, other mammalians and even insects [N.meningitidis are classified through several different schemes, based on serological test (serogroup) or genetic tests (sequence type) [cps), 6 serogroups  caused the majority of invasive meningococcal disease (IMD) globally [abcZ, adk, aroE, fumC, gdh, pdhC, pgm) [https://pubmlst.org/neisseria/). The strains that could not be classified into an existing CC were called unassigned (UA).Meningitis is an inflammation of the protective membranes covering the brain and the spinal cord ( insects . N.menince type) , 3. Amonglobally . In addiN. meningitidis of serogroup A with either CC1 or CC5 were responsible for the majority of IMD cases until 2003 [In China, the strains of til 2003 . In 2003til 2003 . Later otil 2003 . Analysetil 2003 .E.coli protein [Two main strategies have been developed to control the meningococcal disease. Vaccines specific to multiple serogroups have been generated and are globally used . Further protein . The mut protein .gyrA gene from CC4821 strains collected in mainland China between 1978 and 2016. The sequences were compared to gyrA sequences from N,meningitidis with different CCs as well as sequences from other species of Neisseria. Monitoring the evolution of gyrA gene, especially the sites involved in antibiotic resistance is capital to control meningococcal disease.The aim of this study was to assess the evolution of the gyrA sequences of CC4821 N.meningitidis were analyzed in the context of 149 publicly available gyrA sequences  were found on the top of the tree, with no significant bootstrap value  and 1 sequence from N. cinerea, namely N.cinerea-LS483369.1-NCTC10294, which shared a node with N.meningitidis-KF733132\u201359-ST7962-CC77-NG-China-2009-R. However, the N.cinerea sequence featured a long branch suggesting a significant divergence compared to the N.meningitidis sequence. Based on the available data, the sequences from group 6 appeared to be from strains that were resistant to CIP. However, no unique amino acid substitution was shared by these strains suggesting that there was no common marker for the resistance phenotype of these strains  analyzed in this report. The strains were collected in the last 88\u2009years in 13 different countries from 4 continents. Despite the significant time span and geographic spread, these sequences were highly conserved with a p-distance of 0.003. Moreover, these sequences were from strains of 68 STs, 24 CCs, 9 serogroups, including the reference strain 053442. Overall, these observations showed that gyrA gene was highly conserved among most N.meningitidis strains despite different genetic characteristics, geographic locations or collection time.Group 8 contained most of the E.coli, the first region corresponds to the end of the amino terminal domain and the beginning of the carboxy terminal domain. The second region corresponds to the tower domain of the protein based on the 3D model structure  were highly divergent, 40% or more of the 129 sequences were mutated at these positions. For example, 48% of the 129 sequences featured a mutated residue at position 417  and N.cinerea-LS483369.1-NCTC10294 -----. Fig. N.lactamica-CP031253.1-M17106----- and two N.meningitidis strains, N.meningitidis-KJ415206.1\u201354-R6----- and N.meningitidis-CP000381.1\u201353,442-ST4821-CC4821-C-China-2004_R. Finally, a recombination between N.meningitidis strains was featured in Fig. gyrA gene of Neisseria strains is highly prone to recombination.The phylogenetic analysis identified potential recombinants. For example, the group 1 was of particular interest, concerning N.meningitidis strains are endemic in China. CC4821 strains have been first detected in 2003 in Anhui province, China. Since then, CC4821 strains have been detected in more than 20 provinces in China and have been causing deaths and serious disease burden [gyrA gene from CC5 strains from China. It showed 2 main lineages depending on the CIP resistance phenotype. It also showed a quasi-clonal evolution of the strains [gyrA gene independent on their CIP resistance phenotype. Second, multiple gyrA lineages can be identified and no link to CIP resistance can be identified. Finally, gyrA evolution does not appear to be linked to the evolution of any clonal complex. The discrepancy between the gyrA evolution of CC4821 and CC5 strains might be due to the fact that CC4821 strains are spread throughout the country, therefore increasing the chance for genetic exchange with other Neisseria strains.CC4821 e burden . CC4821 e burden , 15. A rE.coli demonstrated that CIP interact with a region called Quinolone Resistance Determining Region located at positions 67\u2013106. Six sites have been reported to be involved in resistance mechanism, namely 67, 81, 83, 84, 87 and 106 [Neisseria gyrA counterpart, 75 to 114. However, as shown in the present study, only the position 91  appears to be linked to a CIP resistance phenotype. Furthermore, the present study identified strains baring the mutation T91I but featuring an intermediate resistance phenotype. Altogether, this suggests that Neisseria might have a different resistance mechanism. An in silico analysis presented in the current study identified 31 positions potentially involved in a resistance mechanism. They are located outside the so-called QRDR. Interestingly, PubMLST, which is a tool to analyze Neisseria strains, is using a 525\u2009nt long region  to identify gyrA allele suggesting that sites outside QRDR are likely to be involved in the resistance mechanism. Further analysis would be necessary to assess the role of these positions in the resistance mechanism. Unfortunately, as far as we know, the 3D structure model does not feature some of these sites as it\u00a0was generated with the GyrA59 region of the protein corresponding to the amino terminal domain of the protein [CIP is one of the most used of the third generation quinolone antibiotics. It has been recommended for the treatment and prevention of IMD worldwide . Studies and 106 . A regio protein .Neisseria species have been previously reported. For example, Wu et al. reported a likely recombination between the genome of N.lactamica and N.meningitidis strains [gyrA gene of Neisseria species. Recombination would mean that the bacteria have been replicating at the same time and same location. Among the reported recombinations, a recombination within gyrA gene between N.meningitidis and N.subflava was identified. Unfortunately, the metadata concerning the N.subflava strain was very limited, only the collection year was identified after contacting the scientists who reported the genome sequence in GenBank. Knowing the country of collection would help to understand a potential recombination with N.meningitidis strain\u00a0collected in China. Recombination between species as well as intra-species is likely to allow the strains to acquire new resistant phenotype however further studies would be necessary to better assess the acquisition of the resistance phenotype.Recombination between the genomes of  strains .The presgyrA gene from CC4821 N.meningitidis strains in the context of other Neisseria species showed that gyrA gene is well conserved among most CC4821 strains. However, significant divergence is observed in a few strains. GyrA gene evolution does not appear to be linked to CIP resistance phenotype. An in silico analysis of amino acid mutations within GyrA protein showed that only mutations at positions 91 were linked to a CIP resistance phenotype. The analysis also suggested that other sites outside the so-called QRDR could be involved in the resistance mechanism. Recombination inter- and intra-species could explain how strains can acquire mutations leading to various resistance phenotype. Analyzing the evolution of gyrA gene is critical to monitor the resistance to quinolone and the acquisition of new resistance markers. Such studies are necessary for the control of the meningococcal disease and the development of new drugs targeting DNA gyrase.A phylogenetic analysis of N.meningitidis isolates have been collected throughout China from IMD patients as well as close contacts and asymptomatic carriers by our laboratory since 1960s. The bacterial strains were propagated on single Petri dish containing Difco\u2122 Columbia Blood Agar Base with 5% Sheep Blood in a 5% CO2 atmosphere at 37\u2009\u00b0C for 18\u2009h. Single colonies were lysed and tested by PCR for the meningococcal-specific contact-regulated gene A (crgA) in order to identify bacterial species [abcZ, adk, aroE, fumC, gdh, pdhC and pgm) were amplified and sequenced. STs and CCs were subsequently assigned by querying the sequence database available at http://pubmlst.org/. Seventy-seven CC4821 isolates were selected as representative isolates based on the typing results (ST and serogroup), isolation year (1978\u20132016) and sample source  for in-depth phylogenetic analysis. The gyrA gene was sequenced by PCR using the primer pair gyrA-F, 5\u2032-GTTCCGCGTCAAAATATGCT-3\u2032 and gyrA-2844R, 5\u2032-GACTATAATCCGCTATATTGT-3\u2032 generating a 2905\u2009bp long amplicon containing the 2751\u2009nt of gyrA gene [More than 4,000\u2009gyrA gene from the reference strain N.meningitidis 053442 (CP000381.1) was used to query the GenBank database and 556 additional gyrA gene sequences were selected from the BLAST output. Identical sequences were deleted unless the strains did not share the same CC, ST or serogroup. Sequences less than 90% of full length (2905\u2009nt) were discarded. The remaining 149 sequences were combined with the 77 sequences generated in this study. The distribution in terms of species, country of collection and year of collection is shown in Additional Figure The gyrA nucleotide sequences were aligned using Mega 6 and a neighbor joining phylogenetic tree was generated using the maximum composite likelihood nucleotide model [Two hundred twenty six de model , 21. Phyde model . Nodes wde model .N.meningitidis strains were incubated at 35\u2009\u00b0C, 5% CO2 in chocolate agar  for 20\u201324\u2009h. A single colony was selected and grown to reach a 0.5 McFarland standard. Escherichia coli ATCC 25922 strain was used as a quality control strain. The bacterial suspension was evenly spread on Mueller-Hinton blood plate . A CIP Epsilometer-test strip  was then attached to the plate. The Minimal Inhibitory Concentration (MIC) was read after incubation at 35\u2009\u00b0C, 5% CO2 for 18\u201320\u2009h. The CIP resistance test was interpreted as established by Clinical and Laboratory Standards Institute (CLSI): S(susceptible)\u2009\u2264\u20090.03\u2009\u03bcg/mL, I(intermediate)\u2009=\u20090.06\u2009\u03bcg/mL, R(resistant)\u2009\u2265\u20090.12\u2009\u03bcg/mL [Additional file1 Figure S1. Maximum likelihood phylogenetic tree of 226 gyrA gene sequences from Neisseria strains. The tree was annotated according to Fig. Additional file 2 Figure S2. Distribution of species, collection country and year of collection for the 226 gyrA gene sequences analyzed in this study. A. Distribution of species. The number of strains for each species is plotted. The dataset contains 192\u2009N.meningitidis strains and 12\u2009N.gonorrhoeae strains. The species are organized based on their host, human specific first. Eikenella belongs to the Neisseria genus. B. Distribution based on collection country. The dataset contains 146 strains from China. C. Distribution based on collection year.Additional file 3 Figure S3. Distribution of ST, CC, serogroup as well as CIP resistance phenotype of the 192\u2009N.meningitidis strains analyzed in this study. A. Distribution of ST among the 88 CC4821 strains. B. Distributions of CC and ST of 86 strains which do not belong to CC4821 and are represented by 2 or more STs in the dataset. ND; Not determined. Strains with no ST are indicated with \u201c-\u201c. C. Distributions of CC and ST of 18 strains represented by only 1 ST in the dataset. D. Distribution of serogroup among the 192\u2009N.meningitidis strains analyzed in this study. Serogroup information was not available for 26 strains. The CIP resistance phenotype is color coded, orange bar for sensitive strains and grey bar for resistance strains. The strains for which the CIP resistance has not been tested are indicated with a blue bar. Intermediate phenotype was considered as resistance in the figure.Additional file 4 Table S1. List of the 226 analyzed strains.Additional file 5 Table S2. Amino acid difference among the 226 GyrA protein sequences.Additional file 6 Table S3. Potential CIP resistance markers among the 226 GyrA protein sequences analyzed in this study.Additional file 7 Table S4. Putative resistance markers based on the analysis of 226 GyrA protein sequences.Additional file 8 Table S5. Alignment of GyrA protein sequence from E.coli K12 and N.meningitidis reference strain 053442."}
+{"text": "Circulating monocytes contribute to inflammatory processes. We here validate abnormal expression of inflammation-related genes in monocytes of a large and well-characterised group of MDD patients, and relate the outcomes to pertinent clinical characteristics. Thirty-two genes of a previously established inflammation-related gene signature were assessed in 197 patients with MDD, and 151 controls collected during the EU-MOODINFLAME project. Monocyte gene- expression data were related to age, sex, BMI, depression severity, childhood adversity (CA) and suicide risk (SR). Three distinct gene profiles were identified within the MDD group . Patients in the merged upregulated groups had a significantly higher prevalence of CA and high SR. Using hierarchical clustering of the genes, we found a cluster of mainly cytokine (production)-related genes; patients with SR had a significantly higher expression of this cluster than patients without SR . Such difference did not emerge for patients with and without CA. A downregulated gene profile was found for patients not exposed to CA and without SR . No inflammatory changes were observed for healthy controls exposed to CA. Our data show that inflammatory activation in MDD is not uniform, and that immunologically discernible phenotypes of depression can be linked to CA and high SR. The absence of monocyte inflammatory activation in healthy controls exposed to CA suggests an inflammatory involvement in MDD-prone individuals exposed to early stressors, but not healthy controls. The exploration of clinical risk factors in combination with biological targets is particularly appealing for identification of MDD subgroups, but ultimately, for tailored treatment of different MDD phenotypes. One such biological target is the immune system. Over the past decades, evidence has been presented, which supports the concept that low-grade inflammation plays a role in the pathogenesis of MDD. An increase in inflammatory serum cytokines4, C-reactive protein5, overall leukocytosis, inflammatory monocytosis, activated glial cell status and a beneficial response to anti-inflammatory therapy have been demonstrated9. Nevertheless, there are also reports showing rises in anti-inflammatory cytokines11, reduced lymphocyte-proliferative response to mitogen7 and overall non-responsiveness to anti-inflammatory therapy12, creating a more complex picture of inflammatory activation in MDD. Recent research has suggested that high levels of inflammatory markers are associated with treatment resistance13, and has shown that particularly those with high levels of inflammatory markers may benefit from anti-inflammatory agents12. In previous research, we have contributed to the understanding of altered inflammatory states in psychopathology by studying gene expression of circulating monocytes in mood-disorder patients17. Blood monocytes are among the principal cells of the innate immune system, and take on important roles in phagocytosis, antigen presentation and cytokine production18. We previously established an abnormal expression pattern of inflammation-related genes (a \u201cgene signature\u201d), that is altered in monocytes of patients with bipolar disorder15, unipolar depression17, postpartum psychosis and endocrine autoimmune disorders such as diabetes19, pointing to a shared inflammatory activation between these disorders. The gene signature was derived by selecting the top up- or downregulated genes in pre-studies using whole-genome analyses. Additional validation was achieved in various confirmation studies using Q-PCR. The signature contains genes that have previously been implicated in the pathogensis of MDD, including interleukin (IL)-6, IL1B, TNF, the glucocorticoid receptor subunits \u03b1 and \u03b2 and the MAP kinase pathway. More information on the identification and validation of the signature can be found in 20, and by others21, lends further independent support to the notion of aberrant activation of innate immune myeloid cells in depressionMajor depressive disorder (MDD) is a highly prevalent disorder24, can have an important impact on the inflammatory-response system. Yet, few current studies focus on these specific clinical characteristics. Secondly, previous studies have a limited sample size of patients or controls, which limits power to detect the additive main and interaction effects. We here tackle these caveats by exploring the relationship of inflammation-related gene expression and pertinent clinical characteristics  and CA) in a large group of MDD patients.However, two important shortcomings hamper interpretability of previous findings. Firstly, it is well established that clinical characteristics, such as depression severity, suicidal ideation and childhood adversity (CA), as well as medication status, age and obesityOur primary aim for this study is to assess whether these clinical characteristics are linked to up- and/or downregulated gene expression in patients with MDD, and can explain (part of) the reported heterogeneity of inflammatory activation. For this purpose, we use in the first approach an unsupervised clustering on the well-defined, previously identified genes, to identify in an unbiased fashion subgroups of MDD patients with predominantly upregulated, downregulated or mixed-up and downregulated inflammation-related genes. We explore the distribution of clinical characteristics between these different MDD subgroups with up- or downregulated gene expression, and focussed in particular on demographic characteristcs, depression severity, CA and SR, as these characteristics have been previously linked to inflammatory activation of the immune system, as described above.In the second approach, we performed a hierarchical clustering of the expression of the genes to identify clusters of genes correlating in expression intensity. We identified three gene clusters, and investigated whether MDD patients with or without the described clinical characteristics  significantly differed in the clusterwise expression of the genes. We hypothesised that upregulated inflammation-related gene expression is not uniform across patients with depression, and that depression severity, CA and SR are linked to specific inflammatory activation in MDD. In addition, as the secondary aim, we also determined serum levels of interleukin 6 (IL-6) and highly sensitive C-reactive protein (hsCRP) to assess whether monocyte gene expression is also reflected in serum markers of inflammatory activity, and whether these serum protein markers are also related to the mentioned clinical characteristics such as CA and SR.Data were derived from the EU-funded MOODINFLAME study (\u201cMOODINFLAME website\u201d 2014) carried out to investigate possible inflammatory biomarkers to advance early diagnosis, treatment and prevention of mood disorders. Participants were adults free of clinical inflammation-related symptoms, including fever and current or recent infectious or inflammatory disease, uncontrolled systemic disease, uncontrolled metabolic disease or other significant uncontrolled somatic disorders known to affect mood. Patients did not use somatic medication known to affect mood or the immune system. Pregnant or postpartum candidates were excluded. This study was set up as a cross-sectional case\u2013control study. Peripheral blood samples of 212 controls and 202 MDD patients were analysed.14, and from as yet unpublished cohorts from Munich (Germany) and Leuven (Belgium). Samples were collected from 2009 to 2012 using uniform clinical selection/evaluation and laboratory techniques. The study was approved by ethical committees of the participating universities , and written informed consent was obtained from all participants.Patients were recruited from three university psychiatry clinics, i.e., M\u00fcnster (Germany), which we partly reported in a previous publication25. The severity of depression was measured by the inventory of depressive symptoms (IDS-C30)26 for patients, for controls with self-report questionnaires (IDS-SR30). The presence of CA was assessed with the childhood trauma questionnaire short form (CTQ-SF)27. Subscales of the CTQ include emotional or physical abuse, and neglect and sexual abuse. Cut-off scores to determine the presence of trauma on any subscale were previously established28. SR status was derived by the MINI section C, where to score high risk, patients need to endorse (1) active suicidal ideation with concrete suicidal plans, (2) have attempted suicide in the past month or (3) have attempted suicide (not during the last month) and currently actively thinking about attempting suicide. Control participants underwent psychiatric assessment with the MINI screening version. In the case of additional questions, the full MINI questionnaire was administered. Assessment of depression diagnosis with the MINI occurred on the same day as the blood sampling.DSM-IV MDD diagnoses were confirmed using the mini-international neuropsychiatric interview (MINI)Determination of IL-6 and hsCRP in serum: For the IL-6 determination, pre-coated microtiter strips were incubated with standards and diluted samples . Unbound serum proteins were removed (washing). Next, a monoclonal biotin conjugate was added to bind directly to the antibody\u2013antigen complex. After incubation , unbound biotin antibodies were removed (washing). Next, a streptavidin poly-HRP conjugate was added and strips incubated . After removal of the unbound conjugate, a TMB-containing solution was added to the wells and incubated . A blue colour developed in proportion to the amount of IL-6 bound. The reaction was stopped (0.5\u2009M stop solution), and signals read at 450\u2009nm.The hsCRP assay was done according to the apDia hsCRP (REF: 740011) manual: 10\u00d7 prediluted standards were diluted 1:100 and serum samples 1:1000. Microtiter strips were incubated with the diluted standards and sera ). Any unbound serum proteins were removed (washing), and a specific HRP-conjugated antibody added to detect the antibody\u2013antigen complex. Strips were incubated . After removal of the unbound conjugate, a chromogen solution containing TMB was added. During the incubation for 10\u2009min at RT, a blue colour develops. The reaction was stopped (0.5\u2009M stop solution), and signals read at 450\u2009nm.17. Shortly, RNA was isolated from CD14\u2009+\u2009purified monocytes; to obtain cDNA for quantitative-polymerase chain reaction (q-PCR), 1\u2009\u03bcg of RNA was reverse-transcribed using the cDNA high-capacity cDNA Reverse Transcription Kit . Then, relative to the housekeeping gene ABL1, the expression of the genes ADM, ATF3, BCL2A1, BTG3, CCL2, CCL20, CCL7, CD9, CDC42, CXCL2, DHRS3, DUSP2, EMP1, EREG, FABP5, HSPA1A/HSPA1B, IL-1\u03b1, IL-1\u03b2, IL1R1, IL-6, IRAK2, MAFF, MAPK6, MXD1, NAB2, PDE4B, PTGS2, PTPN7, PTX3, SERPINB2, STX1A, THBD, TNF and TNFAIP3 was determined, using the comparative threshold cycle (CT) method , yielding \u0394CT values. Due to minor technical differences in leukocyte isolation procedures, we expressed patient data relative to values of controls of the same site by deriving \u0394\u0394CT values before pooling data29. Complete control cases without CA were used as reference group. Data are deposited in the NCBI\u2019s GEO repository (accession number GSE147582-GSE147584).To detect expression of inflammation-related genes in CD14\u2009+\u2009monocytes, collection, purification and determination methods were used as described in previous publicationsn\u2009=\u2009197) and controls with CA (n\u2009=\u200956) was expressed relative to complete control cases without CA (n\u2009=\u200995). Genes with a pre-established threshold of <30% of missing data per site and 20% from merged centres (n\u2009=\u200932) were used for analysis and imputed using median imputation for graphical representations, and multiple imputation using chained equations (mice package)30 for multivariate analysis with covariates. Predetermined power analysis yielded a necessary sample size of 196 to detect even a small group effect  with 80% power.Statistical analyses and graphical representations were performed in R version 3.5.1 and Graphpad Prism 8.0.0. Gene expression of MDD patients  using the Benjamini\u2013Hochberg method. To identify patient subgroups, \u0394\u0394CT values were used for hierarchical clustering, using Euclidean distance and ward\u2019s (ward.d2) agglomeration method31. Optimal numbers of clusters were determined using the package Nbclust in R32. Clinical characteristics of subgroups were compared for the bottom-up approach, with chi-square tests for frequencies, or Wilcoxon\u2013Mann\u2013Whitney test for continuous variables. We also assessed correlated gene clusters. Within these gene clusters, the effects of significant clinical variables were explored in a top-down approach. This included a comparison of patients and controls with CA or SR compared to controls without CA. Due to a relatively small sample size, P values are not reported with correction for FDR. Corrected values are provided in the Supplementary Information. Multivariate analysis of variance with age, sex, BMI, depression severity and CA and SR, as well as an interaction term between SR and CA, was performed on all five datasets arising from the multiple imputation. F values of significant effects arising during multiple imputation were pooled to calculate an approximate overall P value. Pooled P values for single genes were obtained with the mice package30. Separate analyses were performed adding antidepressant medication status. Contrasts for single genes are computed with the no-risk group as reference, and are reported without correction for FDR. Spearman\u2019s rank-correlation coefficient was used to correlate gene-expression levels with serum protein levels. Where appropriate, data were transformed using Tukey\u2019s ladder of power transformations to achieve normality, or if this could not be achieved, non-parametric tests were used.Independent variables for the multivariate analysis were SR and childhood trauma score , and age, BMI and depression severity (continuous) as well as sex . Comparisons for gene expression compared to healthy controls were performed using one-sample \u03c72\u2009=\u200913.36, P\u2009=\u20090.004). This relationship is well described in the literature33 and taken into account in multivariate analysis of covariance (MANCOVA) models, where SR, CA and their interaction were included. Virtually, all patients used a variety of antidepressants , yielding a final sample of 197 MDD patients and 151 controls. Significant differences of demographic data between control and patient groups emerged for depression severity, CA and BMI, all higher in patients, as expected. Serum IL-6 and hsCRP levels were higher in the MDD group, as a trend effect for hsCRP and significantly for IL-6 (Supplementary Table n\u2009=\u200995). Seventeen genes reached statistical significance after correction for FDR , one overall mixed upregulation  and one clear downregulation of monocyte genes  . Subgroups with different gene expression did not differ regarding the distribution of age, sex, BMI, depression severity and medication status. However, more patients in mixed- and combined upregulated compared to the downregulated groups had high SR, and showed higher levels of CA (Table CA and SR are more frequent in MDD patients with (mixed) upregulated inflammation-related genes in monocytes. A closer examination of gene expression revealed high variability within MDD patients, as hypothesised. Three immunologically distinct groups of patients could be identified through hierarchical clustering: one group showing high upregulation  Fig. . In addiCA Table .Fig. 2Hi17 . Amongst the upregulated genes, the well-known genes encoding inflammatory interleukins, IL1A, IL1B and IL-6, were significantly higher in the high-risk compared to both healthy controls   Fig. , and the05) Fig. . Althougn\u2009=\u200956) , out of which 52% significantly, most also after correction for FDR . Age, sex and depression severity had significant effects on single genes, and were taken into account for the final models, but were not significant for overall clusters as explored by MANCOVA. Medication status as a binary variable showed significant effects, but did not alter the effects for single genes. Yet, due to the high variability of medication usage and low number of unmedicated patients, we did not have sufficient power to investigate these effects with certainty. Additional analyses exploring CA subtypes revealed that cluster 1 was linked to emotional neglect (P\u2009= <0.001\u20130.005) and emotional abuse (P\u2009=\u20090.011\u20130.164), cluster 2 gene expression to physical neglect (P\u2009=\u20090.002\u20130.024) and sexual abuse (P\u2009=\u20090.011\u20130340) and cluster 3 gene expression to emotional abuse (P\u2009<\u20090.001\u20130.015) and emotional neglect (0.001\u20130.067).Analysis of covariates showed significant effects of BMI on cluster 1 gene expression with significant levels for TNF expression . In controls, ADM and TNFAIP3 were weakly correlated with hsCRP after correction for multiple testing .P\u2009>\u20090.50). However, for hsCRP, next to significant main effects for CA and BMI, a significant interaction between those factors emerged . Splitting the groups per BMI  showed that for those with a lower BMI, CA significantly correlated with increased hsCRP , while no effect emerged for those in the high BMI group . Of note, CA per se was not associated with a higher BMI . No effect on IL-6 was observed for CA  or SR .When investigating the effect of SR and childhood trauma groups on serum protein levels while taking into account age, sex, depression severity and BMI for MDD patients, no significant effect emerged for SR on hsCRP  in depressed patients. TSPO is usually upregulated in activated (micro-) glia and serves as a proxy for neuroinflammation. The authors found elevated levels in MDD patients at SR, but not those without46, further highlighting the distinction between these subgroups of patients, conceivably also in the central nervous system. It should also be noted that serum protein levels of IL-6 poorly correlated with the monocyte gene expression. A poor correlation can be suspected, given that this acute-phase protein\u2019s production also depends on, among others, IL-6 production by other cell types , and thus probably reflects the diverse origin and interactions that lead to circulating protein levels of hsCRP and IL-6.Similar to the patient group with CA exposure, high SR was associated with clear upregulation of genes in clusters 1 and 2. Inflammatory activation of the immune system in patients with SR has recently been described regarding cytokines in serum, cerebrospinal fluid and postmortem studies47, and accumulating evidence seems to support this notion49. For instance, DUSP2 has previously been identified to link stress experience and depression pathogenesis: DUSP1 and DUSP2 expression was increased in response to chronic stress in rodents and in the brain of two separate cohorts of depressed patients50.In addition to the well-known interleukins, we found an association of high SR and CA with several MAPK pathway-related genes. The involvement of the MAPK pathway in the pathogenesis of MDD has long been suspected52: it was shown that MAPK1 levels normalised after antidepressant treatment with fluoxetine49, and Reus and colleagues have shown that acute blockade of MAPK signalling abolished the antidepressant effects of ketamine in a rat model51. Our results may help to identify more efficacious treatment options, depending on clinical and immune profiles.Many of the cluster 1 and 2 genes previously picked up by whole-genome analysis, belong to the  MAPK pathway. It is hence not surprising that the MAPK pathway shows significant results in the present analysis. However, the fact that this pathway is clearly upregulated in clinically distinct patient groups is intriguing and may provide useful hints as to molecular mechanisms distinguishing  subtypes of depression. Of note, the effect was higher in patients exposed to CA and SR even when depression severity was included in the model, and hence does not merely describe more severe depression. Interestingly, antidepressant actions of regular and rapid-acting antidepressants such as ketamine may depend on MAPK signalling54. Although we here did not observe an interaction effect on specific genes, it is therefore tempting to speculate that CA is linked to suicidal ideation through a long-lasting activation of the monocyte/macrophage system, possibly involving inflammatory compounds and/or the MAPK pathway.Importantly, CA increases the risk for suicidal behaviour55. Monocytes are generally short-lived  in the circulation56. While initially thought to be deprived of any form of immune memory (unlike the T- and B-cell systems), it is now accepted that monocytes/macrophages do build up immune memory by epigenetic imprinting after repetitive exposure to stimulating agents55. One possibility is therefore that the here-described altered regulation of monocyte gene expression in adult MDD patients could be explained by long-lasting epigenetic changes in monocytes in a process of \u201ctrained immunity\u201d.The recently accepted theorem of \u201ctrained immunity\u201d of the innate cell lineage provides a scientific framework that can explain long-lasting changed equilibrium states of steroid-sensitive and inflammation-related genes in monocytes/macrophages14 and endocrine signalling, such as HPA-axis abnormalities57. Indeed, we observed a downregulation of genes in the glucocorticoid-related cluster 3 for patients not exposed to CA and without SR. The genes NR3C1a and HSPA1A/B play important roles in the bidirectional crosstalk between the immune system and the HPA axis59. While NR3C1a encodes the stimulating subunit of the GR, HSPA1A/B encodes the protein HSP70, which regulates GR function60. Downregulation of both may thus alter glucocorticoid sensitivity. Indeed, an altered HPA axis, high levels of glucocorticoids and immune/inflammatory factors have been conceived as offenders in the pathophysiology of MDD59. Patients without SR or not exposed to CA may thus constitute another subtype of depression, characterised by different states of sensitivity for glucocorticoids. Whether these different states of expression of steroid sensitivity-related genes do lead to higher or lower sensitivity for glucocorticoids and impact inflammatory activation, needs further exploration in functional studies.As discussed before, CA per se did not affect monocyte inflammatory state in controls, but it did in patients. This suggests that additional mechanisms confer a vulnerability for depression. These mechanisms might be (a combination of) immune-controlling mechanisms61. Our findings suggest that this practice requires caution: patients with a downregulated monocyte phenotype may not benefit, or even incur harm from further immunological suppression. Stratification of patients based on peripheral markers has proved useful before62. Our results support this notion and urge for further studies to stratify patients in \u201cinflamed\u201d and \u201cnot inflamed/supressed\u201d, when studying the effect of regular antidepressants and anti-inflammatory drugs. When designing future studies, the effect of CA and SR on inflammatory activation should be recognised and accounted for during potential treatment stratification, as to lead to more successful treatment selection. Ideally, future study designs would use a longitudinal design in order to assess the relationship between CA and SR more extensively.The subdivision of MDD in immunologically discernible phenotypes is important in light of current discussions on anti-inflammatory agents to treat patients with MDD63. In addition, we here identified a mixed upregulated cluster, which showed upregulation in ~78% of genes. However, several of the genes did not show a strong pattern of upregulation, and it could therefore also be argued that these patients show no alteration. An important limitation is also that inflammatory gene expression in circulating monocytes does not correspond to peripheral protein levels of the inflammatory compounds. We here found no association of SR with serum protein levels of IL-6 and with hsCRP, this may indicate that our findings are specific to certain cellular compartments of the innate immune system and not generalisable to the overall immune state. Lastly, the results comparing no- and high SR groups were not corrected for FDR. Although we had a large group of MDD patients, subdivision based on SR yielded comparatively small groups. Our analyses should be replicated with particular focus on SR.Firstly, the nature of this study is associative, and although we can probably assume temporal precedence of CA to gene signature overexpression, we cannot assume a causal relationship between the two. We here used an arbitrary yet previously validated cut-off of exposure to CA as a binary variable; however, the grade of exposure to CA may certainly impact depression pathogenesis. Furthermore, as indicated before, we used monocyte inflammation-related genes preselected in earlier and previously published findings and validation studies. MDD patients and controls with and without CA may very well have other monocyte gene-expression changes not directly related to inflammation and not reflected in the here-described set of preselected genes: others also showed the effects of immediate stress exposure on a few of the same inflammation-related monocyte genes as described here , and on genes involved in other pathwaysOur data suggest immunologically discernible phenotypes of depression: while most patients indeed show signs of inflammatory activation, a considerable number of patients show downregulated gene expression of peripheral monocytes. Interestingly, both CA and high SR were linked to increased expression of genes encoding inflammatory compounds. Although no causal assumption can be made based on the presented cross-sectional data, these results highlight the importance of determining clinical variables when assessing biological phenotypes of depression. Future studies should replicate the results in sufficiently large groups, and use other markers of inflammation . If replicated, our results warrant stratification of patient groups by a combination of clinical and immunological characteristics for a more personalised treatment approach.Supplementary NotesSupplementary Table 1Supplementary Figure 1Supplementary Table 2Supplementary Figure 2Supplementary Table 3Supplementary Table 4"}
+{"text": "More critically, these FP@PDA@AgNPs strips could be used as outstanding flexible SERS sensors to quickly collect and detect malachite green (MG) residues on fish scales, crab shells and shrimp skins by a swabbing extraction method. The detection limits for MG residues were calculated to be approximately as low as 0.04635 pg/cm2, 0.06952 pg/cm2 and 0.09270 pg/cm2, respectively. This facile and efficient strategy could to be utilized as a universal approach to fabricating a variety of flexible, cheap and portable SERS sensors for surface contamination analysis, and has great potential in the environmental scientific analysis and food safety monitoring fields.In this work, a series of highly flexible and sensitive surface-enhanced Raman scattering (SERS) substrates were fabricated by the in-situ growth of silver nanoparticles (AgNPs) on polydopamine (PDA) templated filter papers (FPs), based on mussel-inspired surface chemistry. The obtained FP@PDA@AgNPs strips exhibited high sensitivity and reproducibility with Rhodamine 6G (R6G) probe molecules, with a calculated detection limit of approximately 10 Malachite green (MG), as an important triphenylmethane molecule, has been widely used as a fungicidal and antiprotozoal agent in the aquaculture industry . It coulSince surface-enhanced Raman scattering (SERS)can combine ultra-sensitive and unlabeled detection, it has recently garnered considerable attention in the analytical chemistry, biomedical diagnostics, food safety, environmental monitoring and national security fields ,14,15,16In order to solve these problems, some researchers have paid much attention to impregnating noble metal nanoparticles among paper fibers in recent years, and fabricating various paper-based SERS substrates through inkjet printing, screening printing, filtration, dip coating and physical vapor deposition ,24. PapeAs an important type of mussel-inspired molecule, dopamine (DA) has been widely explored for a versatile surface modification approach due to its unique self-polymerization ,30. Unde3)2]+ solutions. The FP@PDA@AgNPs strips demonstrated high SERS sensitivity and uniformity toward Rhodamine 6G(R6G) probe molecules, with a calculated detection limit of approximately 10\u221210 M. More importantly, the as-prepared FP@PDA@AgNPs strips could be utilized as SERS sensors to directly analyze the MG residues on the fish scales, crab shells and shrimp skins by a simple swabbing method. The SERS analysis results showed that the detection limits for MG residues were almost as low as 0.04635 pg/cm2, 0.06952 pg/cm2 and 0.09270 pg/cm2, respectively, and the MG Raman peak intensity at 1616 cm\u22121 showed a great linear relationship with the corresponding logarithmic concentration. Due to excellent flexibility and significant electromagnetic coupling effect, these FP@PDA@AgNPs strips not only could significantly enhance the collection efficiency for MG residues, but also achieve superior SERS sensitivity and reliability. These SERS substrates could serve as sensitive sensors for the fast on-site detection of trace fungicides and demonstrate enormous potential in the aquaculture, environment and bioscience fields.Based on these excellent characters of PDA molecules and FP fibers, FP@PDA@silver nanoparticles (AgNP s) strips were fabricated by using PDA molecules as templates in this research to achieve the in-situ reduction of AgNPs on the filter papers. The growth density of AgNPs on the surface of FP fibers was controlled by adjusting the reaction between the catechol groups of the PDA molecules and the silver ions in + solutions. Subsequently, the FP@PDA strips were totally immersed into the [Ag(NH3)2]+ solutions at room temperature, and the reaction was conducted with a 100\u2013200 r/min stirring speed. By regulating the reaction time at 4, 8 and 12 h, AgNPs with different particle sizes and different coverage could be generated on the surface of FP@PDA strips. Finally, the strips were washed three times with DI water and ethanol to remove the excess AgNPs on the surface of FP @ PDA, and dried for 30 min in the oven at 60 \u00b0C. The FP@PDA strips, after AgNPs growth, were denoted as FP@PDA@AgNPs strips.Firstly, NH\u22123 M concentration was accurately prepared in a volumetric flask and diluted with DI water into a series of solutions in the range of 10\u22124\u201310\u221210 M. Then, FP@PDA@AgNPs strips were immersed into 200 \u00b5L of R6G aqueous solution with different concentrations and dried for 30 min in an oven at 50 \u00b0C before SERS measurement. The Raman spectra of FP@PDA@AgNPs strips were collected on a Renishaw inVia9 with a laser power of 1 mW.The SERS sensitivity and reproducibility of FP@PDA@AgNPs strips were tested using R6G as the probe molecule. Firstly, a standard R6G solution with 10\u22123\u201310\u221210 M) were evenly dropped onto the fish scales, crab shells and shrimp skins, respectively. Then, FP@PDA@AgNPs strips were attached onto the surfaces of fish scales, crab shells and shrimp skins respectively, and gentle swabbing was performed using tweezers to collect the MG residues from the surfaces. After the water evaporated, the FP@PDA@AgNPs strips were detected by Renishaw inVia9 with laser power of 10 mW to collect the Raman spectra. The fabrication procedure for FP@PDA@AgNPs strips and the detection of MG residues in aquatic products through swabbing extraction are illustrated in To simulate real-world SERS applications, the fish, crab and shrimps employed in the experiments were washed thoroughly with DI water and ethanol, and a certain volume of MG solutions with different concentrations  with an accelerating voltage of 5.0 kV. The chemical composition of nanostructures was determined by an energy-dispersive X-ray spectroscope  attached to the SEM. The atomic force microscope (AFM) images were obtained in contact mode with a Multimode 8 atomic force microscope . The crystalline structures of all samples were analyzed with a D8 Advance X-ray diffractometer  using Cu K\u03b1 radiation (\u03bb = 1.54 \u00c5) at a voltage of 40 kV. X-ray photoelectron spectroscopy (XPS), with 200 W monochromatic Al K\u03b1 radiation, was performed, utilizing Thermo Scientific\u2019sESCALabXi+ . Raman analyses were performed on a RenishawinVia9 , using 532 nm laser irradiation, with a 50\u00d7 objective focusing the laser beam onto a spot with a diameter of about 1 \u00b5m, and the collection time for each sample was about 5 s.3)2]+ solutions, abundant AgNPs were spontaneously formed via in-situ reduction and nearly covered the surface of the FP@PDA@AgNPs strips  and atomic force microscope (AFM). From s strips g\u2013i.Accors strips , it appe3)2]+ solution, the silver peak appeared and exhibited a significant peak intensity due to a large amount of AgNPs formation on the surface of the FP@PDA@AgNPs strips. These EDS analyses were consistent with the XRD results and proved that a good number of AgNPs were successfully formed on the surface of the FP@PDA@AgNPs strips.As shown in 5/2 and Ag3d3/2, respectively + solutions on their surface morphology was studied, and the intensity of SERS signal was tested with R6G at a concentration of 10\u22126 M as a probe molecule. As shown in 3)2]+ ions were converted into AgNPs, and the size of AgNPs gradually reached 50\u201360 nm  was 0.9717, which would be very advantageous for being able to determine the concentration of target molecules with these highly sensitive FP@PDA@AgNPs SERS strips.The sensitivity and reproducibility of the SERS substrate were essential to achieve trace and reliable detection for target molecules. ding R6G ,42. As s\u22126 M were collected from 20 randomly selected spots from the FP@PDA@AgNPs strips. As can be seen from \u22121, and the relative standard deviations (RSD) of the Raman vibration at 612 cm\u22121 were approximately 3.98%  was calculated as follows:ISERS and IRS were the peak intensities of the R6G molecule SERS spectrum and R6G molecule normal Raman spectrum at 612 cm\u22121, respectively. NSERS and NRS were equal to the numbers of R6G molecules on the respective substrates within the laser spots [7 for the R6G probe molecules, indicating a Raman enhancement effect superior to those in previous reports [To estimate the SERS performance of the FP@PDA@AgNPs strips, the enhancement factor  were detected from 20 points randomly selected on the FP@PDA@AgNPs strips. As can be observed in \u22121 showed relatively consistent Raman signals, and the RSD of Raman vibration at 1616 cm\u22121 was approximately 7.72% (\u22121 were approximately 7.75%, and these results indicated that the Raman signals on the FP@PDA@AgNPs strips demonstrated good SERS stability. The above results proved that the flexible FP@PDA@AgNPs strips processed superior sensitivity, uniformity and storage stability. In addition to detecting MG residues in aquatic products, these flexible SERS substrates would be expected to be usable as highly sensitive SERS sensors to monitor various pesticides, explosives and carcinogens in the food safety and science fields.In addition to the sensitivity, the uniformity of the FP@PDA@AgNPs strips was also tested, and the Raman signals of MG (10ly 7.72% b. Furthe\u221210 M. More importantly, the FP@PDA@AgNPs strips could be used as flexible SERS sensors to rapidly collect and detect MG residues on the surfaces of fish scales, crab shells and shrimp skins via a simple swabbing method. The detection limits for MG residues were calculated to be nearly as low as 0.04635 pg/cm2, 0.06952 pg/cm2 and 0.09270 pg/cm2, respectively, and the peak intensity at 612 cm\u22121 for the MG residues illustrated a good linear relationship with the corresponding logarithmic concentration. These designed FP@PDA@AgNPs strips would show great application potential for the fast on-site detection of trace target molecules in the aquaculture, environment and bioscience fields.In a word, PDA was used as a versatile molecular platform for the in-situ reduction of AgNPs on the filter papers, based on mussel-inspired surface chemistry, and a series of flexible FP@PDA@AgNPs strips were fabricated. Due to high sensitivity and excellent reproducibility, the FP@PDA@AgNPs strips could produce stable and reliable Raman signals from R6G probe molecules, even if the concentration of R6G was as low as 10"}
+{"text": "Phenacoccus solani (Hemiptera: Pseudococcidae), is known as a thelytokous parthenogenetic species, but there is still debate about the reproductive strategies of this species.The reproductive pattern of most scale insects is ovoviviparity. The solanum mealybug, P. solani and used scanning/transmission electron microscopy and RNA-seq to identify the differences between two types of eggs.Here, we investigated the oviposition characteristics of P. solani laid two types of eggs in one batch, with no significant difference in apparent size: one with eyespots that hatched and another without eyespots that failed to hatch. Furthermore, the physiological and molecular differences between the two types of eggs were highly significant. KEGG enrichment analysis revealed significant enrichment for the JAK-STAT, Notch, Hippo, and Wnt signaling pathways and dorsoventral axis formation, wax biosynthesis, cell cycle, insulin secretion, and nitrogen metabolism pathways. The results suggest that the embryo of the egg undergoes development inside the mother and only a short molting period outside the mother.We found that P. solani can be described as ovoviviparity.Ovoviviparous species produce eggs and keep them inside the mother\u2019s body until they are ready to hatch, and the offspring exits the egg shell during or immediately following oviposition. Therefore, we suggest that the reproductive pattern of  The reproductive strategies of most insects have been described with three main patterns: oviparity, viviparity and ovoviviparity . In ovipPhenacoccus solani (Hemiptera: Pseudococcidae), is native to North America  and transmission electron microscopy (TEM) to distinguish between two types of eggs  laid in one batch. Finally, we further investigated miRNA and mRNA expression in the two types of eggs. We aimed to answer the following questions: (1) What life stage is P.\u00a0solani laying\u2013an egg, a nymph, or something in between? (2) If different reproductive \u201cproducts\u201d coexist, do they differ in appearance, physiology and molecular biology? (3) According to the above findings, what is the reproductive mode of P.\u00a0solani?Here, we investigated the oviposition characteristics of P.\u00a0solani was observed on two transparent, single-well, concave slides  or eggs without eyespots (hereafter eggs without-ES) were further observed in the same batch of eggs under a NikonSMZ1500 zoom stereomicroscope. There were two kinds of treatments: one employed the concave slide method , and the other employed the blade method, i.e., placing a female adult on detached potato leaves, with the petiole wrapped with defatted cotton to maintain leaf freshness and the placing the whole treated leaves in Petri dishes . The oviposition of female adults was observed every 30\u00a0min from 9.00 a.m. to 4.00 p.m, and the numbers of the two kinds of eggs were counted. Each female adult was biometrically tested once, and each treatment was repeated 15 times. A total of 25 eggs were randomly selected from the two kinds of eggs, and their lengths and widths were measured. After 72\u00a0h, the hatching of the two kinds of eggs was observed. Eggs with-ES hatched 114 individuals, while eggs without-ES hatched 45 individuals. The experiment was carried out in an artificial climate chamber with a temperature of 27\u00a0\u00b1\u00a01\u00a0\u00b0C, a humidity of 70%\u00a0\u00b1\u00a05% and a photoperiod of 16 L: 8D.Two treatments were established: (1) eggs with-ES were incubated under the mother\u2019s body , and (2)The surface and internal structure of the two kinds of eggs were observed by SEM  and TEM . The collected eggs with and without ES were pretreated with liquid nitrogen immediately. The follow-up procedures followed the instrument operation methods for the scanning electron microscope. TEM was performed as follows: (1) fixation, eggs were immersed in 2.5% glutaraldehyde fixative solution and then rinsed with buffer solution; (2) gradient dehydration, the fixed samples were dehydrated with an ethanol; (3) gradient osmosis, the samples were permeated with a mixture of acetone and Spurr resin penetrant (1:1); (4) embedding and polymerization, 100% Spurr embedding agent was added, followed by polymerization for 24\u00a0h; (5) ultrathin sectioning, the samples were cut to approximately 90 nm; and (6) observation and photography.https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA554708.Samples of newly laid eggs (with-ES or without-ES) were collected and immediately placed in a 0.5 MlEP tube and then frozen in liquid nitrogen for RNA extraction. Total RNA was extracted from 6 samples, and a library was constructed as previously described . The libp value <0.05 and fold change >2 or <0.5 were set as the thresholds for significant differential expression. Hierarchical cluster analysis of DEGs was performed to explore transcript expression patterns, and KEGG pathway enrichment analysis of DEGs was performed using R based on a hypergeometric distribution.Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. . The functions of the unigenes were annotated by alignment of the unigenes with the NCBI nonredundant (NR), SwissProt, EuKaryotic Orthologous Groups (KOG), Gene Ontology (GO) and Kyoto-Encyclopedia of Genes and Genomes (KEGG) databases. Differentially expressed unigenes (DEGs) were identified using the DESeq  functionP. solani produced eggs in one generation through thelytokous parthenogenesis, and the eggs were long and oval, similar to those described by P. solani, two kinds of eggs with distinct morphological differences appeared   . The eggP. solani belongs to the latter group, secreting a small amount of wax and never forming an ovisac. Thus, it is not surprising that the eggs of P. solani hatched so quickly, especially when the eggs were isolated.To investigate the extent to which the mother\u2019s body affected the hatching of eggs, eggs without-ES were removed from the area beneath the mother\u2019s body after they were laid. Therefore, we further tested whether eggs with-ES hatched after they were removed from the area beneath the mother\u2019s body. We found that eggs with-ES could hatch normally under any treatment \u20132C, but The surface and internal structure of eggs with and without-ES were observed using SEM and TEM. The contour of appendages could be clearly seen across the eggshell, and the bristles, tubular glands and six conical receptors  could be seen on the surfaces of eggs with-ES ; moreovep < 0.05 and fold change > 2 (or fold change < 0.5) , correlation coefficients between samples were calculated and used to estimate biological repeatability and differences between groups. The correlation coefficient of 3 biological replicates in the group with-ES and the group without-ES was >0.8, and the sample correlation coefficient between these two groups was only 0.4, showing an obvious difference between these two groups . DEGs wee < 0.5) /7C. There < 0.5) . KEGG ene < 0.5) .P. solani, but soon emerged 1st-instar nymphs following hatch. Because this hatching process is fast (<30\u00a0min), researchers might think that 1st-instar nymphs are produced by P. solani female adults. A similar phenomenon was reported in another invasive mealybug, Phenacoccus solenopsis, and because both eggs and 1st-instar nymphs were found in oocysts, Coccus hesperidum, naked nymphs appeared from the vulvar orifice, but thin eggshells were shown to remain in the female reproductive tract  after leaving the mother\u2019s body: i.e., this species lays eggs and does not experience live birth. Ovoviviparous species oviposit eggs at an advanced stage of embryological development, and the larva exits the eggshell during or immediately following oviposition (P. solani can be described as ovoviviparity.Although the reproductive mode of position . Therefo10.7717/peerj.9734/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9734/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9734/supp-3Supplemental Information 3The video recording time was 2\u201908\u201d. The preparation before laying eggs ran from 0\u20190\u201d to 1\u201930\u201d; the gradual production of eggs ran from 1\u201930\u201d to 2\u201908\u201d.Click here for additional data file."}
+{"text": "Dactylis glomerata L.) is a popular cool-season perennial grass with a high production value, and orchardgrass seed is the fourth top-selling forage grass seed in the world. However, its yield and quality are often affected by flooding. To date, the molecular responses of orchardgrass to flooding were poorly understood.Orchardgrass (CAT1 and CDS2). The significant positive correlation between RNA sequencing data and a qPCR analysis indicated that the identified genes were credible.Here, we performed mRNA-seq to explore the transcriptomic responses of orchardgrass to a short term flooding (8\u2009h and 24\u2009h). There were 1454 and 565 differentially expressed genes identified in the 8\u2009h and 24\u2009h of flooding, respectively, compared to well control. GO functional enrichment analysis showed that oxidoreductase activity and oxidation-reduction process were highly present, suggesting that flooding induced the response to oxygen stress. Pathways enrichment analysis highlights the importance of glutathione metabolism, peroxidase, glycolysis and plant hormone signal transduction in response to flooding acclimation. Besides, the ROS clearance system is activated by significantly expressed glutathione S-transferase and genes encoding SOD and CAT (In the process of orchardgrass response to flooding stress, multiple differential genes and biological processes have participated in its acclimation to flooding, especially the biological processes involved in the removal of ROS. These results provide a basis for further research on the adaptation mechanism of orchardgrass to flood tolerance. Flooding is a major factor, limiting growth and production of plants on a global scale . AgriculPlants suffer from various biotic and abiotic stresses throughout their life span. Higher plants are aerobic organisms. Flood is a kind of abiotic stress which have a devastating impact on crop growth and survival and thus on food production . The maiZea mays [Nymphoides peltata [Cucumis sativus [Persea americana [Sesamum indie [Taxodium [Cerasus sachalinensis [ERFs, MYB, HSP, and MAPK) and other metabolic processes are involved in the domestication of plants for flooding stress and play an important role. The results provide a better understanding for the adaptation mechanism of plants to flooding stress.RNA-seq technology has higher sensitivity to capture low and high-level gene expression than array technologies . RNA-seqZea mays , Nymphoi peltata , Cucumis sativus , Persea mericana , Sesamumum indie , TaxodiuTaxodium , Cerasuslinensis  have revDactylis glomerata L.) is one of the most important cool-season forage grasses [Orchardgrass , 8\u2009h and 24\u2009h). Sequencing results obtained raw data\u00a0427,211,572 and the raw reads were filtered obtain clean reads was 416,737,748 Table . The orcPearson correlation analysis was performed to validate the gene expression profiles of transcripts from nine different samples Figure . The resTo screen the differentially expressed genes (DEGs), we used DESeq software for differential gene analysis, and q-value (p-adj)\u2009<\u20090.05 was defined as DEGs. Integration the reproducibility of the biological replicates, we found 2019 DEGs in leaves following the flooding stress Table , S3. Amop-value <\u20090.05 can be used as an enrichment project for a certain function. DEGs GO enrichment histogram visually reflected the distribution of DEGs in GO terms of biological processes, cellular components, and molecular function enrichment.To better understand the response mechanism of orchardgrass to flooding stress, we performed GO enrichment analysis on the biological functions of all DEGs. Correction Comparing DEGs in 8\u2009h flooding stress with those in 0\u2009h (CK) treatment, DEGs enriched in biological processes were the most abundant and significantly enriched, mainly involving, single-organism process (GO:0044699), transport (GO:0006810) and establishment of localization GO:0051234); followed by membrane (GO:0016020) in the category of cell components. The DEGs enriched in molecular function were the least, mainly involving oxidoreductase activity (GO:0016491) and transporter activity (GO:0005215)  treatment, the DEGs enriched in the biological process mainly involved the oxidation-reduction process (GO:0055114), and the DEGs enriched in the molecular function were more than the biological processes, including oxidoreductase activity (GO:0016491), co-enzyme binding (GO:0050662), and co-factor binding (GO:0048037) Fig.\u00a0. We founThrough a comprehensive comparison of two flooding stress (8\u2009h and 24\u2009h) DEGs most enriched GO Term Figure A, B, we When plants encounter stress, different genes coordinate with each other to regulate biological functions. The significantly enriched pathway of DEGs can identify the most important biochemical metabolic pathways and signal transduction pathways in plant responses to stress. Here, we used KOBAS (2.0) for pathway enrichment analysis. FDR\u2009\u2264\u20090.05 indicated that the differential gene was significantly enriched in a certain pathway.The key pathways of orchardgrass seedling in response to 8\u2009h flooding stress were glutathione metabolism, selenocompound metabolism, peroxisome, tyrosine metabolism, isoquinoline alkaloid biosynthesis, biosynthesis of secondary metabolites, plant-pathogen interaction, thiamine metabolism, carotenoid biosynthesis, beta-alanine metabolism, glycerolipid metabolism Fig.\u00a0.Fig. 3DThe key pathways in response to 24\u2009h flooding stress were alpha-Linolenic acid metabolism, selenocompound metabolism, phenylalanine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, phenylpropanoid biosynthesis, glutathione metabolism, fatty acid degradation, cutin suberine and wax biosynthesis, plant hormone signal transduction Fig. .The pathways of significant enrichment of DEGs under 8\u2009h flooding stress were different from 24\u2009h stress. Only glutathione metabolism and selenocompound metabolism were found in both (8\u2009h and 24\u2009h) flooding treatments. The DEGs enriched in the glutathione metabolism pathway after 8\u2009h flooding stress was more abundant than that in the 24\u2009h flooding, suggesting that glutathione metabolism more responded to 8\u2009h of flooding stress than the 24\u2009h.KEGG enrichment analysis was performed on all DEGs at 8\u2009h and 24\u2009h compared with 0\u2009h respectively. We screened the significantly enriched 58 DEGs from nine pathways .The cluster heat map clearly showed that the expression patterns of 58 DEGs at different flooding time points were clustered into 6 clusters Fig.\u00a0. Among tPeroxisomes are important organelles equipped with a highly efficient reactive oxygen species (ROS) detoxification system, which is involved in many key metabolic processes such as oxidation of fatty acids, biosynthesis of ether lipids and detoxification of free radicals  Fig.\u00a05)5). It caPEX10 were significantly up-regulated, indicating that it was promoted during peroxisome biogenesis. In abiotic stress, the total amount of intracellular peroxisomes could increase the total number of peroxisomes in addition to up-regulating ROS scavenging enzymes [Arabidopsis, including PEX10 [PEX10 was promoted during peroxisome biogenesis. Studies have shown that peroxidases proliferate in plants under stress conditions [Eleven DEGs were significantly expressed in the peroxisome pathway, of which five were up-regulated and eight down-regulated at 8\u2009h of flooding stress Table . The DEG enzymes . This inng PEX10 , indicatnditions .In recent years it has become apparent that peroxisomes play important roles in reactive oxygen metabolism . Most reMPV17 was significantly down-regulated during ROS metabolism, indicating that ROS metabolism was inhibited under 8\u2009h of flooding stress  genes, in which the SUB1A allele SUB1A-1 limits the growth of plants under flooding and confers flood tolerance [Hormones are important regulators of plant adaptation to environmental changes. Plant hormone signaling plays an important role in the stress resistance of many plants. In response to 8\u2009h of flooding stress Table , SAUR-liss Table , the difolerance .-\u0394\u0394Ct method. The results showed that ten DEGs were differentially expressed between the control group (flooding for 0\u2009h) and the stress-treated group (flooding for 8\u2009h and 24\u2009h)  Figure . The qRT) Figure , confirmIn this study, high-throughput RNA sequencing technology was used to compare the expression profiles of DEGs in the leaves of orchardgrass cv. Dianbei in 8\u2009h and 24\u2009h under flooding stress. This study provided insights into a better understanding of the molecular mechanisms of flooding tolerance in orchardgrass.Taxodium [By comparing the bioinformatics analysis of the obtained transcript data of orchardgrass, we found that many biological processes, cell components and molecular processes in the leaves of orchardgrass were affected by flooding stress. In response to stress, oxidoreductase activity in the GO enrichment term was significantly enriched at both time points, and it was also significantly up-regulated in the GO enrichment entry in cucumber exposed to flooding stress . At 8\u2009h Taxodium .2, ROS and plant toxins [In this experiment, a total of 102 pathways were found in the list of KEGG differential gene enrichment pathways in the 8\u2009h of flooding treatment, and 77 pathways in the 24\u2009h of flooding treatment. The pathways on the top list were glutathione metabolism, peroxisome, selenium compound metabolism, tyrosine metabolism, biosynthesis of secondary metabolites, plant-pathogen interaction, plant hormone signal transduction, suggesting that flooding impacted a wide range of physiological processes. We have found that these processes have been reported in studies of other plant flooding stresses. Flooding stress should be considered as a compound stress consisting of several potential changes in the internal and external sources of ethylene, carbon dioxide, Ot toxins .2\u2212 to superoxide anion, resulting in the production of hydrogen peroxide [ROS is a by-product of basic cellular processes such as plant photosynthesis and respiration, and is produced by the joint action of several enzymes . One of peroxide . ROS areperoxide .CSD2 encoding SOD and up-regulation of ROS scavenging genes (CAT1 and glutathione S-transferase) were observed under flooding stress. SOD is the first line of defense for removing active oxygen in plants [CSD2 might increase ROS levels in the leaves of flooded orchardgrass seedlings. Catalase may be involved in the control of hydrogen peroxide (H2O2) levels by converting H2O2 to O2.This may be the reason why Dianbei is resistant to flooding. Similar to this study, the ROS scavenging genes  were significantly expressed in rape. The difference is that the gene encoding CAT is down-regulated and the gene encoding SOD is down-regulated [Maize [A. philoxeroides [Accumulation of ROS is an important indicator of abiotic stress at the molecular level. Once the ROS removal system is disrupted, the plant will suffer from oxidative stress. In general, plants can detoxify ROS by producing different types of antioxidants. Antioxidants are divided into enzymatic and non-enzymatic antioxidants. Enzyme antioxidants include superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and glutathione reductase (GR), and ascorbic acid, glutathione, tocopherol, and carotenoids are non-enzymatic antioxidants . Many ann plants . At the n plants . Down-reegulated . Similaregulated , Maize [xeroides  floodingADH verified that ADH plays an important role in plant response to flooding. Comparative transcriptome analysis of other plants has also identified glycolysis and upregulation of fermentation genes as common responses to flooding, such as Jatropha [Sesbania cannabina [Glycolysis is one of the important ways of energy production under hypoxic conditions. Maintaining the growth of glycolysis may be the key to plant survival . The glyJatropha , Sesbaniannabina , soybeanannabina . Our res2H4), abscisic acid (ABA), gibberellin (GA), auxin (IAA), and cytokinin (CK). Many genes involved in hormone production and signaling were found to be affected in both roots and shoots of flooded plants [ERF) represented the highest number of significantly expressed TFs under hypoxic conditions. In this study, the expression of EBF1/2 in orchardgrass leaves was significantly increased under flooding conditions, suggesting that flooding is related to ethylene synthesis and perception. This proves that ethylene plays an important role in plant flooding stress. The phytohormone ethylene mediates physiological, developmental, and stress responses by activating ethylene-responsive factors (ERFs) belonging to the multi-gene family of transcription factors [ERF1 plays a positive role in salt, drought, and heat stress tolerance by regulating stress-specific gene, and by integrating jasmonic acid, ethylene, and abscisic acid signals [ERF regulation, but further studies are needed to test other ERFs. ABA has long been recognized as a key factor in abiotic stress response in plants [PYR / PYL / RCAR) responsible for stomatal closure is a soluble protein [PYL2, PYL5, and RCAR3 were significantly expressed in the abscisic acid-mediated carotenoid biosynthesis. This also suggests that ABA plays an important role in responding to flooding stress. IAA is the main biologically active auxin in plants [MAPK signal cascade at intervals, leading to the formation of adventitious roots, which is very important for plants in flooded environments [Plant hormones play an important role in plant resistance to stress. Plant growth regulators are induced to participate in a signal cascade that affects cellular responses under flooding stress, including reduction of ethylene  were significantly induced under water flooding, indicating that these genes regulate GST in an important role in the ROS clearance system. Similar research conclusions have also appeared in the study of resistance to rape  and tallThis study is the first time that a transcriptome analysis of orchardgrass leaves in response to short-term flooding stress has identified a total of 2019 differentially expressed genes. Oxidoreductase activity, cofactor bling and oxidation -reduction process are highly present in GO-enriched terms, suggesting that the leaves of orchardgrass have a complex oxidoreductase process in response to flooding stress. This study also found that peroxidase, glutathione, glycolysis and phytohormone signal transduction play important roles in the domestication of orchardgrass. This study laid a foundation on a further study of genetic control of flooding tolerance for orchardgrass.Dactylis glomerata L.) variety with strong flood tolerance.The test material is \u201cDianbei\u201d, which is an orchardgrass (\u2212\u20092 s \u2212\u20091. Huguelan solution was supplied regularly during shoot growth. Huguelan solution was supplied regularly during the seedling period.Orchardgrass seeds of uniform size and fullness were screened and sown in pots containing sand and soil in a ratio of 1: 2 (V: V)  . After sowing, the pots were placed in a light incubator. The relative humidity in the light incubator is constant, the temperature is set to 25\u2009\u00b0C/20\u2009\u00b0C (day / night), the photoperiod is 14\u2009h / 10\u2009h (day / night), and the photon flux density is about 300\u2009\u03bcmol\u2009m Three months after sowing, select well-grown orchard grasses for testing. The pot was placed in a water tank  filled with room temperature water, and the water level was kept higher than the highest part of the plant as the treatment group, and the control group was a normal-growing orchardgrass plant. The 24\u2009h samples were first flooded, and after 16\u2009h, the 8\u2009h samples were flooded, with three biological replicates per treatment. When all treatments were completed, the leaves of the orchard grass from three treatments  were cut at the same time, put into sterile tubes, quickly frozen with liquid nitrogen, and stored at \u2212\u200980\u2009\u00b0C.Total RNA was extracted from the samples using an RNA prep pure plant kit . (1) Analysis of the degree of RNA degradation and contamination by running agarose gel electrophoresis; (2) Nanodrop testing (OD260/280 ratio); (3) qubit accurately quantification of RNA concentration; (4) Agilent 2100 detecting RNA integrity. Finally, only RNA samples that passed the quality test were selected for RNA-Seq analysis. Transcriptome sequencing was conducted on an Illumina Hiseq 4000  at the Novogene Bioinformatics Institute .http://orchardgrassgenome.sicau.edu.cn/). All of the sequencing data was deposited into the NCBI SRA database.Approximately 3\u2009\u03bcg of RNA for each sample was used for library construction, using the NEBNext\u00ae Ultra\u2122 Directional RNA Library Prep Kit for Illumina\u00ae . Transcriptome sequencing was conducted on an Illumina Hiseq 4000  at the Novogene Bioinformatics Institute . The orchardgrass genome reference was used for read mapping (phred\u00a0<\u2009= 20 bases account for more than 50% of the entire read length of reads). We selected HISAT [Filtering the original sequencing sequence obtained by sequencing: 1) removing the reads with adapters;(2) removing N (N means that the base information cannot be determined) the ratio of reads greater than 10%;(3) Remove low-quality reads  according to the model 3) Finally, multiple hypothesis test corrections were performed to obtain the FDR value (error discovery rate). Analysis of gene differential expression using DESeq [In RNA-seq analysis, we can estimate gene expression levels by counting the number of sequencing reads that are located in the genomic region or exon region. In this experiment, each sample was analyzed for gene expression level using HTSeq  softwareng DESeq  softwareThe software method used in our analysis of GO enrichment analysis was GOseq, which is based on the Wallenius non-central hyper-geometric distribution. Compared to the ordinary hyper-geometric distribution, this distribution is characterized by the fact that the probability of extracting an individual from a certain category is different from extracting an individual from a outside certain category and the probability is different. It is estimated by the length of the gene, so that the probability of GO term being enriched by differential genes can be calculated more accurately. Pathway Significant Enrichment Analysis Using Pathway in the KEGG database, hypergeometric tests were used to find Pathway that was significantly enriched in differentially expressed genes compared to the entire genome background.http://primer3.ut.ee/), for ten selected genes with the sequence data in 3\u2019UTR (Table 2O. Actin was used as the endogenous reference gene. The reaction had three phases. Stage 1 (hold): 95\u2009\u00b0C 30\u2009s 1\u2009Cycle, Stage 2 (PCR reaction): 95\u2009\u00b0C 5\u2009s,60\u2009\u00b0C 20\u2009s,40\u2009Cycles, Stage 3 : 95\u2009\u00b0C 15\u2009s, 60\u2009\u00b0C 1\u2009h,95\u2009\u00b0C 15\u2009s. All PCR reactions were normalized using the Ct value corresponding to the endogenous reference gene . Three P-value\u2009<\u20090.05 was considered to be statistically significant.Data analysis and mapping were performed using IMS SPSS Statistics and OriginPro8.5, respectively. All values are expressed as mean\u2009\u00b1\u2009SD. All the values were expressed as mean\u2009\u00b1\u2009SD. Comparison between control and flooding groups was estimated with One-way ANOVA followed by LSD post hoc test, and Additional file 1: Table S1. Summary of RNA-seq sequence a nalysis of orchardgrass. Table S2. Differential analysis results of all DEGs suffering flooding at WS_8h vs WS_0h in leaves. Table S3. Differential analysis results of all DEGs suffering flooding at WS_24h vs WS_0h in leaves. Table S4. GO enrichment analysis of all DEGs suffering flooding at WS_8h vs WS_0h in leaves. Table S5. GO enrichment analysis of all DEGs suffering flooding at WS_24h vs WS_0h in leaves. Table S6. KEGG Pathway enrichment analysis of all DEGs suffering flooding at WS_8h vs WS_0h in leaves. Table S7. KEGG Pathway enrichment analysis of all DEGs suffering flooding at WS_24h vs WS_0h in leaves. Table S8. The primers used in the qRT-PCR analysis.Additional file 2: Table S9. Analysis of 4 key pathways in response to flooding stress.Additional file 3: Figure S1. Correlation analysis under different time flooding stress. Figure S2. GO enrichment analysis of down-regulated DEGs suffering flooding at WS_8h vs WS_0h and up-regulated DEGs suffering flooding at WS_24h vs WS_0h in leaves. Figure S3. GO functional classification of all DEGs suffering flooding at WS_8h vs WS_0h and WS_24h vs WS_0h in leaves. Figure S4. qRT-PCR verification results of ten DGEs in control and treatment groups. Figure S5. Correlation between qRT-PCR and RNA sequencing for the ten selected genes."}
+{"text": "Despite being the second most common tumor in men worldwide, the tumor metabolism-associated mechanisms of prostate cancer (PCa) remain unclear. Herein, this study aimed to investigate the metabolism-associated characteristics of PCa and to develop a metabolism-associated prognostic risk model for patients with PCa.via single-sample gene set enrichment analysis (ssGSEA). The analyzed samples were divided into three subtypes based on the partitioning around medication algorithm. Tumor characteristics of the subsets were then investigated using t-distributed stochastic neighbor embedding (t-SNE) analysis, differential analysis, Kaplan\u2013Meier survival analysis, and GSEA. Finally, we developed and validated a metabolism-associated prognostic risk model using weighted gene co-expression network analysis, univariate Cox analysis, least absolute shrinkage and selection operator, and multivariate Cox analysis. Other cohorts  were utilized for external validation. Drug sensibility analysis was performed on Genomics of Drug Sensitivity in Cancer and GSE78220 datasets. In total, 1,039 samples and six cell lines were concluded in our work.The activity levels of PCa metabolic pathways were determined using mRNA expression profiling of The Cancer Genome Atlas Prostate Adenocarcinoma cohort TP53 and SPOP), copy number variation, and microsatellite instability were identified in PCa. Eighty-four of the metabolism-associated module genes were narrowed to a six-gene signature associated with DFS, CACNG4, SLC2A4, EPHX2, CA14, NUDT7, and ADH5 (p <0.05). A risk model was developed, and external validation revealed the strong robustness our risk model possessed in diagnosis and prognosis as well as the association with the cancer feature of drug sensitivity.Three metabolism-associated clusters with significantly different characteristics in disease-free survival (DFS), clinical stage, stemness index, tumor microenvironment including stromal and immune cells, DNA mutation (The identified metabolism-associated subtypes reflected the pathogenesis, essential features, and heterogeneity of PCa tumors. Our metabolism-associated risk model may provide clinicians with predictive values for diagnosis, prognosis, and treatment guidance in patients with PCa. Prostate cancer (PCa) is the second most frequent urinary system-associated type of cancer, accounting for 13% of all malignant tumors in men . TherefoDue to the unrestricted multiplicative nature of cancer cells, tumors exhibit different metabolic statues from normal tissue, thus provide a possible way to identify tumors through the difference in metabolism. Recent studies have proven that some metabolisms, such as citrate and choline metabolism, are closely related to PCa . Studiesin vivo . Gene expression data were acquired using the Illumina HiSeq RNA Sequencing platform and expressed as fragments per kilobase of transcript per million fragments (FPKM). The cBioPortal for Cancer Genomics  provided clinical data of the PRAD patients  and tumor tissue from the ICGC (https://icgc.org/). RNA-seq and microarray data of PRAD tissues and clinical information from testing cohorts for prognosis were obtained from the Gene Expression Omnibus (GEO) database\u00a0(https://www.ncbi.nlm.nih.gov/gds/). The GEO search strategy of the GSE datasets was as follows: 1) Include \u201cprostate cancer\u201d and dataset types of RNA-seq or micro-array; 2) Include more than one hundred PRAD samples with survival data; and 3) Include expression information of six risk model genes. Two datasets that met these requirements were identified, GSE54460 and GSE70768. Microarray data of cell lines , and RNA-seq data of melanoma samples were downloaded from the Genomics of Drug Sensitivity in Cancer (GDSC) (https://www.cancerrxgene.org/) and GSE78220. We also acquired the immunohistochemistry (IHC) data for PRAD and normal prostate tissues from the Human Protein Atlas (HPA) data portal (https://www.proteinatlas.org/). Immune infiltrate data for PRAD tissues were downloaded from the Cistrome Project (http://www.cistrome.org/) using the Tumor\u00a0IMmune\u00a0Estimation\u00a0Resource version 2.0 (TIMER2.0)  of prostate adenocarcinoma (PRAD) tissues were downloaded from TCGA (patients . RNA seqIMER2.0) .https://www.gsea-msigdb.org/)   and Kyotdb.org/)  and the db.org/) . The metdb.org/) Table S1Unsupervised clustering analysis using the PAM algorithm was performed based on the ssGSEA score of each sample using the R package ConsensusClusterPlus function . The samTo evaluate the tumor stemness index, we downloaded the mRNA expression-based stemness index (mRNAsi) calculated by machine learning in previous studies . The steAfter selecting the metabolism-associated genes, we generated an adjacency matrix (AM) and topological overlap matrix (TOM) using the gradient method based on power values ranging from 1 to 20. When the correlation between the average degree of connectivity (k) and p (k) reached 0.88, we obtained the optimal power value and constructed a scale-free topology network. Network connectivity of the genes was measured using a TOM transformed from an AM . Modulesvia R package survival 3.1.8. To evaluate the precision of the risk model and nomogram, time-dependent receiver operating characteristic (ROC) analysis was applied using the R package survival ROC 1.0.3. An area under the ROC curve (AUC) >0.60. indicated the prediction ability of the model was meaningful, and an AUC >0.70 indicated an outstanding predictive value of the model. To investigate the function of risk model genes, we performed GSEA of the TCGA cohort according to the high-risk group and low-risk group divided by the risk score medium value. The correlation between clinical variates and the DFS of PRAD was analyzed and calculated using univariate and multivariate Cox with the R package survival v3.1.8. The nomogram was obtained with the R package survival v3.1.8. The C-index analysis was performed with the R package pec v2019.11.3  and staining intensity .All statistical analyses were conducted using R software (version 3.6.1). The Mann\u2013Whitney U-test was used to compare two groups with a non-normal distribution of variables. For comparisons of three groups, Kruskal\u2013Wallis tests of variance were used as nonparametric methods. Correlation analysis was performed using the Person coefficient. All statistical tests were two-sided, and p-values < than 0.05 were considered statistically significant.A schematic of our research workflow is shown in To explore the characterization of each subtype, we described the clustering hot map of the metabolic pathway scores  stage and Gleason scores than patients from C3, but the age and regional lymph nodes (N) stage differences of the patients among these subtypes were not significant . DiffereIn previous studies, cancer stem cell characteristics have been shown to represent the capability of tumor proliferation and are associated with the development and progression of PRAD , 24. To Differential analysis of mRNAsi indicated there were significant differences among the three subtypes  is considered to reflect the sensitivity of tumors to targeted drug therapies . To furtCNV occurring upstream of genes regulates gene expression and influence tumor occurrence and development . To furtPrevious studies have shown that MSI is a crucial indicator of genome instability and is associated with many genetic diseases . In our We selected 2,029 metabolism-associated genes among the TCGA PRAD cohort samples and constructed a co-expression network through co-expression analysis . AverageTo determine the biochemical functions of the metabolism-relevant module genes, we performed GO and KEGG function enrichment analyses. Some metabolic pathways, such as alcohol metabolic process, sulfur compound metabolic process, cellular modified amino acid metabolic process, phenylalanine metabolism, drug metabolism cytochrome P450, and glutathione metabolism were significantly enriched by these genes Table S4CACNG4, SLC2A4, EPHX2, CA14, NUDT7, and ADH5  of chemotherapeutic drugs, risk score, and gene expression data from six PCa cell lines obtained from the GDSC database. As a result, four anti-tumor drugs  were negatively correlated with risk score Table S8Finally, immunotherapies represented by PD-L1 and PD-1 blockades have undoubtedly emerged as a breakthrough in cancer therapy , 37. We With the development of RNA-seq technology, many classical analyses based on gene expression data have been reported for most cancers , 38\u201340. in vitro experiments and single-cell sequencing.Cancer stem cells play important roles in therapeutic responses and the progression of cancer . To furtTME is a remarkable factor impacting the occurrence and development of PCa. Many cancer-promoting factors play a role in the EMT pathway . For insTP53 is the most prominent gene in pan-cancer investigations. For instance, TP53 mutations lead to high-grade cancer and tumor heterogeneity of ovarian granulosa , DFS, metabolism-associated subtypes, and anti-tumor therapeutic effect. Therefore our model can powerfully help clinicians evaluate the prognosis and develop personalized treatment for PCa patients. Although the six prognostic markers still require experimental verification, they may provide insight and a prospect for further investigation and clinical work regarding PCa.https://portal.gdc.cancer.gov/ (PRAD); https://www.ncbi.nlm.nih.gov/gds/ ; https://www.cancerrxgene.org/ ; https://www.gtexportal.org/ (Prostate); https://icgc.org/(PRAD).Publicly available datasets were analyzed in this study. This data can be found here: YZ designed the study and analyzed the data. YZ and RZ revised the images. YZ, RZ, and FL performed the literature search and collected data for the manuscript. XL and LZ revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (No. 81771768).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Training the next generation of industrial engineers and managers is a constant challenge for academia, given the fast changes of industrial technology. The current and predicted development trends in applied technologies affecting industry worldwide as formulated in the Industry 4.0 initiative have clearly emphasized the needs for constantly adapting curricula. The sensible socioeconomic changes generated by the COVID-19 pandemic have induced significant challenges to society in general and industry. Higher education, specifically when dealing with Industry 4.0, must take these new challenges rapidly into account. Modernization of the industrial engineering curriculum combined with its migration to a blended teaching landscape must be updated in real-time with real-world cases. The COVID-19 crisis provides, paradoxically, an opportunity for dealing with the challenges of training industrial engineers to confront a virtual dematerialized work model which has accelerated during and will remain for the foreseeable future after the pandemic. The paper describes the methodology used for adapting, enhancing, and evaluating the learning and teaching experience under the urgent and unexpected challenges to move from face-to-face university courses distant and online teaching. The methodology we describe is built on a process that started before the onset of the pandemic, hence in the paper we start by describing the pre-COVID-19 status in comparison to published initiatives followed by the real time modifications we introduced in the faculty to adapt to the post-COVID-19 teaching/learning era. The focus presented is on Industry 4.0. subjects at the leading edge of the technology changes affecting the industrial engineering and technology management field. The manuscript addresses the flow from system design subjects to implementation areas of the curriculum, including practical examples and the rapid decisions and changes made to encompass the effects of the COVID-19 pandemic on content and teaching methods including feedback received from participants. Training the next generation of industrial engineers and managers is a constant challenge for academia. Course contents must be up-to-date and cover all current and predicted development trends. In addition, they must always be relevant with the end-to-end product chain, from client requirements to maintenance via design and production. The COVID-19 pandemic has induced a significant number of challenges to industrial activities and their management. Graduates of today\u2019s academic programs will have to handle tomorrow\u2019s challenges from the technological, economic, medical, and industrial perspectives. Therefore, to be relevant, program contents had to be adapted to the new reality in a short time. In this manuscript, we expose the ante and during COVID-19 pandemic upgrade of the curriculum of the Bachelor degree in Industrial Engineering and Management (IEM) at the faculty of Industrial Engineering and Technology Management (IETM) of the Holon Institute of Technology (HIT), Israel. This manuscript focuses on the academic issues related to the Industry 4.0 requirements, which are at the forefront of the needs for constant adaptation [Moreover, we center our attention on the challenges encountered while delivering technological courses during the COVID-19 pandemic and the need to transfer to a regime of remote-teaching to comply with regulations that were imposed in real time and support the urgent need for social-distancing.One specificity of the B.Sc. program at HIT we must present up front, since it has a significant impact on our methodology, is the fact that two parallel tracks co-existing during the first three years of the B.Sc., the regular track and the evening track. The first one offers five days a week teaching during the two regular semesters . Generally, regular track students are not working. The evening track relies on participants working in parallel with their degree where courses are given two evenings a week and on Friday morning (6 h each time) all over the year (two regular semesters and summer semester). The fourth-year is unified for both regular and evening tracks students learning evenings and weekends.During the four years of the programs, the students are progressively trained for starting their careers in positions such as industrial engineers, data analysts, and information system managers with an emphasis on the technology management aspects. Moreover, these positions require close interaction and cooperation with other specialists such as electrical and software engineers, designers, trainers, domain-experts , and the ability to manage multidisciplinary projects. Therefore, the IEM curriculum must be updated continually to provide theoretical knowledge and practical experience. The focus is on ubiquitous technologies such as Industrial Internet of Things (IIoT), Automation, Robotics and Embedded Systems, and Applied Informatics  ,3. The sMore particularly, the nowadays-trained next generation of industrial engineers that will manage Industry 4.0 systems and related-projects will need to understand how these new production environments are impacted and reciprocally influence others  by clearA \u201clearning by experience\u201d policy has been implemented since 2018 to encourage the IEM students to develop their soft and practical skills. One of the main objectives is to develop the students\u2019 project-based learning capabilities by involving them in real-world-oriented tasks and projects during each course and over time by linking projects in different courses. Moreover, this policy facilitates the interactions between the faculty\u2019s laboratories for operating an integrated cluster of educational and research laboratories , and as During the first months of 2020, the SARS-CoV-2 virus spread worldwide and induced the COVID-19 pandemic. According to the World Health Organization (WHO), one year later, as of 31 January 2021, there have been 101,561,219 confirmed cases of COVID-19, including 2,196,944 deaths over 220 countries, areas, or territories . This diIn March 2020, to limit the spread of infection, social distancing was enforced ,16, inclA large number of solutions have been implemented to limit the pandemic\u2019s spread and try to return society and economic activities to previous \u201cnormal\u2019. For example, the healthcare system moved to teleconsultation-based services using Internet of Medical Things (IoMT) tools such as digital medical remote consultation . AnotherThe Israeli population is massively taking the COVID-19 vaccine . By 31 JThis situation creates additional challenges for the educational system, higher education staff, and business managers. During quarantines and lockdowns ,26, remoThe case study presented in the paper aims at sharing with the Industrial Engineering and Management academics and professionals one Israeli experience of teaching and training the next generation of professionals in a context of complex and worldwide crisis.Understanding the modernization of a part of the IEM curriculum in combination with its migration to a fully-based online landscape must help to understand how blended learning should be efficiently used when the engineering training involving, hardware manipulation, face to face group dynamics, and leadership skill development, will be back to normal without imposed social distancing.The higher education system has been intensely engaged in the effort of emergency risk management and mitigation of the COVID-19 pandemic involving scientific research of delivering solutions from the health and business perspectives to overcome the crisis. One of the main contribution fields relates to the \u201con the move\u201d innovation of the online education models . One cruMoving from face-to-face teaching/learning has induced a global reaction in the overall education systems. Over 31 countries have a joint reflection run during spring 2020 for sharing feedback on emergency remote education. The main focus was more on how social injustice, inequity, and the digital impacted learners and why the (higher) educational community must support each one for reducing the impact of being alone . AdditioIndustrial Engineering and Management departments worldwide moved their teaching both fully online for theoretical courses and in a blended way for the one involving practices ,33 or moThis study\u2019s leading objective is to propose new ways to promote online and cloud-based technologies for teaching cyber-physical systems, both at the initial training and continuous one during the professional career. An additional objective is to recommend practical and real-world experimented ways to increase the quality and effectiveness of teaching and training engineering students to Industry 4.0 management and manufacturing technologies in regular and crisis times.The COVID-19 pandemic allows for accelerating industrial engineering and management changes, such as blended teaching, practice, and mentoring as a standard.Moving to successful simulation or online cyber-physical systems as a part of industrial engineering and management education and training depends on reactivity and adaptability of the teaching staff and the students.Industry 4.0 deals with distant actuation monitoring and management. Therefore, Online-based learning that covers all aspects of the domain is fundamental to train the next generation of engineers and managers and provide the needed skills.Three hypotheses lead the observational research (case study) presented in the following sections:This manuscript focuses on relating the real-world experience of upgrading the IEM curriculum before and during the COVID-19 pandemic. The goal is to point out the importance of involving the students in their training in a tangible way. This involvement is the critical way the studies occur in a dematerialized context, but similarly to the new reality of remote working and multi-site exploitation. This research characterizes changes needed in teaching and learning Industry 4.0 theories, concepts, and systems to facilitate graduates\u2019 integration in the employment market.As a modern discipline, Industrial Engineering and Management (IEM) is at the crossroad of many fields such as engineering and technology, formal and natural science and management science, and human sciences.Forecasting, looking for innovation, future potential of market development, imagining scenarios that may occur, and defining actions for handling new challenges.Planning, defining, deciding, and scheduling actions to deal with the previously forecast challenges and bring them to goals and action plans.Organizing by allocating human and non-human resources for acting according to the plan.Coordinating, achieving the different steps of the action plan and goals.Controlling, checking that research, development, and production activities fit the disruptive goals and progress accordingly to the strategy previously defined.Management, as a transverse component of IEM, is organized over five central concepts , which aEvaluating the syllabus, in regard to the current and predicted evolution of the industrial and business environments;Proposing improvements taking into account both, the expected changes in the industrial and business environments, and the academic budget and infrastructure constraints;Getting previous years students and graduates\u2019 feedbacks, and understanding their needs in regards of their involvements in none-academic projects and positions.Getting peers and industrial practitioners\u2019 feedback, by submitting the syllabus proposal to faculty and institutional committees involving these people giving suggestions for consolidating the new syllabus;Implementing the new syllabus during the next relevant academic semester;Getting feedback and suggestions during course run for the students and eventually teaching assistants;Industrial engineering is commonly defined as a domain attending to developing and improving different kinds of business and industrial tasks, e.g., describing a modus operando; and handling milestones of a production. Offering an up-to-date and cutting-edge curriculum is challenging. It is required to consider the curriculum of other engineering specialties to give the students the ability to communicate and be productive with other specialists efficiently. Furthermore, as IEM is a very dynamic field and with the continuous development of new technologies, the students\u2019 training must fit industry needs both locally and globally. Thus, the methodology for developing, evaluating, and maintaining a set of syllabi dealing with Industry 4.0 and smart manufacturing must consider the following in regular times:Considering the recently upgrade courses before COVID-19 implemented for the first time during the fall semester 2019\u20132020;Moving to emergency-mode management and upgrading relevant syllabi for delivering the most similar course content during COVID-19 lockdowns;\u25cbBefore the COVID-19 pandemic, the student had to complete at the end of the course a survey;\u25cbDuring COVID-19 lockdowns, to reduce the risk of student\u2019s churn, the faculty\u2019 management frequently interviewed students for getting their feedbacks in near-real-time;Semestrial students\u2019 feedbacks (before and during COVID19 pandemic);Learning from experience and moving forward by preparing the post-COVID-19 teaching/learning era .Taking into account the above and dynamically updating the course content in the COVID-19 crisis context and the forced move to full-online-based teaching, practice, and mentoring, this framework has been adapted at the faculty level in the shortest downtime of one week. The methodology we used for adapting, enhancing, and evaluating the learning and teaching experience under the urgent and unexpected challenging request to move from face-to-face university courses to be taught distantly and online  is as foIt is important to highlight that focusing on the COVID-19 pandemic higher education crisis, an equivalent case study focusing on a set of syllabi dealing with Industry 4.0 and smart manufacturing has, to our current knowledge, not been published.Before the pandemic and the needed social distancing rules , the overall curriculum was taught in a frontal manner.The curricula were initially drafted and approved before the beginning of the pandemic. Accordingly, these \u201ccontracts\u201d between the teaching staff and the students cannot be deeply revised at short notice. If not carefully designed and monitored, they may induce unexpected changes in the achievement of educational objectives and goals. Distant, online, synchronous/asynchronous teaching and learning is a new standard in the COVID-19 era.The following section describes the courses and the specific adaptations during the last year (since March 2020) to respond to the urgent COVID-19 demands. Given the present situation worldwide at March 2021, it is reasonable to forecast that these changes will have a mid-and long-term impact on teaching IEM as a part of the business and industrial ecosystem-induced changes that now encourage remote working when possible ,36.Introduction to algorithmic and Python (Python);Computer Integrated Manufacturing (CIM);Introduction to Internet of Things (IoT);Models of Business Intelligence (MBI);This section focuses on four courses in the IEM curriculum at HIT and the graduation project that are at the heart of the educational requirements of the fourth Industrial Revolution:These four courses and the graduation capstone project (GP) are linked to one another and are a part of the third and fourth years of the curriculum. Their related project-based learning approach emphasizes the viewpoints of both standards, \u201cReference Architecture Model Industry 4.0\u2014RAMI 4.0\u201d and \u201cIndustrial Internet Reference Architecture\u2014IIRA\u201d ,38,39. RIntroduction to Programming in Python is an introductory course for undergraduate students in the Industrial Engineering and Management program. The course introduces basic principles of modern programming techniques and problem-solving logic. The course deals with the practical aspects of programming and imparts Python programming skills. The course includes an introduction to the concept of an algorithm, basic variable types, operators, courses, conditions, loops, regular and recursive functions, methods for passing parameters, data structures , and interacting with text files. The programming language we use in this course is Python, which is a popular programming language. Python is a dynamic, object-oriented language and has an elegant and friendly syntax for the novice programmer. Before the COVID19 era, the course had been delivered in four academic hours: two hours of theory (using slides and whiteboard) and two hours of practice in a dedicated laboratory. During the practice course, the students solved given exercises and then compared them to the slides\u2019 solutions. During the independent work, the students asked questions and asked for direction.Moving to emergency-mode management at the beginning of the pandemic, the course is taught using video conferencing software. The theoretical part of the course required only minor adaptation by adding further explanation slides to the presentation.The main challenge was the practice part of the lessons, which required significant changes. First, more solved examples were provided and explained in the plenary session. Next, during self-practice, the students got exercises, and they were allocated to breakout rooms, 3\u20134 students in each room. The lecturer moved between virtual spaces, where the students could share their screen, and provided assistance and direction. Besides, the students in the room discussed the exercise and helped each other.The feedback was very positive about the theoretical part of the online course. The students claimed that the remote mode is more efficient, in the sense that more material was covered and more examples given. However, the students lamented about the practical part of the course. They commented that in remote mode there is not enough personal attention and help with code bugs, that is possible in frontal lessons.Regarding the lack of practice, Moodle offers some modules, such as \u201cVPL\u2014Virtual Programming Lab\u201d  is an acThe CIM course gives the students the fundamental tools for designing, implementing, and managing automated production environments and their computerized control systems. CIM is a semester course (13 weeks) of three lecture hours a week and one laboratory hour a week, combined with Project-Based Learning involving teams of two to three students. This course is articulated around three main components:Draft and implement specification of discrete and continuous control applications;Use information technologies for processing industrial data and supporting decision making, for example, for handling troubleshooting of production lines;Use Supervisory Control and Data Acquisition (SCADA) systems for integrating the control of discrete and continuous (sub-)systems;Smartly use at the production plant and end-business mobile asset tracking technologies  codes, near field communication (NFC), and radio frequency identification (RFID).At a first step, the students are getting an overview of industrial process and control, in parallel with introducing the history of the different industrial revolutions and their impacts on day-to-day life. This part is associated with refreshers on essential mathematical tools needed to deal with automation and introductory physics to understand how sensors operate. The second principal component of the CIM course focuses on teaching \u201chow-to\u201d:These two first parts of the course were before the COVID-19 pandemic events, teachable in a non-frontal way, and mainly involving the students by using different simulation software environments. In the third part of this course, the students discover \u201crobotics\u201d as an automation kind and how it can be integrated into the production processes at small and large scales. The first lessons are herein mainly dealing with the history of robots, ethics, the safety of use, and physics concepts such as direct and indirect kinematics, motion limitations, and the integration with machine vision tools.All these concepts are more easily gained when the students are participating in lab workshops. Thus, they are actively involved by using an arm robot such as Dobot Magician  and its To avoid meetings and respect social distancing, the robotics workshop used an education robot simulator . MoreovBefore the COVID-19 crisis, the students\u2019 feedback mainly pointed out the need to concentrate the laboratory and practice time on larger slots (2 h every two weeks instead of one hour per week). The largest part of the students was highly and intensely engaged in this course\u2019s PBL experience. It allowed them to prepare themselves (according to their feedback) for the graduation project and industry (when they were regular track students).This course has successfully moved to an online version, getting positive feedback from students with and without a prior background in automatics and electronics . However, practice with hardware has this course\u2019s PBL experience been reported as a missing part of the course; even the use of simulation software was strongly expected and supported by the teaching staff.The course is optional and is the continuation of the CIM course, which is mandatory.It allows students to study in-depth some critical components of the fourth industrial revolution, the IoT. IoT brings together smart objects, cloud computing, and automated analysis of large amounts of data. This introduction to IoT mainly deals with the hardware and cloud computing aspects .The course presents the history of IoT, the status of the field today, key initiatives and key players in IoT, and teaches about the significance of the implementation of smart and interactive devices.An introduction to IoT  and its An overview of key enabling technologies ;Service-Oriented Architecture for the design of IoT systems ;Applications in the industry ,54,55.A more in-depth analysis than in the CIM course of the mode to operate of the different sensors, cameras, and actuators;Prototyping IoT systems .The main subjects of the course are:A large part of this introduction to IoT is dedicated to practicing. Therefore, students are working with Raspberry PI kits which make the hardware more accessible to the students. Raspberry PI is a friendly single-board computer that students can quickly turn into IoT product prototypes . Moreove(1)could not use the dedicated IoT laboratory(2)Systems modeling: Unified modeling language (UML) was added to the curriculum  and was Simulation: since the students were not enabled to build an IoT product using dedicated software, they learned how to simulate an IoT product using Python programming language and particularly by using the \u201cFaker\u201d package that generates random users and random data with the programmer  defined constraints .Cloud services: besides essential AmazonWeb Services (AWS) as S3, Simple Notifications Service (SNS) and Simple Email Service (SES), additional services have been introduced as AWS Lambda and AWS Rekognition.Projects with emphasis on COVID-19: during the course, the students must design, use UML and SOA models, and develop an IoT product using cloud services and python. Students were instructed to design and develop a project related to COVID19 or lockdown-associated issues.could not take home the IoT equipment for practice. Therefore, significant adaptations have been performed to the course curriculum, increasing the focus on:Since the COVID-19 crisis, the course was delivered online by using a video conferencing platform. The new reality impacted the program by introducing two main and strong constraints, the students:The course was offered during the winter semester; the third one taught during the pandemic. Therefore, the students already have had experience with online courses.The fourth and senior students of this course expressed their preference for the online mode. They were delighted with theoretical lessons, and practical experiments run remotely using their computational resources. We received feedback such as \u201cThe course was exciting and delivering innovative content\u201d and \u201cDespite the online and challenging learning, the course trained us to new technologies\u201d.However, the student lacked real-hardware practice. They mainly had previous experience working according to the PBL methodology, in the third-year course dealing with CIM, and engaging them in regular time (pre-COVID-19 era) with hardware .Business intelligence  supportsDuring spring 2020, this course has been run entirely in an online version in webinars format and recorded tutorials. The flexibility of Microsoft Power BI as a free tool and a low-cost online platform allowed students to work on their project efficiently and professionally, giving senior students just before graduation an advantage on their resume, particularly in troubled times COVID-19 pandemic.In the previous years (in the pre-COVID-19 era), the students\u2019 feedback was very positive, and the engagement and involvement in the PBL were significant. In their last semester of B.Sc. studies, the students received inputs regarding the course utility in their position search in the industry and more about the recruiters\u2019 interests during the course and its integration in the graduation one. Indeed, the PBL activity of the NBI course is, in most cases, the last part of the graduation project, and the students are using both as a part of their project\u2019s portfolio.The \u201cModels of Business Intelligence\u201d course is one of the first courses that moved online in the shortest delays regarding prior experience in delivering a part of it online in synchronous and asynchronous ways. From the students\u2019 perspective, the online version of the course got positive feedback. Moreover, to help the students benefit efficiently from completing the PBL activity, the extra-hours dedicated to supporting their training to Power BI have been increased with specific time and assistance to each project.All the courses discussed in this manuscript give a strategic and central position to PBL. PBL is a fill-component of the new pedagogical approach of the upgraded curriculum. Moreover, students are developing their skills over different courses by starting their PBL journey in one course, for example, CIM, and go deeper during another one, such as the IoT, and complete the same project as a part of the final graduation project requirements.Before the COVID-19 pandemic, social distancing, and the different confinement waves, the faculty lab cluster for teaching Industry 4.0 specialties  ran flexibly. The regular, evening, and visiting (from aboard) students could schedule with a research assistant to develop and implement their PBL and graduation projects.AURIS\u2014Automation, Robotics, IoT, and Analytic Intelligence for Smart Industrial Engineering,IIoT\u2014Industrial Automation and IoTBig Data\u2014Big Data,BIA\u2014Business Intelligence and Automation,ERP\u2014Enterprise Resources Planning,HumFa\u2014Human Factors.(1)Data acquisition from different kinds of inputs, such as sensors and their related small board computer, robots, social networks, and more,(2)Data cleansing,(3)(Big) Data Management,(4)Knowledge Discovery with Data Mining, Text Mining, Process Mining, Machine Learning, Statistics, Natural Language Processing, to(5)Knowledge Management, and(6)Decision Support making.As it is presented in The cluster activities relate to industrial and business applications such as automation, industrial manufacturing , serviceSmall robots  ,Single Board Computer assemblies  and their related sensors and actuators ,Cloud technologies  .The AURIS laboratory is the most recent addition to the cluster. It integrates the Industrial Engineering and Management faculty curriculum\u2019s overall educational activities with a strong focus on Industry and Automation 4.0. The robotics lessons in the CIM and IoT courses are involving the use of low cost educational and production systems combining, for teaching the end-to-end computational and Information Management processes:Accordingly, the IIoT laboratory focuses on sensors, controllers, actuators, a device-to-device connection, wireless technologies, mesh communication networks, and low-cost computerized manufacturing systems  , as wellThe BIA laboratory research emphasis on artificial intelligence and business intelligence applications, mainly from the data science perspective, with a focus on data mining, machine learning, language processing, knowledge engineering, management, and representation , data quality and quality in information systems. The BIA laboratory researchers are involved in teaching activities related to research such as analysis and design of databases, data mining, machine learning, big data and cloud technologies, business intelligence models, quality engineering for information technology, and digital health.Students\u2019 increasing interest in carrying out their final graduation projects in an academic framework and applied technological research in the domains investigated by each lab alone or overlapping. Some of these projects have a strong potential for becoming industrial developments.International cooperation in research, teaching, and student mentoring.Enhancement of research, teaching, and mentoring actions intersecting between the cluster and partners\u2019 labs inside and outside HIT.The creation of the lab cluster was raised by significant developments in the faculty activities such as:Moreover, this lab cluster and its continuous enhancement for considering the recent advances and the new perspectives in the Industrial Engineering domain aim to deliver an end-to-end vision and understanding of the Organizational Information systems and Knowledge Management resources . AccordiSmart household pet feeding monitoring system\u2014A design and implementation of a pet feeding station prototype. Additionally, it provides health and behavior monitoring services, gathering health\u2019 pet indicators such as feeding habits and weight .Smart parking system design\u2014Design a network of cameras and motion sensors that collect data about parking lots throughout the city. All devices are powered by solar energy and are connected wirelessly to a local Raspberry Pi. The Raspberry PI uses cellular communication and transmits the data to Amazon servers. The system\u2019s purposes are on first-hand billing drivers for legal parking around the city and to send out fines for illegal parking; on the second-hand monitoring available parking spots for the benefit of drivers.Smart baby monitor\u2014A design and implementation of a monitor embedded with a microphone and camera connected to a Raspberry Pi. The monitor detects noise in the room and sends a short video of the baby to parents through Amazon SNS (Simple Notification Service).Smart garbage can collector\u2014A system design of weight sensors that are attached to garbage cans. The sensors weigh the can and, using a connected Raspberry PI and cellular connectivity, report the remaining garbage can capacity (or lack thereof) to the local municipality.Upgrading the IEM curriculum specifically from the automated systems perspectives  was associated with an increased central position given to the project- and problem-based learning approaches. Below two examples of students\u2019 accomplishments in this framework, as graduation projects:having a training continuity,reduce the least possible disruption of the educational activity,preparing the students to massively remotely work and dealing with non-materialized resources such as models and simulations.As previously highlighted, the B.Sc. The COVID-19 pandemic has deeply impacted IEM studies. All the teaching activities moved online, such as practicing and developing engineering and managerial capabilities. Accordingly, by taking into account that the future graduates will have to start their career in the challenging COVID-19 era or post-era, the PBL framework has been adapted for:Following, two examples of third and fourth-year (courses and graduation) projects specifically handling issues related to the COVID-19 pandemic.monitoring of temperature, light intensity, humidity, vibrations, falls, and openings of the said unexpected container;position tracking services, alert generation, and push in case of unexpected deviation of a predefined route;user identification ;real-time reporting of the status, state, and position of the container.COVID-19 vaccine containers: the project consisted of following the dispatching of COVID-19 vaccine doses. It has been defined as a complex logistic end-to-end process. Indeed, these vaccine doses need to be stored at a low temperature continuously, and they are exposed to a hijacking risk. Similarly, to the solution proposed to the vaccine producers, some of our students have designed and simulated computationally and  a container with sensors and actuators connected to communication systems and providing:Transportation and hygiene: This project intended to design an online application for hygiene reports in the public domain, like malls and public transportation, and private venues such as restaurants. During COVID-19 times, when maintaining hygiene and social distancing is very important, technological and collaborative tools can contribute significantly to this common goal. The relevant existing technological platforms are not developed enough . This apThis project involved acquired technical knowledge and skills during the different courses disclosed in the present manuscript. This kind of PBL allowed the students to understand this kind of product\u2019s potential genericity, which may have other applications such as transplantation organs or sensitive samples transportation.the organization and clarity of the teaching material;the way for delivering the course in an interesting manner;the way to involve and induce active participation of the students;the quality of the communication between the teaching staff of the course and the students;the feelings about the contribution to the learning experience of the home-work or course projects;the overall satisfaction about the course.The students\u2019 satisfaction with the teaching process is measured in regular time at the end of each semester for each course. The questions of this survey are the same, each semester, for all the courses taught in our institution. The survey focuses on:During 2019\u20132020, only at the end of the fall semester (February 2020) a computed and anonymized survey has been run. During the spring and summer semesters, instead of running students\u2019 satisfaction surveys, in order to increase the numbers of contacts between the teaching and management staff with the students, the faculty\u2019 staff frequently interviewed the students\u2019 representatives for getting their feedbacks in near-real-time about each courses and the way to facilitate their learning experience. The feedbacks of these interviews are reported above for each course.-Things\u201d (around 15 students each year) courses taught during this period. At the end of the fall semester of the 2020\u20132021 academic year, a computed students\u2019 satisfaction survey ran. This survey is relevant for the \u201cComputer-Integrated Manufacturing\u201d (around 70 students each year) and the \u201cIntroduction to the Internet-ofp < 0.05). It is critical to highlight, that even \u201cComputer-Integrated Manufacturing\u201d and the \u201cIntroduction to the Internet-of-Things\u201d are engineering courses which are in regular time hardware-based the students report their overall high satisfaction. From an engineering perspective training the next industrial engineers and managers to Industry 4.0 during the COVID-19 era and so mostly online in our case is both challenging. Nevertheless, it is opening opportunities to look at the future of the whole Industry 4.0 and the online, distant and cloud-based operations [These results shows surprisingly and globally a satisfaction increase regarding these courses. Indeed, even the courses were delivered online and the course material organization in the learning management system has not change year-over-year, the students satisfaction increased (before vs. during COVID-19 pandemic: +8.16%). Similarly, the students which learned online have feel the courses contents more interesting that the one which learned it in-class (+4.93%). The same trend appears regarding the perception of the \u201cproject-based learning\u201d approach (+8.70%). More, even than the courses were taught online, the students reported feeling a more appropriate and fair communication that the in-class students (+2.84%). However, we can notice a small decrease in the involvement and active participation feeling (\u22122.65%). The overall satisfaction has significantly increased .(1)Classroom exercises contribute to understanding.Add a short explanation in presentations in a situation of someone who did not come to class.Many examples were presented throughout the course.are useful).Presentation of a topic by students (Examples before the pandemic:(2)required).More practical examples, more practice (knowledge) while answering homework give amazing tools for study even outside of school hours.Remote teaching and the possibility of repeating the lesson when you want to deepen  remotely. This must allow students and staff to interact efficiently to acquire practical skills equivalently to frontal lab work. Research related to the virtualization of laboratory resources for encouraging and facilitating the study of technological processes has been described in the literature, in different modes, for the last two decades ,81,82,83In the context of the upgrading actions of the industrial engineering and management curriculum for fitting with the needs of Industry 4.0 and the post-COVID-19 era, the authors have set up a challenging postgraduate project at the beginning of 2021 to produce a working prototype of a virtual lab that can be implemented from the home computer of staff and students. The project is underway as we write this manuscript. In brief, the setup will be operated from a home computer. It will include a connection via the network to a remote gateway at HIT to which microprocessor endpoints will be connected wirelessly to sensors collecting data such as temperature, humidity, noise/sound, and images and, not less critical, robotic actuators that can simulate the human operations in the lab. The technological solution will work through IoT-compliant communication protocols and add-on hardware to the existing equipment that allows the entire network to work in a close-loop without human presence in the lab. This is aligned with the higher education regulation in Israel that emphasizes a digital ecosystem transformation to a system expected to adapt to a continuously increasing number of students and limited resources such as infrastructure and staff. Moreover, the project is performed in a framework of international cooperation with universities in Europe and Argentina that have identified similar urgent needs. It is expected that a prototype will be working for the coming 2021 summer semester to enable the first pilot run of the IIoT course in a blended virtual and physical mode. Upgrading the industrial engineering and management curriculum for Industry 4.0 and handling the numerous challenges of the COVID-19 era pushed the boundaries forward at the next level. The education of the 21st century Industry 4.0 engineers has to perform in a continually changing environment.As a next step, and for enhancing the usages rate of the lab cluster infrastructures, we are investigating existing solutions for sharing our local resources and getting access to devices and resources not available on our side. For example, platforms such as LabsLand , WebLab-The case-study reported in this manuscript addresses the flow from system design to implementation areas of an industrial engineering and management curriculum, including practical examples, feedback received, and the rapid decisions and changes that have been made to encompass the effects of the COVID-19 pandemic on content and teaching methods.Three hypotheses drove this observational research:Firstly, we demonstrated that the COVID-19 pandemic (more than any other significant events) allowed accelerating changes to educate and train the next generation of industrial engineers and management. As a concrete example, the proportion of staff teaching online (at least in a blended approach) jumped, at the faculty of Industrial Engineering and Technology Management at the Holon Institute of Technology, Israel, from 10% before the COVID-19 pandemic to 95% during the first-week s of the crisis then 100% today. Moreover, when teaching returns to normal , all the students will have to learn at least two online courses during their curriculum. Furthermore, and this the most critical point herein, the students were delighted with the online framework, allowing them access to recorded lessons and giving them a higher time management flexibility.p < 0.05) more satisfied by online teaching than in-class. This must be due to the flexibility of the teaching/learning process that can fit with familial and professional constraint during quarantine or confinement times. It is also possible to envisage that the virtual lab we have started to implement may also improve the indicator of \u201cinvolvement and participation\u201d that was the only one with negative tendence in results in Secondly, we highlighted that moving efficiently to simulation or online cyber-physical systems as a part of the industrial engineering and management education and training to smart manufacturing and Industry 4.0 depends on reactivity and adaptability of the teaching staff and the students. Therefore, the students were significantly . Besides, the use of blockchain technology must provide a layer that enables enhancing traceability of actions over these platforms and systems, increasing the security of access , and so The results of the use-case presented in this manuscript can be generalized to any industrial engineering related domain, such as electrical and electrical engineering, systems engineering, software engineering, and more by considering that online teaching based on simulation and remotely shared resources allows students to by efficiently trained to the new business and industry features that will emerge in the post-COVID-19 era. Moreover, the curriculum update process before and during the COVID-19 pandemic allowed to point-out the crucial necessity to develop \u201cdisaster preparedness plan\u201d curriculum fitting with the  engineering needs at the present and expected future Industry needsFinally, training the next industrial engineers and managers to Industry 4.0 is challenging over the COVID-19 triggered chances. Nowadays and in the next few years, Industry 4.0 will be led by the artificial intelligence, blockchain, cloud/cybersecurity, and data analytics (ABCD) paradigm. These four technologies converge to a multidisciplinary focal-point orchestrating innovation in many businesses and industries . The lab"}
+{"text": "BackgroundDespite ample evidence of continuing preoperative aspirin to improve coronary artery bypass surgery outcomes, practice for the routine continuation of preoperative aspirin is inconsistent due to concern for increased postoperative bleeding. The purpose of this study was to investigate preoperative aspirin use and its effect on postoperative bleeding after off-pump coronary artery bypass grafting (OPCABG).MethodologyThis cohort study involved patients (n = 74) who underwent OPCABG at a single center between August 2017 and January 2018. After considering the inclusion and exclusion criteria, the patients were divided into two groups: one (n = 37) received tablet aspirin 75 mg till the day of the surgery, and for the other group (n = 37) aspirin was stopped five days before the surgery. Postoperative bleeding was recorded in both groups. After considering preoperative, intraoperative, and postoperative variables, statistical analysis was performed.ResultsThere was no significant difference between the two groups concerning peroperative and postoperative variables. In addition, no significant difference was observed between the two groups in chest tube drainage at one, two, three, twenty-four, forty-eight, and seventy-two hours . No significant difference was seen in the transfusion requirement of blood and fresh frozen plasma (FFP).ConclusionsContinuation of preoperative aspirin till the day of the surgery is neither associated with an increase in chest tube drainage, reoperation for bleeding complications nor transfusion of blood and FFP. Coronary artery disease (CAD) is among the leading causes of morbidity and mortality worldwide, and its prevalence and adverse impact continue to increase . CoronarThis prospective cohort study involved 74 patients who underwent OPCABG at a tertiary cardiac center between August 2017 and January 2018 to observe the effect of preoperative continuation of aspirin on postoperative bleeding by assessing the amount of bleeding, use of blood products, and incidence of re-exploration. The study protocol was approved by the ethics committee of the institute. All patients admitted under different cardiac surgery consultants for elective CABG were included in the study. Patients on oral or parenteral anticoagulation, or an antiplatelet drug other than aspirin; known allergy or intolerance to aspirin; a history of bleeding diathesis, liver failure, renal failure; and on-pump CABG, redo CABG, or needing other procedures with CABG were excluded from the study. A study of a continuous response variable (amount of postoperative blood loss) from OPCABG with preoperative aspirin and without aspirin was planned. In a previous study , the resThe baseline characteristics of the 74 patients are outlined in Table During surgery, transfusion of fresh whole blood (recently collected unrefrigerated blood rich in platelets and coagulation factors) and stored blood (collected from standard blood donation and refrigerated) was 78.4% and 32.4% in the aspirin continued group and 64.9% and 37% in patients who stopped aspirin preoperatively, respectively . None of the patients required transfusion of fresh frozen plasma (FFP). The amount of peroperative fluid infusion was also similar in both groups (p = 0.167)   concludeSeveral studies in which preoperative administration of aspirin caused increased postoperative bleeding used a higher dose of aspirin (>150 mg). For example, Sethi et al. (1990)  and FerrBlood transfusion after surgery is now discouraged due to several adverse effects. However, increased bleeding might increase transfusion requirements. Goldhammer et al. (2015)  investigRemoval of the chest drain tube is associated with early mobilization, improved respiratory effort, shorter intensive care unit (ICU) stay, reduced postoperative pain, and accelerated overall recovery. The chest drain tube is usually removed on the second or third POD after CABG ,9. Most Furthermore, no difference was noted regarding preoperative and postoperative hemoglobin, hematocrit, and platelet count among the two groups. However, both groups showed a significant decrease in all these parameters noted postoperatively in comparison to their preoperative values. A similar observation was noted by Lee et al. 2017 .Surgical re-exploration for bleeding is associated with a significant increase in postoperative mortality as well as an increase in ICU and hospital stay. While Wu et al. (2011)  and JacoThe use of antifibrinolytic drugs became popular for their role in reducing postoperative bleeding. Pleym et al. (2003)  reportedFor ensuring the reduced risk of thrombotic accidents during the peroperative period, Spadaccio et al. (2015)  analyzedThis study has limitations including smaller sample size, focus only on postoperative bleeding, and purely observational. Furthermore, peroperative blood loss was not measured. As the study population was selected from only one hospital in Dhaka city, the results of the study may not reflect the exact picture of the country.The continuation of\u00a0aspirin throughout the surgery period is not associated with an increase in chest tube drainage, reoperation for postoperative bleeding, or increased requirement of transfusion of blood and blood products. Therefore, our recommendation is to continue aspirin during the perioperative period of OPCABG without the concern of postoperative bleeding and bleeding-related complications."}
+{"text": "Many clinicians increasingly use dry needling in clinical practice. However, whether patients' intake of antithrombotic drugs should be considered as a contraindication for dry needling has not been investigated to date. As far as we know, there are no publications in analyzing the intake of antiplatelet or anticoagulant agents in the context of dry needling techniques. A thorough analysis of existing medications and how they may impact various needling approaches may contribute to improved evidence-informed clinical practice. The primary purpose of this paper is to review the current knowledge of antithrombotic therapy in the context of dry needling. In addition, reviewing guidelines of other needling approaches, such as electromyography, acupuncture, botulinum toxin infiltration, and neck ultrasound-guided fine-needle aspiration biopsy, may provide specific insights relevant for dry needling. Based on published data, taking antithrombotic medication should not be considered an absolute contraindication for dry needling techniques. As long as specific dry needling and individual risks are properly considered, it does not change the risk and safety profile of dry needling. Under specific circumstances, the use of ultrasound guidance is recommended when available. Worldwide, clinicians are using dry needling (DN) to reduce pain , 2, incrIn an Irish study of the possible adverse events of DN, 39 physiotherapists used trigger point DN 20 times per month within a 9-month time frame for a total of 7,629 treatments. Mild adverse events, including bleeding, bruising, and pain during and after treatment, were reported in 19.18% of the treatments. While no significant adverse events occurred, the risk of significant adverse events for 10,000 treatments was estimated to be less than 0.04% .There are many specific factors to consider before using DN, such as the actual needling procedures, the area to be needled, and the characteristics of the needles. Specific factors to be considered are the diameter and type of needle, the technical procedure, the target of the intervention, and the anatomical structures in proximity of the needling site. Much can be learned from studies of electromyography (EMG), acupuncture, botulinum toxin (BTX) infiltration, and neck ultrasound-guided fine-needle aspiration biopsy (USGFNAB), realizing the obvious differences between these procedures and DN with respect to clinical practice or the type of needles used.Dry needling is used mostly by physiotherapists, but other disciplines have also started to use DN, such as occupational therapy, athletic training, chiropractic, and acupuncture. Since these clinicians may not have a solid working knowledge of antithrombotic drugs and their specific risk factors, it is imperative to review the different kinds of antithrombotic drugs before reviewing guidelines from other procedures that use needles.There are two classes of antithrombotic drugs: antiplatelet agents and anticoagulants. The main antiplatelet agents are aspirin and P2Y12 receptor blockers. Anticoagulants include vitamin K antagonists (VKAs) and direct oral anticoagulants (DOACs), which are administered orally, and heparins, which are administered subcutaneously or intravenously.Aspirin, the first-line antiplatelet, prevents the formation of blood clots and is used in patients with angina, previous heart attack or stroke, coronary artery stents, after coronary artery bypass graft surgery, and in patients undergoing surgery for hip fracture . Other aThe main types of oral anticoagulant agents are VKAs and DOACs. Warfarin is the most widely used VKA worldwide, while in Europe acenocoumarol and phenprocoumon are the most frequently prescribed VKAs . They arFinally, unfractionated heparins (UFH) and low molecular weight heparins (LMWH) have been used for decades. UFH are administered subcutaneously or intravenously. LMWH, derived from UFH, are administered subcutaneously and have gradually replaced UFH for most clinical indications. UFH and LMWH are prescribed, along with dual antiplatelet therapy in patients with acute coronary syndrome (ACS) and they reduce VTE complications of high-risk medical conditions, such as heart failure or prolonged immobilization in bed, or in knee or hip arthroplasty .  Table During VKA therapy, the risk of bleeding is assessed with the International Normalized Ratio (INR), which is a standardized method for reporting results of the prothrombin time (PT assay) . TypicalDOACs have equivalent or improved therapeutic profiles compared to warfarin, and they are associated with significant reductions in stroke, intracranial haemorrhage, and mortality . As the Factors influencing blood clotting must also be considered see . The FDAIn case of major bleeding, it is recommended to withhold antithrombotic drugs and provide mechanical hemostasis and hemodynamic support . NumerouMoreover, other factors such as age , gender In relation to the interaction between food and anticoagulants, Violi et al. concluded that the available evidence does not support current advice to restrict dietary vitamin K intake while taking VKAs. However, they recommended maintaining a stable diet avoiding wide variations in vitamin K .Leite et al. analyzed the interferences of medicinal plants with blood hemostasis and warfarin anticoagulation and found a total of 58 different plants that may alter blood clotting and anticoagulation with warfarin . RegardiFurther research is needed to assess the influence of physical activity on the pharmacokinetics of antithrombotic drugs. Shendre et al. demonstrated that regular physical activity in patients on chronic anticoagulation therapy with warfarin is associated with higher dose requirements and a lower risk of haemorrhage , which wConsidering the type of needle, the degree of potential tissue damage is dependent on whether the needle is bevelled. The potential for tissue damage and bleeding is greater with a hypodermic needle than with a solid filament needle used with DN. Other needling approaches use different types of needles. For example, the needle diameter used with DN is smaller than the typical gauge of needles used for diagnostic EMG or hypodermic needles, ranging from 35\u2009G to 28\u2009G or 0.14\u2009mm to 0.35\u2009mm, respectively, compared to gauges of 30\u2009G (0.30\u2009mm), 26\u2009G (0.45\u2009mm), or 23\u2009G (0.60\u2009mm) with EMGs . InteresAnother factor is the technical procedure itself. Typically, DN involves manipulating the needle within a muscle and fascia to elicit the so-called local twitch responses . TheoretSeveral EMG studies have evaluated the incidence of hematoma and the risk of bleeding after the procedure in patients taking antithrombotic medication . Two casGertken et al. established the incidence of Magnetic Resonance Imaging- (MRI-) detectable hematomas after EMG of the paraspinal muscles. The sample included patients taking antithrombotic agents and controls. A total of 431 spine segments and a total of 370 patients were reviewed with 139 patients taking aspirin, eight taking clopidogrel, ten taking warfarin, two taking heparin, and two taking LMWH. In this study, no paraspinal hematomas were observed in any group, which suggests that EMG of paraspinal muscles is a relatively safe procedure . These rLee et al. found that increasing the diameter of acupuncture needles significantly increased the incidence of bleeding-related adverse events in patients taking anticoagulants or antiplatelet therapy, especially more in the head, face, and feet regions . A prospA retrospective chart review of 242 patients and 4,891 acupuncture treatments concluded that acupuncture appeared safe for patients taking warfarin or antiplatelet medications . The WorStudies of botulinum toxin (BTX) injections and potential bleeding complications must be considered with some caution as the infiltrations are generally made with larger and bevelled-edged needles compared to the smaller solid filament needles used with DN. Nevertheless, a review of possible BTX bleeding complications may help as injections are commonly used for the management of spasticity in patients taking antithrombotic agents. Only a few studies have attempted to determine the rates of bleeding complications, the particular physicians' practice of performing the injections, and common preferences to control the risk of bleeding prior to the injections. For example, a Korean survey of physiatrists found a high variability between physicians with respect to injecting patients on anticoagulant therapy, and generally, there was a tendency to avoid BTX injections all together. Seventy percent of the respondents considered the INR value prior to performing the injections in patients taking anticoagulants. The majority of respondents thought that INR values between 2 and 3 were optimal. However, many others replied that an INR value lower than 2 was the preferred range. Most respondents used an INR value measured 2\u20137 days prior to the injection and acknowledged that they did not have access to standardized prevention protocols for performing BTX injections. Thirty-one percent of the respondents used some preventive measures, such as applying prolonged compression at the injection site and observing signs of swelling or bruising after the injection .In a 2012 study by Schrader et al., 20 patients on the oral anticoagulant phenprocoumon with optimal INR values received BTX therapy using a 27\u2009G (0.40\u2009mm) needle without producing an increase in hematoma formation . In relaIn summary, there are no established and validated criteria, recommendations, or consensus for BTX injections for patients taking anticoagulants , 77. WitUSGFNAB is the most accurate technique to evaluate thyroid nodules, and many patients who are candidates for this procedure are on anticoagulant treatment . The neeThere are no studies comparing the incidence and prevalence of adverse events associated with DN in subjects taking antithrombotics and subjects who do not take these medications. It is also not known whether some of the relatively mild adverse effects in general populations may become more severe for patients taking anticoagulants.Clinicians must always be cautious when applying DN techniques, considering the use of medications that affect coagulation. Besides, it is necessary to have other competencies, especially excellent anatomical knowledge. It is mandatory to complete a thorough clinical evaluation, which should include an inspection of the skin prior to DN looking for signs of excessive bruising . AccordiAlthough not directly related to DN, some professional associations have recommended specific considerations that may be considered for DN. For example, AANEM has advised that an EMG evaluation should start with small superficial muscles before proceeding to deeper ones, although according to Boon et al., standard needle EMG of potentially high-risk muscles in patients taking antithrombotic drugs does not imply an increased risk of hematoma formation , 80. CurIn conclusion, with DN, the pressure is usually maintained for about 5\u2009seconds for nonanticoagulated patients; for patients taking anticoagulants the pressure should be applied for about 10\u201315\u2009seconds. Despite the low risk of bleeding and bruising with DN in patients who are taking medications that affect coagulation, it is important to be cautious when needling close to major blood vessels, or when applying hemostasis is not an option. The patient's individual risks must be considered as previously described, similar to EMGs and other needling procedures .The use of sonography in Doppler mode is recommended, but not essential, with DN of particular muscles such as the tibialis posterior, lateral pterygoid, or psoas major muscle, which not only reduces the chance of penetration major vessels but also increases the accuracy of the technique. Although sonography is increasingly used in clinical practice, most clinicians do not have access to sonography.Clinicians must always inform patients about the possible risks of DN and obtain at least an oral informed consent prior to DN. When specific information about the patient's coagulation status, such as the INR, is not readily available, clinicians should determine the potential risk factors by including specific questions during the clinical interview, such as the patient's experience with bleeding following venipuncture. When a given patient presents with diminished coagulation, clinicians may consider initially reducing the intensity of the DN techniques. Although the bleeding response for every patient may vary between days or even on the same day, it is recommended to observe the patient's bleeding response and initially avoid DN in deeper muscles until its safety has been established with more superficial muscles, especially with patients on anticoagulant therapy. Applying prolonged hemostasis following DN is recommended.Venipuncture and other invasive procedures, such as USGFNAB and botulinum injections, are performed regularly in patients who are taking anticoagulants, and they have been proven to be safe. They do not cause a major bleeding event. Therefore, taking antithrombotic medication should not be considered an absolute contraindication for DN techniques. If specific dry needling and individual risks are properly considered, antithrombotic medications do not change the risk and safety profile of dry needling. For improving the safety of DN practice in these patients, the use of ultrasound guidance is recommended, but not essential, for DN in locations close to major blood vessels or in deeper muscles where hemostasis cannot be applied."}
+{"text": "Epulopiscium spp., and their close relatives within the Firmicutes phylum, are intestinal symbionts of surgeonfish (family Acanthuridae). Each of these giant (up to 0.6\u2009mm long), cigar-shaped bacteria contains tens of thousands of chromosome copies. Epulopiscium spp. do not use binary fission but instead produce multiple intracellular offspring. Only \u223c1% of the genetic material in an Epulopiscium sp. type B mother cell is directly inherited by its offspring cells. And yet, even in late stages of offspring development, mother-cell chromosome copies continue to replicate. Consequently, chromosomes take on a somatic or germline role. Epulopiscium sp. type B is a strict anaerobe and while it is an obligate symbiont, its host has a facultative association with this intestinal microorganism. Therefore, Epulopiscium sp. type B populations face several bottlenecks that could endanger their diversity and resilience. Multilocus sequence analyses revealed that recombination is important to diversification in populations of Epulopiscium sp. type B. By employing mechanisms common to others in the Firmicutes, the coordinated timing of mother-cell lysis, offspring development and congression may facilitate the substantial recombination observed in Epulopiscium sp. type B populations.Most studies of bacterial reproduction have centered on organisms that undergo binary fission. In these models, complete chromosome copies are segregated with great fidelity into two equivalent offspring cells. All genetic material is passed on to offspring, including new mutations and horizontally acquired sequences. However, some bacterial lineages employ diverse reproductive patterns that require management and segregation of more than two chromosome copies.  Epulopiscium sp. type B maintains tens of thousands of copies of its genome throughout its lifecycle. These highly polyploid bacteria reproduce solely by the formation of internal offspring. A limited number of genome copies is passed on to each offspring, but most copies remain in the mother cell and are needed to support metabolism of the mother cell and growing offspring. Since not all copies of the genome are inherited, Epulopiscium populations run the risk of losing genetic diversity acquired in the somatic genome copies. Epulopiscium sp. type B populations bypass this loss by the coordinated release of DNA from dying mother cells and recombination of these fragments in genome copies within newly emerged offspring cells.The giant bacterium Most scientists do not think about bacteria when considering germline dynamics. That is entirely reasonable because most of our models for bacteriology use binary fission for reproduction. In binary fission, a bacterium grows to about twice its starting size, while it replicates and then segregates copies of its genome to distal parts of the cell. Reproduction culminates in division of the cytoplasm at the midcell to produce two equivalent and self-sufficient offspring cells, each with a complete complement of the parent cell\u2019s genomic material. All genetic material is preserved in this process.population or colony of cells.Several, perhaps less-familiar bacterial models have communication systems used to coordinate multicellular behaviors and regulate alternative reproductive programs . A numbeMyxococcus xanthus, exhibit multicellular behaviors for finding prey and for dispersing myxospores when nutrients become limited  differentiates into myxospores (Members of the myxobacteria (phylum Proteobacteria), such as  limited . Myxobacbacteria . When prbacteria . The M.\u00a0xospores . The strStreptomyces coelicolor, and related genera in the phylum Actinobacteria, keeps cells of the colony physically connected to one another throughout vegetative growth and arthrospore development  appeared to contain one or two well-defined nuclei , a symbiont of the surgeonfish Naso tonganus  and within its mother cell  could be located in the Epulopiscium sp. type B genome , in specialized cells  and in cancer cells (supporting aberrant growth and seen in cells responsible for recurrence), just to name a few examples . PolyploEpulopiscium sp. type B cells contain an astonishing amount of DNA. When examined under high magnifications, the stained DNA appeared to be the equivalent of thousands of bacterial nucleoids interconnected in a web-like structure , large Epulopiscium cells could contain thousands of genome copies  yielded significantly higher copy numbers than the other markers tested. The results supported the model that these giant cells contained a typical-sized genome for a bacterium, and moreover Epulopiscium sp. type B is highly polyploid throughout its life cycle  targeting single-copy house-keeping genes, revealed that e copies . Copy nue copies . Quantitfe cycle . More refe cycle . A high-oriC. Bacterial chromosome replication generally begins at this single site, which is flanked by binding sites for the replication initiation protein DnaA and for proteins involved in partitioning the chromosomes  corresponds with the position of the locus on the chromosome  remains associated with an individual fish or what proportion of the population is expelled on a regular basis. Surgeonfish, including N. tonganus, are long lived and may reach ages of 20\u201345\u2009years  chromosomes. Here \u201csimple\u201d modifications of a developmental program, and a lifestyle that affords predictable and reliable access to usable carbon and energy, can lead to the evolution of giant microorganisms with complex germline dynamics.Multiple essential cellular mechanisms and external factors collaborate to provide a means of overcoming genetic bottlenecks that have the potential to endanger the diversity of"}
+{"text": "Sintered silver paste is widely used as the die-attachment material for power semiconductors. However, sintered silver joints encounter problems, such as severe coarsening of sintered pores and oxidation issues, in harsh high-temperature environments. These lead to the deterioration of the die-attachment joints. In this paper, a novel method of sintering silver joints is demonstrated, where silver\u2013indium alloy paste is used to improve the reliability of sintered Ag joints. The silver\u2013indium (Ag\u2013In) alloy paste was fabricated through mechanical alloying using the ball-milling technique. The well-bonded sintered Ag\u2013In alloy joints inhibited pore coarsening better than pure sintered Ag joints and significantly enhanced the mechanical properties at high operating temperatures. Lastly, an oxidation mechanism for the sintered joint was proposed, and strategies to prevent such high-temperature oxidation were discussed. In the age of pursuing energy savings and reducing carbon emissions, the popularization of electric vehicles (EVs) has become the primary objective of next-generation transportation technology because the use of EVs can significantly reduce carbon emissions, which will diminish the greenhouse effect and global warming. Consequently, many countries advocate that internal combustion engine vehicles should be phased out and replaced by EVs or hybrid EVs within the next decade . TherefoInverters need to withstand a considerable amount of current in EVs, which leads to a substantial amount of heat generation and a harsh operating temperature of above 200 \u00b0C . Given tFor power chips to function well, packaging materials with high-temperature reliability are essential for the die attachment, and they should provide power chips with excellent mechanical support and heat dissipation. However, the conventional Pb-free Sn-based packaging materials used in consumer electronics cannot withstand the high temperatures generated during power semiconductor operation due to their intrinsically low melting points and creep resistance ,11,12. THerein, a novel method of directly sintering the Ag\u2013In alloy pastes using a homogeneous solid solution is proposed. Through the employment of the Ag\u2013In alloy paste, the additional step of adding In foil can be eliminated, thereby simplifying the fabrication process and bringing sintered Ag\u2013In joints closer to practical industrial application. Such Ag\u2013In alloy pastes can retain the benefits of In foils but at much-reduced manufacturing cost. A proper powder production method needs to be developed for the fabrication of homogeneous Ag\u2013In alloy pastes. Powder production methods such as gas and water atomization are common in industrial applications because they can mass-produce metal and alloy powders ,27. HoweIn this study, a novel Ag\u2013In alloy paste was developed by directly alloying In with Ag powder through the ball-milling process and mixing with an organic solvent. The Ag\u2013In alloy paste was utilized to fabricate sintered joints by a hot-pressing process. In addition, we compared the bonding and sintering results of the fabricated Ag\u2013In alloy joint to the pure Ag joint. Furthermore, the high-temperature reliability of both sintered joints was evaluated using high-temperature storage (HTS) and die shear tests, after which the microstructure and phase were investigated. Moreover, the correlation between the mechanical properties of both sintered joints during HTS was established. Lastly, the oxidation mechanism in the sintered joint and strategies to prevent oxidation were discussed.2) grinding balls, and 0.2 g of stearic acid were mixed into one of the ball-milling jars, as illustrated in 2 grinding balls, and 0.2 g of stearic acid were mixed, as illustrated in 2 with a diameter of 5 mm was chosen for the grinding balls because ZrO2 ceramic is less prone to metal powder contamination during ball-milling. Stearic acid was added to inhibit the cold welding and agglomeration of ductile Ag and In powders during the high-energy ball-milling process. Furthermore, the atmosphere in both ball-milling jars was replaced with high-purity argon gas to prevent oxidation of the Ag and In powders. The milling speed in both jars was 600 rpm for 10 h, after which the jars were opened in a nitrogen glove box  with an oxygen concentration of less than 0.1 ppm. Then, the ball-milled Ag and Ag\u2013In alloy powders were passed through a 500-mesh sieve. After sieving, Ag and Ag\u2013In alloy powders with particle sizes less than 25 \u00b5m were homogeneously mixed with a polymer solvent to complete the preparation of the pastes.HTS and die shear tests were performed to examine the reliability and mechanical properties of the sintered Ag and Ag\u2013In alloy joints. The HTS test temperature was 300 \u00b0C in a furnace with an atmospheric environment to test the short- and long-term high-temperature reliability of both sintered joints. After the HTS test, die shear tests were performed using a shear tester  to measure the shear strengths and conduct the assessment for the mechanical reliability of both sintered joints. + ion gun . Moreover, an electron probe microanalyzer  was utilized for elemental mapping and compositional analysis.The morphology and microstructure of the ball-milled Ag and Ag\u2013In alloy powders were characterized using a scanning electron microscope  equipped with an energy-dispersive X-ray spectrometer . The phases of the Ag\u2013In alloy powders were identified using X-ray diffraction  with Cu K\u03b1 radiation (\u03bb = 0.15418 nm). A transmission electron microscope  was used to observe the crystal structure of the ball-milled powders. In addition, the size distributions of the powders were examined using a laser-diffraction particle size analyzer , with an examination range from 0.04 \u03bcm to 2000 \u03bcm. After the hot-pressing bonding process and the HTS test, bonded samples were mounted with epoxy resin and polished using abrasive sandpapers. For SEM observation, an artifact-free cross-section was created by using an ion-milling system equipped with an ArThe XRD patterns for the raw Ag and Ag\u2013In alloy powders before and after the ball-milling process are shown in 50 indicates that 50 vol.% of the powder had a diameter less than this value. Herein, we regarded D50 as the average particle size and compared the D50 of powders before and after ball-milling. The D50 of the raw Ag powder was 28 \u03bcm, while the D50 of the ball-milled Ag and Ag\u2013In alloy powders was 19 \u03bcm and 13 \u03bcm, respectively. Hence, both the ball-milled Ag and the Ag\u2013In alloy powders were refined through the fracturing effect during the milling. The agglomeration and coarsening of ductile Ag and In powders caused by cold welding could be effectively inhibited by the addition of stearic acid, which was consistent with previous studies [The particle size distributions (PSDs) for the Ag and Ag\u2013In alloy powders after the ball-milling process are shown in  studies . The Ag 2) can invade the porous structure [2. Meanwhile, isolated and interconnected pores coexist at the sintered joint in the theoretical porosity range from 3% to 20%, while fully interconnected pores exist to form a 3D porous network at the sintered joint for a theoretical porosity above 20%. Hence, when the porosity of the sintered joint is beyond or close to this theoretical limit of 20%, the 3D porous network provides abundant pathways for O2 to invade and penetrate the sintered joint.Cu oxidation above 200 \u00b0C should have a considerable effect on the pure sintered Ag joint. Although it is not deeply understood yet, many researchers have discussed that the sintered Ag joint would suffer from Cu oxidation at high temperatures, which detrimentally affects the mechanical properties of sintered Ag joints. However, the Cu oxidation phenomenon was not observed in the sintered Ag joint during the HTS at 300 \u00b0C in this study. The main reason is the porosity of the as-bonded sintered Ag joint. There is a positive correlation between the porosity of the as-bonded sintered joint and the occurrence of such high-temperature oxidation, which is described by percolation theory. Specifically, the high porosity of the as-bonded sintered joint facilitates the oxidation process during HTS. Assuming that the pores are compact and uniformly distributed, three different theoretical porosity ranges can be calculated using percolation theory to describe whether external media  layer near the interface between the sintered Ag joint and the Cu substrate owing to the high affinity of Cu and O2. By contrast, a relatively high bonding pressure was utilized for the flake-like Ag sintering in this study. As a result, a low porosity of 4.1% was achieved in the as-bonded sintered Ag joint due to densification during the sintering process, and most of the pores in the sintered joint were unconnected and isolated. Consequently, O2 was effectively prevented from invading the sintered joint, reacting with Cu, and forming the Cu2O layer. Although the porosity of the sintered Ag joint significantly increased during the HTS and oxidation should occur, the tendency of the pores to coalesce instead of maintaining a uniform distribution caused most of the pores to remain isolated despite the relatively high porosity. However, although the oxidation issue during the HTS could be effectively addressed by increasing the applied pressure, the sintered pore coarsening phenomenon in the sintered Ag joint would still cause degradation of the mechanical properties.In previous studies, a low-pressure or pressureless Ag sintering process was attempted to reduce the production cost. However, the high porosity occurring from low-pressure bonding resulted in interconnected pores in the as-bonded sintered Ag joint; therefore, OThe porosity of the Ag\u2013In alloy joint did not increase as dramatically as that of the pure Ag joint after the HTS at 300 \u00b0C for 50 h and 100 h, as shown in 2O layer could be identified between the Ag\u2013In alloy joint and the substrate near the side regions of the joint in 2O layer converged and ended near the central region of the joint, as shown in 2 entered the Ag\u2013In alloy joint through the interconnected pores from the edges of the joint. At the beginning of the HTS, the O2 concentration was too low to form a lamellar Cu2O layer near the central region of the joint. Instead, Cu2O clusters with dark-gray contrast were formed around the sintered pores in the central region of the joint, as shown in 2O3 clusters with light-gray contrast were also found around the sintered pores. However, In2O3 clusters were identified around the pores in all sintered regions of the joint after the HTS, which was not the case for the Cu2O clusters. Furthermore, owing to the interdiffusion of Ag and Cu at the wetting interface, the 300 nm thick sputtered Ag was transformed into the Ag-based Ag\u2013Cu solid solution phase, labeled (Ag)\u2013Cu in 2 could reach the central region of the Ag\u2013In alloy joint and form a continuous lamellar Cu2O layer. The formation and growth of the thick Cu2O layers generated cracks at the interface between the continuous Cu2O layer and the substrate, which negatively affected the mechanical properties.The EPMA elemental mapping of the Ag\u2013In alloy joint after HTS at 300 \u00b0C for 100 h is shown in 2. After 50 h to 2000 h of HTS, as shown in 2O layer was found in the sintered joint, which indicated that the amount of O2 entering the joint bonded at 20 MPa was significantly reduced compared to the joint bonded at 10 MPa. Nevertheless, clusters of Cu2O and In2O3 could still be observed around the sintered pores after the HTS; interconnected pores were still present in the as-bonded Ag\u2013In alloy joint, which provided pathways for O2 penetration. Although the Cu2O and In2O3 clusters were formed in the Ag\u2013In alloy joint, the oxide clusters around the sintered pores had a negligible effect on the mechanical properties of the joints compared to the lamellar oxides and cracks. More importantly, the pores in the sintered Ag\u2013In alloy joint bonded at 20 MPa did not coarsen, which was consistent with the results at 10 MPa.The shear strength variations of the sintered Ag and Ag\u2013In alloy joints with the HTS treatment time are illustrated in 2O layers and cracks. The red dashed line in 2 from entering the interior of the sintered joint and forming fragile Cu2O layers and cracks. Furthermore, the addition of In was proven to help prevent sintered pore coarsening. Consequently, the Ag\u2013In alloy joint with the optimal bonding condition was the most mechanically reliable at high temperatures because it simultaneously overcame the oxidation and pore coarsening issues.The blue dashed line in The surface fracture analyses of the sintered Ag joints are shown in 2O layers mainly formed near the edges of the sintered joints. Hence, the Cu surfaces were near the edges of the fracture plane, which was consistent with the cross-sectional SEM analysis. This indicated that cracks tended to propagate along the fragile Cu2O layers near the edges, rather than along the sintered joints, as illustrated in 2O layers were formed throughout the sintered joint, and the cracks only propagated along the Cu2O layers, as shown in The fracture surface analysis of the sintered Ag\u2013In alloy joints bonded at 10 MPa and 20 MPa is shown in 2O and one type of In2O3 were observed in the Ag\u2013In alloy joint bonded at 10 MPa after the HTS, including a Cu2O layer, Cu2O clusters, and In2O3 clusters, as shown in 2; therefore, O2 could still attack the sintered Ag\u2013In alloy joint through interconnected pores and react with Cu and In. This resulted in the formation of a Cu2O layer, which led to severe reliability issues.Two types of Cu2 from the environment entered the Ag\u2013In alloy joint via the interconnected pores at the edges of the joints; the high porosity favors the penetration of O2. Consequently, the concentration of O2 near the edges would be higher than that near the middle of the joint at the beginning of the HTS. Therefore, continuous Cu2O layers were observed between the Ag\u2013In alloy joint and the Ag-sputtered Cu substrate near both edges of the sintered joints, as shown in 2 and immediately reacts to form the Cu2O layer near the wetting interfaces at both edges of the joint. From the EPMA analysis, it was found that the 300 nm thick sputtered Ag layer was transformed into a Ag-rich Ag\u2013Cu solid solution phase, labeled (Ag)\u2013Cu, which was the evidence for the Ag\u2013Cu interdiffusion during the HTS. In addition, near the middle region of the Ag\u2013In alloy joint at the early stages of the HTS, the convergence of the Cu2O layer can be seen, as shown in 2 was not sufficient to form a Cu2O layer in the middle region of the joint at this stage. Therefore, Cu from the substrate needed to diffuse a relatively long distance before it formed the Cu2O layer near the middle region of the join, as illustrated in 2O also existed in the form of clusters located around the sintered pores in the middle region of the joint. The Cu2O clusters were mainly formed there in the early stages of the HTS because O2 that had reached the middle region via the interconnected pores was not sufficient to form a continuous Cu2O layer. However, Cu2O clusters could not be found near the edges of the joint; the Cu2O layer caused the Cu diffusing from the substrates to continue to react with O2, forming a thick layer. In addition to the two types of Cu2O contained in the joint, In2O3 clusters were found in all sintered regions of the joint after the HTS. In2O3 clusters precipitated around the sintered pores in the Ag\u2013In alloy joint because of the O2, which entered the joint through the interconnected pores, similar to the case for the Cu2O clusters. Additionally, mixed In2O3 and Cu2O clusters precipitated in the middle region of the joint because O2 reacted with both In from the joint and Cu diffusing from the substrates. After the long-term HTS in 2O layers and cracks were formed at both interfaces between the Ag\u2013In alloy joint and the substrate. At this stage of the HTS, a considerable amount of O2 diffused into the middle of the joint over a long period, causing the Cu2O layers near the edges to merge and form continuous Cu2O layers. Having developed a complete understanding of the oxidation mechanism in the sintered joint, it is now important to find solutions for overcoming oxidation issues.Herein, a mechanism for the oxidation process in the sintered Ag\u2013In joint was established, as illustrated in Sintered nano- and micro-Ag materials are promising for die attachment of power chips. In recent years, many studies have focused on pursuing low-pressure or pressureless bonding processes for Ag sintering to achieve cost reductions. However, the porosity of the sintered joint would unavoidably increase when a low-pressure or pressureless bonding was attempted. Consequently, this would significantly increase the risk of high-temperature oxidation, which affects the mechanical properties of the sintered joint. Therefore, developing a strategy for overcoming oxidation problems is a critical and urgent issue for sintered joints. Meanwhile, it is also crucial to inhibit the sintered pore coarsening phenomenon at high temperatures because the pore coarsening also decreases mechanical strength. Alloying the Ag joint has become the primary strategy to combat this issue in pure sintered Ag joints.2O layer in the sintered joints is insufficient. Lastly, another potential strategy is to introduce a surface finish as a diffusion barrier to prevent the diffusion of Cu from the substrate at high temperatures.In this study, we found a positive correlation, described by percolation theory, between the porosity of the as-bonded sintered joint and the occurrence of such high-temperature oxidation. In other words, the sintering porosity of the as-bonded sintered joint is an important reliability evaluation metric for high-temperature oxidation. According to the predictions of the percolation theory, high-temperature oxidation can be completely prevented when the porosity is below approximately 3%. Hence, reducing the porosity of the as-bonded sintered joint as much as possible helps prevent high-temperature oxidation. Previous studies have proven that the TLP bonding reaction can help prevent oxidation because the introduction of low-melting-point liquid metals during bonding can fill the gaps between the nano- and micro-Ag particles and form isolated pores. This not only resolves the oxidation problem but also addresses the sintered pore coarsening phenomenon. However, the TLP bonding method is not favored in industrial applications. Hence, in this study, the sintering behavior of the Ag\u2013In alloy paste developed by ball-milling was investigated. The results demonstrated that increasing the bonding pressure is another effective method to lower the porosity. The low-porosity Ag\u2013In alloy joint with the optimized bonding condition exhibited excellent reliability because it simultaneously avoided the oxidation and sintered pore coarsening problems. Consequently, the Ag\u2013In alloy joint could retain its outstanding mechanical properties at high operating temperatures. In addition to reducing the porosity, enhancing the heat dissipation of power chip modules is another way to avoid oxidation. If the heat dissipation is sufficient to keep the working temperature from exceeding 200 \u00b0C, then the oxidation problem can also be avoided because the driving force to form the Cu2 to enter the joint from the edges and deteriorated the sintered joint. Increasing the bonding pressure to 20 MPa significantly reduced the porosity of the Ag\u2013In alloy joint, blocking external O2 at high temperatures. Therefore, the robust Ag\u2013In alloy joint possessed the most stable shear strength and reliable mechanical properties by maintaining high shear strength of above 76 MPa without deterioration in a high-temperature environment because it inhibited the sintered pore coarsening and oxidation issues simultaneously. Lastly, the mechanism for the oxidation process in sintered joints was established in this study and used to formulate strategies to prevent such oxidation. In conclusion, a novel ball-milling method was employed in this study to produce the Ag and Ag\u2013In alloy pastes for die attachment through mechanical alloying, and a promising and reliable Ag\u2013In alloy material was developed for power electronic packaging. It is considered that the materials and technologies developed in this study have great potential for practical application in power-chip attachment and can meet the urgent requirements of future industries for highly reliable power semiconductor packaging materials for EVs or aerospace technology.In this study, pure Ag and In powders were refined to make flake-like Ag and Ag\u2013In alloy powders using a high-energy ball-milling process. Homogeneous Ag\u2013In alloy powders in solid solution could be obtained via mechanical alloying. Moreover, Ag and Ag\u2013In alloy pastes containing particle sizes less than 25 \u03bcm were synthesized for power chip attachment after sieving and mixing with an organic solvent. The graphical summary of the sintered Ag joints and Ag-In alloy joints is exhibited in"}
+{"text": "H-1,9-diazaphenothiazine and 10H-3,6-diazaphenothiazine, exhibited very potent action with the IC50 values less than 1 \u00b5g/mL and 1 \u00b5M against selected cancer cell lines. The strength of the anticancer action depends both on the tricyclic ring scaffolds and the substituents at the thiazine nitrogen atom. The review discusses the kind of the substituents, nature of tricyclic ring scaffolds with the location of the azine nitrogen atoms, the types of the cancer cell lines, and the mechanism of action.Azaphenothiazines are the largest and most perspective group of modified phenothiazines, and they exhibit variety of biological activities. The review sums up the current knowledge on the anticancer activity of isomeric pyridobenzothiazines and dipyridothiazines, which are modified azaphenothiazines with one and two pyridine rings, respectively, against 10 types of cancer cell lines. Some 10-substituted dipyridothiazines and even 10-unsubstituted parent compounds, such as 10 This system, as the phenothiazinium salt, was for the first time synthesized by Caro in 1876 as a dye called methylene blue . The last modification is the most perspective because it allows the formation of not only a new phenothiazine scaffold , but also allows to introduce new substituents. The introduction of the azine ring such as pyridine, pyridazine, pyrimidine, pyrazine, or quinoline in the place of the benzene ring, leads to new derivatives of phenothiazines, azaphenothiazines. Over 50 types of azaphenothiazine scaffolds are known which belong to varied heterocyclic ring systems ,19.The anticancer activity of classical and modified phenothiazines was summarized for the first time by Motohashi in the book chapter in the late 1980s . Later, The modified phenothiazines exhibit not only anticancer activity, but a wide range of other activities such as the reversal of multidrug resistance, anti-inflammatory, antibacterial, antiviral, antiplasmid, cholinesterase inhibitory, antioxidant, and antihyperlipidemic and are considered potential agents in the treatment in Alzheimer\u2019s and Creutzfeldt\u2013Jakob diseases, published in hundreds of original papers and patents, and summarized in numerous comprehensive review papers and chapters ,36,37,38The modification of the phenothiazine structures with the pyridine ring leads to different pyridobenzothiazines and dipyridothiazines, also named x-monoazaphenothiazines and x,y-diazaphenothiazines, the largest but also most diverse family of azaphenothiazines. The aim of this review is to present comprehensively anticancer activities of those azaphenothiazines.Pyridobenzothiazines represent linearly condensed tricyclic ring systems where the 1,4-thiazine ring is fused with the benzene and pyridine rings along the C-C bonds. As they differ from classical phenothiazines in the presence of the additional nitrogen atom instead of the carbon atom, they are named azaphenothiazines .3), called prothipendyl (4\u20139)  .Prothipendyl, the most useful pyridobenzothiazine, was synthesized in 1960  and is k50 value of 23.2 \u00b5g/mL and 28.1 \u00b5g/mL, respectively. The T47D and the SNB19 cells lines were the most resistant for the tested compound . Prothipendyl was found to be non-toxic (IC50 > 50 \u00b5g/mL) against the normal human fibroblast (HFF-1) cell line in comparison with cisplatin used as a reference (IC50 = 8.2 \u00b5g/mL) [The anticancer activity of prothipendyl was investigated recently using the cultured glioblastoma SNB-19, melanoma C-32, breast cancer MCF-7, and ductal carcinoma T47D cell lines with the normal human fibroblast (HFF-1) cell line used as a control. The cell lines used showed a different sensitivity to prothipendyl. The MCF-7 and the C32 cells lines were found to be the most sensitive with the IC2 \u00b5g/mL) ,46,47.4\u20139) containing aminohexyl and bromohexyl groups together with a nitro group in the pyridine ring (A set of twenty-one novel 10-substituted 1-azaphenothiazines (ine ring  were inv50 < 10 \u00b5g/mL) and were a few times more effective in comparison to a standard drug actinomycin D. Compound 6 (with the piperidinylhexyl group) showed the highest activity against the lung cancer H460, the malignant brain cancer T98G, and the thyroid cancer SNU80 with the IC50 values of 2.27\u20133.8 \u00b5g/mL, respectively. Similarly active were derivatives 9 (with the pyrrolidinyl-piperidinylhexyl group) against the H460 and SNU80 cell lines (IC50 = 2.1 and 2.3 \u00b5g/mL), 7 (morpholinylhexyl group), and 5 (pyrrolidinylhexyl) against H460 line (IC50 = 2.5 and 2.7 \u00b5g/mL). The derivative 4 was active against those three cell lines showing the IC50 values of 3.8\u20136.2 \u00b5g/mL [Eleven of the compounds tested exhibited distinct inhibitory action against selected cancer lines  inducing CHIK fever ,50.Other derivatives of 1-azaphenothiazine exhibited mainly antihistaminic, anticholinergic, anti-emetic, antitussive, neuropharmacological, antibacterial, and antitubercular activities and were the subject of many publications and reviews ,60,61,62H-2-azaphenothiazines (10) (Despite the fact that 10nes (10)  was syntH-3-azaphenothiazine (11) (Although the synthesis of 10ine (11)  was descH-4-Azaphenothiazine (12) (10ine (12)  was obtaAs was discussed above, pyridobenzothiazines are not intensively explored nowadays, despite their wide range of biological activities.The information provided on anticancer activity of pyridobenzothiazines was summarized in Dipyridothiazines mean linearly condensed tricyclic ring systems where the 1,4-thiazine ring is fused with two pyridine rings along the C-C bonds. As they contain two additional nitrogen atoms (instead of two carbon atoms) in comparison with classical phenothiazines, they are named diazaphenothiazines. Out of ten theoretically possible dipyridothiazine systems, only six have been known as 1,6-, 1,8-, 1,9-, 2,7-, 3,6-, and 3,7-diazaphenothiazines. This small amount of well-known dipyridothiazine types is a result of the difficulties in their synthesis involving orto disubstituted pyridines .H-1,6-Diazaphenothiazine (13) (10ine (13)  was prep50 in the range of 3.9 to 49.1 \u00b5g/mL) depending on the type of substituent and the type of cell line. The parent compound, 10H-1,6-diazaphenothiazine (13), and compounds 14 and 15 (containing the propargyl and nitropyridinyl substituents at the thiazine nitrogen atom) were found to be more active  in relation to cisplatin as a reference compound (7.4 \u00b5g/mL) against the breast cancer MCF-7 cells. The derivative 16 (containing the methylpiperazinylbutynyl substituent) was as effective as cisplatin (7.5 \u00b5g/mL). 10H-1,6-Diazaphenothiazine (13) and compound 17 (containing the diethylaminoethyl substituent) were slightly more effective (7.5 and 6.6 \u00b5g/mL) than cisplatin (7.8 \u00b5g/mL) against the C-32 cells. The derivative 18  was the most effective against the SNB-19 cells with the IC50 values of 18.9 \u00b5g/mL. The most anticancer active compounds were nontoxic with reference to normal fibroblasts HFF-1 with the IC50 values exceeded 50 \u00b5g/mL [A large family of 1,6-Diazaphenothiazine derivatives  with the50 \u00b5g/mL .Further studies looking for the structure\u2013activity relationship (SAR) related to lipophilicity analysis were performed; however, they did not show the absolute dependence of the anticancer activity on the lipophilic properties .p-substituted benzyl substituents at the 1,2,3-triazole ring were obtained. The compound 19 (p-chlorobenzyl substituent showed an extremely promising anticancer activity (IC50 = 0.25\u20134.66 \u00b5M) in relation to the glioblastoma SNB-19, the colorectal carcinoma Caco-2, the lung cancer A549, and the breast cancer MDA-MB231 [Recently, novel 1,2,3-triazole-dipyridothiazine hybrids containing the 1,6-diazaphenothiazine system and selected pound 19  with theDA-MB231 .50 > 100 \u00b5M. This derivative was selected to study the mechanism of anticancer activity using RT-qPCR method. The influence on the gene transcriptional activities of proliferation marker (H3), the cell cycle regulator (TP53 and CDKNIA), and the intracellular apoptosis pathway (BCL-2 and BAX) were analyzed. The compound greatly affected the mRNA copy number of histone H3 gene in all cancer lines, which had an influence on a modification of the chromatin structure in the cells and significantly interfered with the amount of mRNA copies of the TP53 in all investigated cancer lines. There was also a strong decrease in the CDKN1A copies in all cancer cells, which can suggest a possibility of participation in cell cycle arrest. The derivative reduced remarkably the expression of the BCL-2 in SNB-19, A549, and MDA-MB231 cancer lines, but in the Caco-2 cell line there was a slight increase. The analysis of expression of H3, TP53, CDKN1A, BCL-2, and BAX genes revealed that this compound inhibited the proliferation in all cells and activated mitochondrial events of apoptosis [The compound was nontoxic against the normal fibroblasts NHDF with ICpoptosis .H-1,8-Diazaphenothiazine 20  were screened against the leukemia L-1220 and the colon carcinoma SW-948 cell lines. The most effective compound turned out the parent compound 20, exhibiting similar effect as cisplatin against the cancer cells of carcinoma SW-948 and leukemia L-1210 at 5 and 10 \u00b5g/mL, respectively.A group of 1,8-diazaphenothiazines was first reported in 2015. The biological potential of the compounds was the subject of both the publication and the patent ,71. 10H-azine 20  and its 23) (50 in the range of 26 to 46 \u00b5g/mL) which was dependent on both the type of substituent and the type of cell line. The most active compound was 10-diethylaminobutynyl-1,8-diazaphenothiazine (24) (IC50 = 26.1 \u00b5g/mL) against the melanoma C-32 [Another group of 1,8-diazaphenothiazines was obtained by a transformation of the 10-propargyl-1,8-diazaphenothiazine (23)  into theoma C-32 .In order to search for quantitative relationships between physicochemical and pharmacological properties of the investigated compounds, preliminary QSAR (Quantitative Structure-Activity Relationship) studies were undertaken for anticancer and immunosuppressant 1,8-diazaphenothiazines. For this group of diazaphenothiazines, some correlations between the TNF-\u03b1 inhibition, anticancer activity, and lipophilicity were observed .50 = 1.8\u201349 \u00b5M). The compound 25 (p-chlorobenzyl substituent showed a strong promising anticancer activity (IC50 = 1.82 \u00b5M) in relation to the lung cancer A549. However, this activity was lower than that of the isomeric 1,6-diazaphenothiazine 19 (IC50 = 0.25 \u00b5M) [A group of novel hybrids of the 1,8-diazaphenothiazine and 1,2,3-triazole systems were obtained and tested for their cytotoxicity against the glioblastoma SNB-19, colorectal carcinoma Caco-2, lung cancer A549, and breast cancer MDA-MB231 cell lines. The anticancer activity of this group of derivatives was differential (ICpound 25  with the0.25 \u00b5M) .H-1,9-diazaphenothiazine (26) (The structure of 10ine (26)  was firsH-1,9-diazaphenothiazine (26) was described, along with a complete structural analysis, as well as new 10-substituted derivatives containing alkyl, alkynyl, and dialkylaminoalkyl substituents. These compounds with various substituents were screened for their anticancer activity against using the glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB-231 cell lines [H-1,9-diazaphenothiazine (26) was very active against melanoma C-32 (IC50 = 3.83 \u00b5M), even more potent than cisplatin (IC50 = 13.2 \u00b5M), but inactive against other lines. These derivatives induced a varying anticancer activity. The most promising derivatives in this group were compounds 27 and 28 with the propynyl and diethylaminoethyl groups  was highly active against the glioblastoma SNB-19 (IC50 = 3.85 \u00b5M) and the melanoma C-32 (IC50 = 3.37 \u00b5M). 10-Diethylaminoethyl-1,9-diazaphenothiazine (28) was even more active against the glioblastoma SNB-19 with an IC50 value of 0.34 \u00b5M, and the breast cancer MDA-MB-231 cell line with IC50 = 2.13 \u00b5M. For those two compounds, the expression of H3, TP53, CDKN1A, BCL-2, and BAX genes was detected by the RT-qPCR method. The analysis of the gene expression revealed that both compounds selectively reduced the expression of H3 and TP53, and enhanced the expression of CDKN1A in the examined cell lines. The gene expression ratio of BAX/BCL-2 indicated the induction of mitochondrial apoptosis in two cancer cell lines (SNB-19 and MDA-MB-231). In the melanoma C-32 cell line, the transcriptional gene activity suggests a different way of cell death. The proteome profiling study showed the most probable compound action on SNB-19 cells through the intrinsic mitochondrial pathway of apoptosis [10-Propynyl-1,9-diazaphenothiazine  and its methyl derivative 30 , and its selected 10-substituted derivatives containing various substituents, were also screened for their anticancer activity in the National Cancer Institute, Bethesda, MD, USA. In a preliminary experiment, the most promising compound turned out to be parent compound 29, which was screened on about 60 cell lines including leukemia, melanoma, non-small cell cancer, colon cancer, CNS cancer, ovarian cancer, renal cancer, prostate cancer, and breast cancer. The strongest action was observed with relation to the non-small lung cancer cell lines HOP-62 and HOP-92 with IC50 values of 0.3 and 1.7 \u00b5g/mL, respectively. Other types of cancer cell lines were also sensitive to compound 29 showing the following IC50 values: 2.4 and 3.6 \u00b5g/mL (colon 205 and HCT-116), 3.1, 3.9, and 5.4 \u00b5g/mL ; 4.1 \u00b5g/mL (leukemia HL-60(TB)); 5.9 \u00b5g/mL (breast HS 578T), 6.5 \u00b5g/mL (melanoma M-14); 6.8 \u00b5g/mL (CNS SF-539 and SNB-19); 7.1 \u00b5g/mL (ovarian OVCAR-8); and 8.4 \u00b5g/mL (prostate PC-3). 10-Substituted derivatives were less active in the preliminary test [10ary test .H-2,7-diazaphenothiazine (29) and its nitrophenyl and pyrrolidynyloethyl derivatives exhibited a meaningful antioxidant activity (IC50 = 64\u2013125 \u00b5M) in the experiment of non-enzymatic peroxidation of hepatic microsomal membrane lipids [10e lipids .31 with the methylpiperazinylbutynyl substituent . This derivative was also the most effective in relation to the glioblastoma SNB-19 (21.2 \u00b5g/mL). The remaining derivatives of this group showed a moderate activity with IC50 values in the range of 24.8 to 29.2 \u00b5g/mL. To understand the anticancer mechanism, the effect of derivative 31 on the expression of genes encoding TP53, CDKN1A, BCL-2, and BAX in the tested cancer cells was investigated. The RT-qPCR experiment showed the heightened number of CDKN1A copies in the T-47D and SNB-19 cells which implied the ability of the cell cycle arrest. Examination of the ratio of gene expression BCL-2/BAX revealed the activation of mitochondrial apoptosis in the T-47D cells [Another group of 2,7-diazaphenothiazine derivatives was obtained by a transformation of the 10-propargyl derivative of 2,7-diazaphenothiazine into the dialkylaminobutynyl derivatives. The anticancer activity was evaluated against the cell lines of human glioblastoma SNB-19, melanoma C-32, and breast cancer T-47D. The most active compound was the derivative stituent  against 7D cells .50 = 0.26\u201349 \u03bc\u039c. The most active compound was the derivative with benzyl substituent 32 in the 1,2,3-triazole ring showing the IC50 values of 0.26 \u03bc\u039c against the Caco-2 and A549 cancer cell lines and 0.77 \u03bc\u039c against the MDA-MB231 cancer cell line. A similarly high antiproliferative activity exhibited the derivative 33 with p-fluorobenzyl substituent 33 in 1,2,3-triazole ring against the MDA-MB231 (IC50 = 0.64 \u03bc\u039c) and against the A549 (IC50 = 0.65 \u03bc\u039c) cancer cell line [The hybrids of 2,7-diazaphenothiazine and 1,2,3-triazole systems were obtained and examined for their cytotoxicity against the glioblastoma SNB-19, colorectal carcinoma Caco-2, lung cancer A549, and breast cancer MDA-MB231 cell lines . The antell line .Further studies looking for the structure\u2013activity relationship (SAR) related to lipophilicity analysis were performed; however, they did not show the direct dependence of the anticancer activity on the lipophilic properties of 2,7-diazaphenothiazines .The 3,6-diazaphenothiazine system with the nitro group in position 1 and the chlorine atom and methoxy group in position 7 was described in the 1960s, but the identification of these compounds was based only on the logic of the conducted chemical syntheses. The author drew attention to the chemical properties of these molecules, while the biological activities were not described ,87.H-3,6-Diazaphenothiazine (34)  exhibited similar action to cisplatin (6.3 vs. 7.8 \u00b5g/mL) against the melanoma C-32 and moderate activity (11.3 \u00b5g/mL) against the breast cancer MCF-7. The family of these diazaphenothiazines showed non-toxic or almost non-toxic action against the normal fibroblast HFF-1 cell line [In 2016, a novel group of 3,6-diazaphenothiazines was described both due to interesting chemical and structural properties, but above all anticancer properties . 10H-3,6ine (34)  and its H3, TP53, CDKN1A, BCL-2, and BAX) supported the antiproliferative action of diazaphenothiazines 34 and 35 and revealed the activation of the p53 pathway in cancer cells inducing the cell cycle arrest. Examination of the ratio of gene expression BAX/BCL-2 indicated the activation of mitochondrial apoptosis in the MCF-7 and SNB-19 cells [The RT-qPCR experiment of gene expressions  was also studied on the A2780 ovarian cancer cells by an investigation on cytotoxicity profiles, the mechanism of apoptosis, and cell invasion. This parent compound induced a dose-dependent inhibition on the A2780 cancer cells (IC50 = 0.62 \u03bcM), with a significant less cytotoxicity towards the normal kidney HEK293 cells and the normal heart H9C2 cells. It was presented that the generation of reactive oxygen species (ROS) and the polarization of mitochondrial membrane potential (\u0394\u03a8m) suggested that compound 34 induced cell death through the oxidative damage. This compound elicited an upregulation of caspase-6, -3, and -7, which are actively involved in the formation of cell shrinkage, chromatin condensation, and the fragmentation of DNA. Additionally, the activation of caspase-3 brought about an increased enzymatic activity of DFFA (DNA fragmentation factor-\u03b1). This compound induced apoptosis via an intrinsic (mitochondria-dependent) and an extrinsic (cell death receptor-dependent) pathway. The inhibition of NF-\u03baB and the subsequent inhibition of BIRC6-XIAP complexes reduced the invasion rate of the A2780 cancer cells penetrating through the MatrigelTM Invasion Chamber. The investigated compound exhibited a cytostatic action and significantly arrested cell proliferation at the G2/M phase. The presented results suggested that the 10H-3,6-diazaphenothiazines (34) would become promising chemotherapeutic agents in the future [Anticancer activity of 10e future .37 with the diethylamino-2-butynyl substituent (50 = 0.11 \u03bcg/mL). The analysis of genes expressions  using the RT-qPCR method indicated the induction of mitochondrial apoptosis in the SNB-19 cells [Very recently, another group of new 10-substituted derivatives of 3,6-diazaphenothiazine, containing the triple bond linker ended with tertiary cyclic and acyclic amine groups was synthesized. This group exhibited varied anticancer activities against the human glioblastoma SNB-19, melanoma C-32, and breast cancer MDA-MB231 cancer lines, depending on the nature of the substituents. The most active compound was the derivative stituent  against 19 cells .p-substituted benzyl substituents at the 1,2,3-triazole ring were synthesized and screened for the anticancer activity. In this group of derivatives, the compound 38 with the p-fluorobenzyl substituent showed a very promising anticancer action (IC50 = 0.25 \u00b5M) in relation to the colorectal carcinoma Caco-2 and the lung cancer A549  and its 10-derivatives containing the diethylaminoethyl and dimethylaminopropyl substituents  route is suggested. The structure\u2013activity relationship of dipyridothiazines led to the conclusion that the strong anticancer action is dependent on both kinds of substituents at the thiazine nitrogen atom and the nature of the dipyridothiazine systems. These dipyridothiazine scaffolds are crucial for the anticancer activity as some N-unsubstituted dipyridothiazines were found to be very potent, for example, 10H-1,9-diazaphenothiazine and 10H-3,6-diazaphenothiazine. It also means this activity is relying on the location of the two azine nitrogen atoms in the tricyclic ring systems. The authors hope that this review highlights the importance of the pyridine modified phenothiazines in the search for lead compounds in the anticancer therapy.The review summarizes the current knowledge on the anticancer activity of isomeric pyridobenzothiazines and dipyridothiazines, the modified azaphenothiazines with one and two pyridine rings against 10 types of cancer cell lines. Whereas pyridobenzothiazines were synthesized as modified successors of the classical neuroleptic phenothiazines and their anticancer activity was found only recently, dipyridothiazines were mostly obtained with the aim at the anticancer activity. The last type of compounds  exhibited very impressive activity with the IC"}
+{"text": "Given the common problem of missing data in real-world applications from various fields, such as remote sensing, ecology and meteorology, the interpolation of missing spatial and spatio-temporal data can be of tremendous value. Existing methods for spatial interpolation, most notably Gaussian processes and spatial autoregressive models, tend to suffer from (a) a trade-off between modelling local or global spatial interaction, (b) the assumption there is only one possible path between two points, and (c) the assumption of homogeneity of intermediate locations between points. Addressing these issues, we propose a value propagation-based spatial interpolation method called VPint, inspired by Markov reward processes (MRPs), and introduce two variants thereof: (i) a static discount (SD-MRP) and (ii) a data-driven weight prediction (WP-MRP) variant. Both these interpolation variants operate locally, while implicitly accounting for global spatial relationships in the entire system through recursion. We evaluated our proposed methods by comparing the mean absolute error, root mean squared error, peak signal-to-noise ratio and structural similarity of interpolated grid cells to those of 8 common baselines. Our analysis involved detailed experiments on a synthetic and two real-world datasets, as well as experiments on convergence and scalability. Empirical results demonstrate the competitive advantage of VPint on randomly missing data, where it performed better than baselines in terms of mean absolute error and structural similarity, as well as spatially clustered missing data, where it performed best on 2 out of 3 datasets. Under perfect lab conditions, a data scientist can train models, infer variables of interest and discover new knowledge from neatly organised, consistent and complete datasets. However, in real-world scenarios, one is rarely so lucky. Whether it is random measurement noise, inconsistent annotation, missing data or another problem, real-world data can be messy, and tricky to process in such a way that downstream models and processes can use it effectively.In this work, we aim to address the problem of missing data in the specific case of spatial gridded data by proposing a computational method for spatial interpolation. Prominent examples of such missing data in real-world scenarios include satellite imagery in remote sensing data  Jiang , tend to Anselin  or convo Anselin , rely on Anselin , can usesystem-oriented perspective, illustrated in Fig.\u00a0Markov reward processes , also known as Krige   ha Anselin . Moving  Anselin , but can Anselin . Highly  Anselin . Apart f Anselin . This la Anselin , varying Anselin , ambient Anselin  and an a Anselin . The spaNeural networks and deep learning Deep learning techniques, and convolutional neural networks (CNN) in particular, have been used to great effect in many computer vision applications . Moreover, like Gapfill, it offers a computational alternative to existing methods with an emphasis on explicit statistical spatial modelling.n corresponds to the number of rows and m to the number of columns. At every cell c in i and j corresponding to the row and column indices in c in Let us define a spatial grid f denotes the number of features per cell. Thus c in c in We further define a feature grid In this section, we will describe our proposed interpolation method in four steps. The general procedure and main philosophy will first be illustrated, after which we introduce some background for our update rules, and propose the two concrete variants of our method that we implemented. Finally, we will discuss our approach for ensuring efficient computation allowed by parallel matrix operations.estimated value of its neighbours c. Thus, by iterating this procedure, our algorithm recursively propagates known values throughout chains of estimated values in The core of our proposed method relies on iterative element-wise updates to an estimation grid. We first instantiate S is a set of states s and R is a set of rewards s. MRPs extend Markov chains, which do not incorporate rewardsR, and have been successfully used to model the behaviour of a single variable over time  absorbing state is reached from which no further transitions are possible: s to s, and are unaffected by the history of previous states in the trajectory. If a reward s, this gives information about the desirability of state s. However, aside from this immediate reward state values, where the rewards of all possible successor states v(s) for all c (at a certain time) can be seen as a state s, with the set of spatial neighbours v(s), then, would be the target variable c, such that our spatial interpolation algorithm takes the form of:c. While in principle, it is possible to add any user-defined aggregation function, we opted to stay close to the canonical Bellman equation, by taking the mean  of Our update rule is based on Markov reward processes (MRPs). MRPs Bellman  are mode Bellman , where tOne could also consider generalising this approach to spatio-temporal interpolation problems. In that case, the algorithm cannot solely rely on Eq.\u00a0We propose two variants of our value propagation interpolation method. The first, SD-MRP (static discount-MRP), uses a single spatial weight parameter entclass1pt{minimaentclass1pt{minimadecrease over subsequent recursive calls, making known values reminiscent of a light source in the fog, radiating values around itself and merging with other light sources, but decaying over distance. In the example of spatial interpolation of temperatures, it would propagate the known temperature values over the grid, at an intensity decreasing with every recursive call, like a heat source dissipating over distance. This can be seen in Fig.\u00a0k is the number of times Eq.\u00a0The most basic variant of our proposed method stays closest to the canonical form of the Bellman equation in Eq.\u00a0entclass1pt{minimaIn an ideal case, rather than using a single static weight entclass1pt{minimaIn the case of machine learning models and pipelines, in order to train the model, we use the available cells with known true values in y indicates this tensor contains values, and i indicates the stage of operations the data is currently in, with the accompanying neighbour weights in For the efficient processing of the main iterative loop of lines of our algorithms as in lines 9-22 Algorithm\u00a01, we reformulated our update function as a series of matrix operations, allowing updates to be carried out in a highly parallelised manner through vectorisation. This approach does, however, necessitate the use of weighted averaged  as an aggregation function. We will illustrate the procedure on the simpler case , but the approach can be generalised to spatio-temporal MRP as well. The main idea of this approach is to shuffle neighbouring values around in matrices and tensors d denotes the maximum number of neighbours c, the entries along the d-axis of c. Concretely:c, rather than the values:c in a single dimension, although the information on the original columns of Concretely, let d becomes equal to 6, as any cell can now have up to 6 neighbours. Since all neighbours are already included in the fourth dimension, there is no reason to keep the spatial and temporal dimensions separate. Thus, we can still generate the 2D matrices Using this vectorised approach, we found that the complexity of our algorithms in terms of wall-clock time improved by a factor between 10 and 100. In order to adapt this approach to spatio-temporal MRP interpolation, which adds an extra dimension for time, In this section, we will share the details of our experiments. We will first introduce the research questions we were interested in, after which we will list the baselines we compared our method to and the datasets used in our experiments.R1: How does VPint compare to baseline methods in terms of mean absolute error, root mean squared error, peak signal-to-noise ratio and structural similarity?R2: Does VPint converge to stable prediction values?R3: Can VPint be generalised to spatio-temporal problems?R4: Can WP-MRP leverage spatial features to perform better than SD-MRP, given sufficiently informative features?R5: How do VPint and baseline methods scale as the size of the dataset increases?In addition to these main research questions, we were also interested in whether different patterns of missing data would give different results.We were interested in answering the following research questions with our experiments:Ordinary Kriging (OK), using an implementation by the Python library PyKrige , also using PyKrige\u2019s implementation. UK is highly similar to OK, but it compensates for the possible existence of a trend in the data. For both OK and UK, while more advanced methods exist, such as local approximate Gaussian processes , moving average (MA) and autoregressive moving average (ARMA) models, using auto-sklearn to find the best performing regression model. Canonically, these models are ordinary least squares (OLS)-based linear regression methods, with extra spatial (SAR) or error (MA) terms (both in the case of ARMA). However, since we use auto-sklearn, though OLS is also a possible model, the final model will generally have a different formula, such as the potentially non-linear support vector regression models. For SAR, the spatial term is based on a spatial weight matrix c into a single vector  Haining . Its forConvolutional neural networks (CNN), optimised using automated neural architecture search (NAS). The CNN regression predicted Our selection of baselines was aimed at including competitive interpolation and regression methods used for spatial and geo-spatial modelling in practice. The selection we made consists of:Our main experiments involved a synthetic spatial dataset as well as two real-world datasets (GDP and COVID-19 trajectories), with an additional synthetic spatio-temporal dataset used to address R3. The implementation of our data generation algorithms used to create the experimental synthetic datasets is included in our public code repository; likewise, the real-world datasets are available for public use at their respective sources, allowing others to reproduce our results.Spatial targets For this synthetic dataset, based on a parameterised mean c was assigned a base value c as a weighted average  GDP per capita for residents in the area covered by a grid cell, or the sum of COVID-19 incidence at that location. The granularity of these datasets thus introduces a trade-off: a high granularity increases the computational cost and may result in relatively sparse datasets (as was the case in our COVID-19 dataset), but does provide a high level of detail. Meanwhile, a low granularity may result in data too low-grained to draw meaningful conclusions from, or cells that simply all regress to a global mean due to the erasure of local spatial patterns, but will be faster to compute and likely results in a higher density dataset. There is no minimal or maximum granularity cutoff point at which an interpolation method becomes infeasible. However, when gauging how applicable an interpolation method is to a users\u2019 gridded dataset, this trade-off merits consideration.Gross domestic product (GDP) targets For GDP data, we used a gridded spatial dataset containing worldwide GDP estimates sourced from World Bank , feature selection  and feature normalisation . In accordance with the design philosophy of programming by optimisation (PbO) (Hoos bO) Hoos , we did bO) Hoos , to seleThe following section will explain the procedures and experimental conditions necessary to carry out our experiments.In order to evaluate our methods, we required data that was fully available to compute error metrics, while also having access to grids with missing data. To this end, we introduced two methods for \u2018hiding\u2019 known values, resulting in different patterns of missing data.Random missing values This missing value approach was straightforward. Given a proportion of known values p, for all other cells there is a probability of being randomly obscured if a number p. That is, for every cell p was set to 0.8.Spatially clustered hidden values Much like the spatial data itself, the missing data points in a grid may not be independent, and instead subject to spatial autocorrelation themselves. For example, some locations may have missing data due to natural barriers making measurements difficult, or due to local phenomena such as clouds obscuring parts of the measurements. In this missing value approach, we were inspired by optical satellite data, where clouds are the biggest source of missing data in the field. This approach is also why algorithms like Gapfill  30 times for two types of missing data  on every dataset , including both synthetic data and real-world datasets and addressing R1 and R3. The performance of the methods was compared according to their ranks based on the Wilcoxon signed-rank test Wilcoxon , which if in Eq.\u00a0n and m allowed us to see how well all methods scaled to larger datasets (R5). Thus, in addition to the general performance results, we used the synthetic data to run additional experiments to address research questions 2 through 5. Conversely, we used the real-world datasets to gauge how well the performance on synthetic data, and the analysis thereof, would generalise to real-world cases. For the scalability analysis we set n ranging from 20 to 200 in steps of 20. The spatially clustered missing data was generated using For spatial synthetic data we set the size of the grid to For every combination of a dataset with a type of missing data, all algorithms were run 30 times, and used automated algorithm configuration . These results were computed as the mean of 30 runs per algorithm and dataset for every performance metric. After covering the dataset-specific performance metrics, we investigate other properties of VPint: qualitative visual plausibility, the convergence of Eq.\u00a0Since multiple properties can be desirable in an interpolation method, we evaluated our method based on 4 performance metrics. The first of these was the mean absolute error (MAE):The results presented in this subsection are dedicated to answering R1. We ran detailed experiments on the synthetic spatial dataset as well as the real-world GDP per capita and COVID-19 datasets.Synthetic spatial dataThe results for synthetic spatial data are shown in Table GDP per capita The results for GDP data are shown in Table COVID-19 trajectories The results for COVID-19 trajectories are shown in Table We now present the results of the experiments addressing the remaining research questions, R2\u2013R5, exploring various properties of our proposed method.Visual plausibility An example of hidden synthetic data (Algorithm convergence (R2)An example of the convergence of WP-MRP over iterations can be seen in Fig.\u00a0Generalising to spatio-temporal problems (R3)Addressing R3, to gauge whether our method could also be applied to 3-dimensional spatio-temporal problems, we ran an additional set of experiments on synthetic spatio-temporal data. The results of this experiment are shown in Table Performance of WP-MRP compared to SD-MRP as a function of feature correlation (R4) To address R4, we ran an additional experiment on synthetic data with settings of the feature correlation coefficient f in Eq.\u00a0f, and conversely, SD-MRP appears more successful for low feature-target correlations. However, there appear to be diminishing returns for higher f after 0.4, and already at a correlation of 0.1 WP-MRP performed better than SD-MRP on the synthetic data. In conclusion for R4, this experiment shows that WP-MRP leverages spatial features to perform better than SD-MRP in situations where the features are sufficiently informative.Scaling to larger datasets (R5)  Addressing R5, we ran a scalability analysis by running every algorithm once on synthetic data for grid sizes ranging from 20 to 200 (height and width) in steps of 20. The results of these experiments, based on the total running time of methods  can be seen in Fig.\u00a0atasets R  AddressWe can also see in both figures that universal Kriging scales very poorly to larger datasets; in fact, its runs timed out after grids of the size In conclusion for R5, our methods scale better than Kriging to larger datasets, on par with non-spatial regression, SAR, MA, and ARMA, though SD-MRP did take longer than these methods on randomly missing data. Generally, our methods use substantially less memory than ordinary Kriging and universal Kriging.on average, when they do fail to produce good results, the errors will be more extreme than those of baseline methods. This was seen especially clearly in runs where WP-MRP obtained error values orders of magnitude higher than all other methods.Tables On spatially clustered missing data, in terms of MAE, SD-MRP still performed best on the COVID-19 dataset, but was outperformed by belief propagation on GDP data and by ARMA on synthetic data. WP-MRP also failed to significantly outperform baseline methods on any of the datasets for this type of missing data. However, in terms of SSIM, both VPint variants performed significantly better than all baselines on GDP and COVID-19 data, and SD-MRP tied with SAR and ARMA for synthetic data. Since it seems that SD-MRP preserves the spatial structure of spatially clustered hidden data better than baseline methods on all 3 datasets, and WP-MRP did so on 2 out of 3 datasets, we conclude that VPint would be a better option for this type of missing data if the spatial structure of the interpolations is important. Interestingly, SSIM is higher for all methods for spatially clustered data; this is likely caused by this type of missing data being considered a substantial structural element, thus affecting SSIM less than random missing data.f of around 0.1 for synthetic data, implying that a high correlation between features and targets is not required for the feature data to have added value to the method. Finally, Fig.\u00a0Regarding our additional experiments, we found that the convergence of VPint tends to progress smoothly and has very little variance between runs , the latter of which exploits spatial features describing the characteristics of the grid. In our experiments, VPint was found to perform significantly better than baseline methods in terms of mean absolute error and structural similarity on randomly missing data in 3 datasets, and 2 out of 3 datasets for spatially clustered missing data. Overall, whether VPint is the appropriate choice of algorithm appears to depend on the type of data in question, and the goals of the user. In the common case where a low error rate is the objective, particularly in a way that preserves the spatial structure of a grid, VPint  will generally be the best option for randomly missing values. On spatially clustered missing data, SD-MRP is usually a better option, though other methods are more competitive on this type of missing data. Despite these advantages offered by VPint, if the user is looking for a method that does not suffer from outliers of particularly bad predictions, and is willing to accept higher average errors as a result, other methods may be a better option.In future work, it would be interesting to focus on exploring the performance of our methods on other real-world datasets, particularly when using other sets of features not derived from map data. Furthermore, we see value in further analysis of the spatio-temporal variant of VPint, focussed on the circumstances under which it will perform well. Alternatively, a different approach to spatio-temporal interpolation could be a temporally layered version of WP-MRP, using a representation similar to the tensor-based approach adopted by Corizzo et\u00a0al. . Such an"}
+{"text": "The adjacent cranial and caudal EPA showed by far the most frequently significance in the prediction of all parameters used. Anthropometric and spino-pelvic parameters showed sparse impact, which was frequently in the lower lumbar regions. On average, a very good prediction was found. For two mEPA  and two bEPA , the R2 was >0.8. However, the predicted EPA differed for individual patients, even in these very effective prediction models\u2014roughly around \u00b110\u00b0 as compared to the measured EPA. Conclusions: In general, the prediction showed good to perfect results. In the supine position, the spinopelvic and anthropometric parameters show sparse impact on the prediction of mEPA or bEPA.Background: The correction of malposition according to vertebral fractures is difficult because the alignment at the time before the fracture is unclear. Therefore, we investigate whether the spinal alignment can be determined by the spino-pelvic parameters. Methods: Pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), adjacent endplate angles (EPA), age, sex, body weight, body size, BMI, and age were used to predict mono- and bisegmental EPA  in the supine position using linear regression models. This study was approved by the Ethics Committee of the Medical Association of Saxony-Anhalt Germany on 20 August 2020, under number 46/20. Results: Using data from 287 patients, the prediction showed R Approximately 80% of spinal fractures are located in the lumbar or thoracic sections of the spine, of which 50% are located at the thoracolumbar junction Th11-L2) [1-L2 [1,2There are efforts and investigations to elucidate the sagittal profile of the spine using pelvic parameters ,4,5. TheHowever, it is unclear whether there is a relationship between the sagittal spine profile and the pelvic parameters, even in the supine position, and whether the pelvic parameters are suitable for estimating the sagittal spine segment angles.Therefore, the aim of this study is to investigate whether the spinal alignment, captured by the mono- and bi-segmental sagittal Cobb angles, can be determined by the spino-pelvic parameters and the Cobb angles of the adjacent segments.This study is a retrospective analysis of cross-sectional data, and it was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Medical Association of Saxony-Anhalt Germany on 20 August 2020, under number 46/20. Initially, polytrauma CT scans of 1826 patients were examined and evaluated for suitability according to the inclusion and exclusion criteria. The CT images were acquired using the Aquilion PRIME  in the supine position. Included in this study were patients with intact spine, pelvis and femur. Patients under 18 years old, with spine deformity or spondylolisthesis with a Meyerding grade greater than 1 ; spine, The following spino-pelvic parameters were determined in the CT images: pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS). The spino-pelvic parameters were measured according to the specifications of Roussouly et al. and Le Huec et al. ,4.The PI was measured as the angle between the line perpendicular to the midpoint of the cranial endplate of the sacrum  measured for each segment from Th8/Th9 to L4/L5 and bi-segmentally (bEPA) for the segments of Th8 to L4, respectively. The mEPA is the angle between the cranial endplate of the cranial vertebra and the caudal endplate of the caudal vertebra of the segment. The bEPA is the angle between the cranial endplate of the nearest cranial vertebra and the caudal endplate of the adjacent caudal vertebra . However, the procedure for the prediction of the caudal segments of the lumbar spine L3/5, L4/5 (mEPA) and L3, L4 (bEPA) were different due to the issue that no complete mEPA or bEPA could be measured as described above. Therefore, the segment angles of L4/S1 and L5/S1 were used as caudal segments for the prediction and were measured between the cranial endplates of L4 or L5 and S1, respectively.Lordotic angles are described by positive values and kyphotic angles by negative values.To identify differences between older and younger patients, the cohort was divided in older and younger patients, according to the median age. Differences regarding each mEPA and bEPA between the age groups were examined using a linear general model for repeated measures. The mEPA and bEPA of the segments were used for repeated measurements. The impact of sex, body weight, body size, BMI, and spino-pelvic parameters  on the segmental angles were adjusted by using them as covariates. Differences in spino-pelvic parameters were examined using a multivariate general linear model. Sex, body weight, body size, and BMI were used as covariates. Sex was coded with 1 for men and 2 for women.The results of the Bonferroni-Holm post hoc pairwise comparison between the younger and older group regarding mEPA and bEPA for each segment, as well as the spino-pelvic parameters, are reported as means, standard deviation (sd), lower and upper limit  of 0.95 confidence interval (CI), and minima and maxima .To predict the mono- and bisegmental angles from Th9/10 to L4/5 (mEPA) and Th10 and L4 (bEPA), linear regression models were used for each segmental level, respectively. For the prediction of mEPA, the adjacent cranial and caudal segments were chosen in the following way. For example, if the mEPA of Th10/11 is the focus for the prediction, it can be predicted using Th8/9 and Th11/12, or using Th9/10 and Th12/L1 as the cranial and caudal segments, respectively. In other words, Th8/9 and Th11/12 can be used as the adjacent mEPA for the prediction of the mEPA of Th9/10 and Th10/11, respectively. In an iterative process, the dependent variables were successively reduced to such an extent that all variables ultimately used in the regression model showed significance. 2 was used to assess model quality of the linear regression. Model quality with an R2 greater than or equal to 0.8 was set to define the prediction as valid. Predictions of valid EPA were subsequently compared with the measured EPA, using difference plots. Therefore, the measured EPAs were used, and the difference between the predicted and the measured EPAs was calculated. The limits of agreement were set using a 1.96-fold standard deviation of the differences between both methods. Linear regression lines were fitted to this data in order to reveal the tendency impact, depending on the values. SPSS V.28  was used for statistical analyses, with p = 0.05.Corrected RIn total, the data of 208 male and 79 female adults were be included in this study. Due to the median split (40 years), a group of 143 younger adults  and a group of 144 older adults  was generated. p = 0.017).The younger group included significantly more males  compared to the older group . The mEPA differed significant between the segmental levels (p < 0.001), except between L4/L5 and L5/S1 (p = 0.121), Th12/L1 and Th8/9 (p = 1.000), Th9/10 (p = 0.660), Th10/11 (p = 1.000), between Th11/12 and Th8/9 (p = 1.000), and between Th10/11 and Th8/9 (p = 1.000). The covariates showed a significant effect for sex (p < 0.001), PT (p = 0.015), and SS (p = 0.009). Body weight (p = 0.584), body size (p = 0.389), BMI (p = 0.509), and PI (p = 0.051) showed no significant effect.In general, no differences could be found for mEPA between the younger and older group (p = 0.469) and the segments (p < 0.001). No significant differences in bEPA could be found between Th10, Th11, and Th12 (p > 0.535). Sex was the only covariate which showed significance (p = 0.001). The other covariates showed no significance .For the bEPA, similar results can be found for the main effects regarding age group (p = 0.188), PI (p = 0.725), and SS (p = 0.121). The covariates showed no significant effects on spino-pelvic parameters . Descriptive data for mEPA, bEPA, the spino-pelvic parameters, and the results of the pairwise comparison of the younger and older group are given in The multivariate general linear model showed no differences between age groups for PT (2 0.092 (L2/3) up to 0.953 (L4/5). Valid model quality (R2 \u2265 0.8) was reached for the prediction of L3/4  and L4/5 (R2 = 0.953). The closest prediction, which failed the validity threshold of 0.8, was L2/3, with an R2 of 0.712.Using the iterative process to reduce the number of variables to predict mEPAs using the linear regression with only significant variables led to the exclusion of body weight, body size, BMI, and SS. None of these showed a significant impact on explaining the variance. The coefficients and the model quality of the logistic regression are shown in It can be observed that when predicting the mEPA, for nearly every case, the cranial and caudal mEPA had a significant impact. Age showed significance in just two cases, and seemed to have a low impact. Only in the caudal segments of the spine (L3/4 and L4/5), sex, PT, and PI showed significant impacts. 2 = 0.899) and L4 (R2 = 0.962) met the validity criteria of R2 \u2265 0.8. For both predictions, the adjacent caudal bEPA showed significance, while the cranial bEPA did not.For bEPA the body weight and BMI showed no significant impact on the prediction. The adjacent bEPA showed the most frequently significant impact. The model quality ranged from 0.294 (L1) up to 0.972 (L4). The prediction of L3  and for the prediction of bEPA, significance was shown in only 6 of 21 possible cases (29%), respectively.2 \u2265 0.8) and the measured mEPA (L3/4 and L4/5) and bEPA (L3 and L4), shown as difference plots, is given in 2 of 0.07. However, the direction of the general trend of the linear regression is comparable in each figure, showing a positive trend. Visually, the scatter clouds show that the variability of the prediction seems not to be dependent on the value of the predicted EPA.A visual comparison of the predicted , with negative coefficients found for PT and positive coefficients for PI, respectively. The coefficients, and thus, the actual impact on the prediction, is distinctly stronger for the PT than for the PI.For the PI, the influence on the spinal profile, and thus, on these segments, is easy to imagine. The more the sacrum is tilted, the greater the PI, the greater the lordosis, and thus, the larger the individual segment angles. The less tilted it is, the more it is parallel to the horizontal plane in supine position. Regarding the geometric similarity for the PT, the more the pelvis is retro verted, the higher the PI and the more pronounced the lordosis.Our data and analyses indicate that the closer the segments are located to the sacrum, the stronger the impact of the PT and the PI on the segment angles. In the cranial segments of the spine, their influence seems to completely disappear. For the SS, we would have assumed a similar relationship, compared to the PT and PI. However, no significant impact on the prediction can be determined for the monosegmental segments. We assume that the influence of the three spino-pelvic parameters and their effects interact and thus, cannot be reduced at a one-dimensional level. Additionally, other variables, including the position in which the parameters are determined, may interact and affect the segmental angles . In our prediction, we considered body weight, height, and BMI as anthropometric. However, none of these variables were found to affect the segment angles in the supine position. The body weight, and thus, the BMI, have an effect on the spino-pelvic alignment in the standing position ,17,18; hAge and gender show only a rare significance. These findings affect the predictions of the mEPA and bEPA in a comparable manner. In investigating the effect of age on the spino-pelvic parameters, no differences could be found between age groups. Various authors have so far, been able to demonstrate age effects on the spino-pelvic parameters ,12,17,19Different authors have been able to show differences between men and women with regard to the spino-pelvic parameters in the standing position ,17,20. ONaturally, effects of positional changes\u2014standing versus supine\u2014are to be expected. Radiological diagnosis is also possible in the standing position, but this is not possible for every patient, depending on the injury. Additionally, not every hospital has the technical equipment for this type of diagnostic method. It would be interesting to investigate the prediction of mEPA and bEPA for a population in which radiological diagnostics were performed in both a standing and a supine position in order to compare the results of both predictions. Nevertheless, the consideration of spino-pelvic parameters in fracture correction is recommended . FurtherIn this respect, the correction or non-correction of fractures not only has an impact on the spino-pelvic alignment, but also has a secondary effect on the patient\u2019s quality of life.In this study, we did not examine lumbar lordosis per se. The aim of this study was to determine valuable parameters to predict segment angles. These parameters should not have a great deal of influence if a fracture has occurred. In a fractured spine, it can be assumed that depending on the severity and location of the fracture, the thoracic kyphosis or lumbar lordosis might have changed, and it is likely unclear how the fracture has affected the other segmental angles. Differences between mEPA or bEPA in the lower lumbar region are greater than in the more cranial segment angles, respectively. This might certainly be a reason why there is a better prediction in the lumbar area, and why the influences of the adjacent segment angles are greater here. In the more cranial regions, the differences in segment angles between may be too small to differentiate between segments.If the surgeon orientates the fracture reduction on the lumbar lordosis from S1 to the inflection point, however, in our experience, the inflection point appears to be more cranial when lying down than when in a standing position, which makes lumbar lordosis less suitable as a target parameter in the supine position.During the surgical reduction of the fracture, the avoidance of insufficient or excessive reduction of fractures, lacking sufficient information regarding the individual physiological profile, is also in surgical focus. With precise individual adjustment of the fracture, less additional compensation may be necessary, resulting in better rehabilitation after surgery. Particularly after spondylodesis and existing malalignment, unfavorable loads on the adjacent movement segment are to be expected. Misalignments due to poor fracture position likely diminish the success of subsequent rehabilitation and can lead to pain, adjacent fracture, or other complications. The combination of nutritional supplements and rehabilitation programs can reduce the patient\u2019s pain and improve the quality of life . HoweverFinally, only a few of the parameters used in this study are suitable for predicting segment angles in general. Overall, the caudally and cranially adjacent segment angles seems to be sufficient parameter for use in predicting the segment angles between them. The prerequisite for this, however, is that the adjacent segments are intact. Looking closer at our results, the predictions achieve a wide range, which is shown by the relatively large limits of agreement in the difference plots. Regarding the mean values of the mEPA and bEPA, plus/minus 10 degrees is too great to provide a good individual recommendation for the scope of correction. Ultimately, further investigations must be carried out in order to predict the segment angles in the supine position, considering the spino-pelvic parameters. Questions also arise regarding the amount of malalignment for which the human body can physiology compensate, as well as the way in which this compensation changes with age."}
+{"text": "Frailty is a geriatric syndrome, a clinical state of vulnerability for developing dependency and/or death. Due to its multidimensional nature, Comprehensive Geriatric Assessment (CGA) constitutes the best strategy to evaluate frailty in older patients. Accumulation of deficits model synthesizes the global assessment of geriatric domains in the Frailty Index (FI) score. Muscle Ultrasound (MUS) has been employed to evaluate muscle mass wasting as tool to assess sarcopenia in late life. The present study aims to evaluate the association between CGA-based FI and MUS measures in a population of hospitalized older adults.Patients aged \u226565 years underwent CGA for the evaluation of the domains of health and functional status, psycho-cognition, nutritional status, socio-environmental condition. Following standard procedure, a CGA-based FI was elaborated, taking into account 38 multidimensional items. Muscle thicknesses (MT) of rectus femoris plus vastus intermedius were measured through MUS axial cross-section. Multivariable regression analysis was employed to determine factors associated with FI.The study population consisted of 136 older patients, 87 men (63.9%), with median age of 74 (70\u201381) years, FI of 0.3 (0.21\u20130.46), and MT of rectus femoris plus vastus intermedius 29.27 (23.08\u201335.7) mm. At multivariable regression analysis, FI resulted significantly and independently associated with age and MT.Muscle thicknesses of rectus femoris plus vastus intermedius, measured through MUS, resulted to be significantly related to FI in a population of hospitalized older patients. In the CGA-based assessment of frailty, MUS may constitute an additional imaging domain. Frailty is a multifactorial geriatric syndrome with multiple causes and contributors, constituting the most complex expression of population aging . It reprIn the last decades, several definitions of frailty have been proposed and multiple instruments have been developed for its assessment for both clinical and scientific purposes. Although several scores have been proposed for frailty assessment, the gold standard tool has not been identified yet, thus the selection of a specific frailty instrument depends on the clinical setting, the study purpose, and the geriatric domains of interest . In thisA growing piece of scientific research has focused on muscle ultrasound (MUS) to evaluate muscle mass wasting in older populations, assessing both muscle quantity and quality . MUS hasFried's frailty phenotype shows close overlap with sarcopenia, which constitutes a key contributor to frailty, facilitating the development of disability and being responsible for several adverse outcomes. Indeed, sarcopenic patients suffer increased vulnerability, negative adaptation to external stressors, and disability for basic activities of daily living (BADL) . NeverthTherefore, the central hypothesis of the present study is that MUS may constitute an additional \u201cimaging\u201d domain of multidimensional frailty, and to this aim, we explored the association between frailty and MUS measures in a population of hospitalized older patients.The participants have been recruited among patients aged \u226565 years referred to the Geriatric division of Department of Translational Medical Sciences of the University of Naples \u201cFederico II\u201d. The specific procedures of the study, described below, were performed at the resolution of the acute clinical condition that led to hospital admission. Exclusion criteria were: cachexia, extreme obesity, dialysis-dependent kidney failure and/or end-stage organ failure, central and peripheral nervous system diseases, myositis and diseases inducing muscular atrophy, major surgery on the lower limbs, and the presence of scars at the measurement sites.All patients underwent medical history collection, clinical examination, and evaluation of the main demographic/clinical factors. The results of the main biochemical blood tests were also registered. All participants were carefully informed and signed a written consent to participate in this study. The research protocol was reviewed and approved by the Local Ethics Committee (124/17) and was conducted in compliance with the ethical principles stated in the Declaration of Helsinki.All patients underwent Comprehensive Geriatric Assessment (CGA), with the evaluation of the domains of health and functional status, nutrition, psycho-cognition, and socio-environmental condition. A CGA-based Frailty Index (FI) was created following a standard procedure as proposed by the Rockwood's research group , taking The participants were assessed in a supine position, with the knees resting in extension for 30 min. The rectus femoris and vastus intermedius of dominant thigh of each patient were assessed at mid-point between greater trochanter and proximal border of patella, following proposed standards . A lineaSD) or median and interquartile range (IQR) and compared using Student's t-test or the Mann\u2013Whitney U-test. The Normal distribution was assessed using the Shapiro\u2013Wilk test. The categorical variables were expressed as a percentage and compared using Pearson's \u03c72 test. Descriptive comparisons between groups were conducted according to gender and frailty status. The multivariable regression analysis was used to identify factors associated with continuous dependent variable FI. Parsimonious selection criteria were used to avoid overfitting bias. The analysis considered: age, gender, and BMI as independent variables. An alternative model was developed with subcutaneous fat thickness as an independent factor, instead of BMI. The regression model was employed to determine the impact of the MUS parameters on FI. All analyses were performed using the STATA statistical software , and a p-value < 0.05 was considered as the statistical significance threshold.Continuous variables were expressed as mean \u00b1 standard deviation  mm, and FI of 0.3 (0.21\u20130.46). Dividing the population according to the predetermined FI cut-off, 91 (66.9%) subjects were \u201cfrail\u201d (FI \u2265 0.25) and 45 \u201cnon-frail.\u201d Characteristics of the overall study population and of subgroups divided according to the frailty status are reported in The study population consisted of 136 older patients, 87 men (63.9%), with median age of 74 (70\u201381) years and mean BMI of 26.01 \u00b1 4.51 kg/mp = 0.007] and less predominantly male (58.2 vs. 75.6% p = 0.041). Of note, no other relevant differences emerged between the two groups in terms of BMI, kidney function, hemoglobin, and serum protein levels. As expected, frail patients presented worse scores in the great majority of tests and tools included in the CGA, compared to non-frail ones. While subcutaneous fat thickness did not statistically differ between the two groups, frail patients presented significantly lower thickness values of all examined muscles compared to non-frail ones, in particular MTs of rectus femoris plus vastus intermedius were 33.4 (26.8\u201338.5) and 26.4 (21.9\u201333) mm, respectively (p < 0.001). After stratification according to gender, the groups did not differ for age and BMI, but female participants showed higher subcutaneous fat thickness assessed through ultrasound, worse scores at physical performance tests , and significantly higher FI  (p < 0.001)]. MT values stratified according to gender and frailty status are shown in At univariate analysis, frail subjects resulted to be significantly older than non-frail ones  . Consistp < 0.01), while no relevant association emerged with BMI and gender. Notably, the contribution of MT to the overall R2 of the employed model was remarkable and superior to chronological age . Similar results were obtained by replacing the independent variable BMI with subcutaneous fat (not shown).The multivariable regression analysis included as independent variables the binary predictor gender and the continuous predictors age, BMI, and MT of vastus intermedius plus rectus femoris . ImportaThe main result of the present study is represented by the identification of a significant association between frailty, assessed through accumulation of deficits model, and MT of vastus intermedius and rectus femoris, measured using MUS, in a population of older patients. To the best of our knowledge, this is the first study demonstrating a role of bedside MUS as marker of frailty evaluated through the CGA-based FI.Along with the increasing interest of scientific community on sarcopenia, MUS has been proposed as valuable potential diagnostic tool to perform estimation of muscle mass. Compared to the current gold standard , MUS represents a promising portable, accessible, cheap, cost-effective, and non-invasive imaging tool, particularly suitable for assessing older adults . BioelecSeveral previous studies reported MT measurements of lower extremities, obtained through MUS, to be positively correlated with muscle strength and sarcopenia in older subjects . The relFrailty is a geriatric syndrome widely investigated in landmark studies through several valid models of assessment. Irrespective of the adopted tools, frailty has been associated with adverse health outcomes in different settings of care, thus increasing the scientific interest of geriatric research. Progressively more clinical decision processes are considering frailty status when selecting people to the most appropriate procedure  or drug treatment . Among tFollowing these premises, the aim of the present study was to evaluate whether the measurements of MT, obtained through an imaging technique increasingly employed in clinical research for sarcopenia assessment , were correlated with frailty, assessed through validated instruments of CGA, in a population of hospitalized older adults. The central hypothesis was that MUS may constitute an additional imaging tool of the geriatric multidimensional CGA-based approach.The MUS values of MT of vastus intermedius plus rectus femoris came out to be significantly and independently correlated to FI in the study population, as emerged both at univariate and multivariable analyses. Importantly, the results of the latter analyses were corrected for potential confounding factors, such as BMI, gender, and age, aiming to avoid that such biological and anthropometric measures could influence the result and consequently condition MT impact on frailty. Furthermore, taking into account of the redistribution of body adiposity with age , we havePrevious studies have focused on MUS as measure of frailty, with a specific interest on muscle strength and sarcopenia. The research group of Miron-Mombiela has demonstrated both MT and echo intensity of quadriceps to be correlated with grip strength in a subpopulation of adult outpatients aged 60 years and older. Moreover, the authors reported these measures to constitute imaging biomarkers of frailty, assessed according to Fried's criteria . A very Besides these pieces of literature that are consistent with the findings of the present study, our results are not in line with the previous evidence reported by Madden and collaborators, which performed point-of-care MUS of vastus medialis to test for association between MT and frailty in older adults. The authors detected only a weak correlation of MUS measurements with frailty, assessed through the Frailty Phenotype and the Clinical Frailty Scale (CFS), a 9-point judgment-based measure of frailty . OtherwiThus, taking into account the multifactorial nature of FI and considering the great heterogeneity which characterizes older subjects, the promising results of the present study allow to speculate on the potential role of MUS in detecting phenotypic characteristics of aging other than those canonically captured by the consolidated CGA tools.The study participants were recruited from a single geriatric medicine clinic, a population that tends to be frailer than the general population, due to multiple chronic illnesses. No control group was included in the protocol. The sample size calculation is burdened by the lack of evidence and reference values in the method and by the specificity of the population in question. Larger studies are needed to confirm our findings, even considering MT controlled by definite physical indicators which may affect its measures, not yet established by the scientific community. Even though the comparison with sarcopenia was not an aim of the present research, the lack of ascertained diagnosis of muscle mass decline does not allow a comparison between the MT measurements and the result of other reference methods.Frailty is a multifactorial geriatric syndrome; CGA-based tools are valid instruments for its diagnosis and management. MUS measures of MT of vastus intermedius plus rectus femoris resulted to be significantly correlated to FI in a population of hospitalized older patients, independently from other considered covariates. Further studies are needed to confirm this association and determine the clinical impact of these findings, aiming at defining MUS as an additional imaging domain of frailty.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Comitato Etico per le attivit\u00e0 biomediche - Universit\u00e0 degli Studi di Napoli Federico II. The patients/participants provided their written informed consent to participate in this study.LB and GR conceived this study. LB, FP, and CR extracted the data. LB, DV, and KK designed and performed the statistical analyses. LB, LF, FR, IS, GG, and GF wrote the first draft of the manuscript. NF and GR reviewed and modified the final manuscript. All authors read, critically reviewed, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Remarkable improvement in the perovskite solar cell efficiency from 3.8% in 2009 to 25.5% today has not been a cakewalk. The credit goes to various device fabrication and designing techniques employed by the researchers worldwide. Even after tremendous research in the field, phenomena such as ion migration, phase segregation, and spectral instability are not clearly understood to date. One of the widely used techniques for the mitigation of ion migration is to reduce the defect density by fabricating the high-quality perovskite thin films. Therefore, understanding and controlling the perovskite crystallization and growth have become inevitably crucial. Some of the latest methods attracting attention are controlling perovskite film morphology by modulating the coating substrate temperature, antisolvent treatment, and solvent engineering. Here, the latest techniques of morphology optimization are discussed, focusing on the process of nucleation and growth. It can be noted that during the process of nucleation, the supersaturation stage can be induced faster by modifying the chemical potential of the system. The tailoring of Gibbs free energy and, hence, the chemical potential using the highly utilized techniques is summarized in this minireview. The thermodynamics of the crystal growth, design, and orientation by changing several parameters is highlighted. Hybrid halide perovskites (HHPs) have appeared in a supporting role as visible light sensitizers  in the sDue to the interdependence of material properties, they are entangled in a greatly complex way. Major complexity arises due to ion migration which is the main reason for the degradation in perovskites. One of the highly used strategies to minimize ion migration is the reducing defect density in the crystal structure by fabricating the high-quality perovskite thin films. In this review, several reports focusing on understanding and controlling the crystallization process to achieve some best quality perovskite films are discussed. The perovskite film morphology is a key factor in deciding the carrier lifetime, the carrier diffusion lengths, and ultimately the device performance . For theIn the last decade, a vast amount of research to maximize the efficiency of the HHP-based devices is based on the optimization of the hybrid perovskite crystal design, size, and growth orientation. A plethora of strategies and methods have been used to obtain uniform, pinhole-free, full coverage, and smooth perovskite layers. These methods involve changing the perovskite precursor solution concentration and temperature, optimizing the coating parameters such as the spin speed and time, tuning the annealing temperature of thin films. In addition to these methods, new techniques such as composition  and therIn any solution processable technique, such as spin coating, crystal growth can be considered as a two-step process; the first step is the nucleus formation (nucleation), and the second step is the growth of nuclei into larger crystals. The nucleation of crystals can be of two types: homogeneous and heterogeneous nucleation. Homogeneous nucleation occurs spontaneously without any external assistance within the solution. However, heterogeneous nucleation requires an external site to occur . Both of these types of nucleation can occur in the solution on the colloidal particle sites. The produced nuclei can assemble into bulk clusters and grow into exaggerated film morphology when dropped on a substrate, leading to incompact films with incomplete coverage and a large number of defects. Therefore, nucleation on the substrate is the ideal approach for a high-quality perovskite film.Nucleus formation on the substrate is initiated when the solution is supersaturated and the concentration of the precursor solution is sufficiently higher than its solubility. The solution in the supersaturated state is prerequisite to yield pure perovskite phases. This supersaturation stage can be achieved sooner by preconditioning the precursor or the coating substrate. One of the commonly used techniques to assist this process is the temperature treatment. The input of thermal energy facilitates a faster nucleation process, leading to uniform perovskite thin films. In addition, the intermediate phases or the perovskite precursor solvate (solvent\u2013solute interaction) significantly influences the final perovskite film formation. These always occur in the thin film during the processing methods such as antisolvent treatment or solvent engineering.Nucleation and growth are best understood in terms of chemical potential (\u00b5), which in the simplest term is the energy released or absorbed by the addition  of a particle into (from) the system. At a constant pressure and constant temperature, which is the archetypical lab conditions for solution processing, the chemical potential (\u00b5) can be calculated from a thermodynamic potential called Gibbs free energy (G).The process of nucleation and growth can be tuned by regulating the chemical potential of the thermodynamic system. There are several thin film processing methods by which the chemical potential of the nucleic site can be altered. Few of the latest techniques being followed these days include the variation in the substrate or solvent temperature and variation in the rate of solvent extraction at the time of spin coating or by changing the vapor pressure of the ink, which have to be used in order to fabricate perovskite films. Here, we focused on how the crystallization kinetics is modulated and the crystals are designed to yield a high-quality perovskite thin film by changing the substrate temperature before film fabrication, antisolvent treatment of the film during spin coating, and solvent engineering of the precursor solution.3) films coated on substrates with different temperatures were examined. It was noted that preheating the substrate affects the degree of surface-induced nucleation. With the increase in temperature, the number of nucleation events increased, leading to a larger number of smaller crystals and hence better coverage. In another study, Zheng et al. demonstrated the thermally induced Volmer\u2013Weber growth mechanism during the hot casting of methylammonium lead iodide (MAPbI3) perovskite films (2) and methylammonium chloride (MACl) solution onto the substrate maintained at temperature of up to 180\u00b0C followed by a subsequent spin coating of the precursor. They have attributed that the excess solvent present on the substrate at a temperature above the crystallization temperature for the formation of the perovskite phase allows the prolonged growth of the perovskite crystals and hence yielding large crystalline grains.We have already discussed earlier that the formation of perovskite crystals is a two-step process: nucleation and growth. In addition, during the complete process, the temperature plays a very significant role. The chemical potential (\u03bc) and, hence, Gibbs free energy (G) of the system can be significantly tuned by modulating the temperature of the process. The temperature of the system can be changed either during nucleation or during growth. The fabrication of the perovskite thin films using spin coating involves mainly two steps. The first is spin coating the precursor solution onto the substrate (nucleation) and annealing the coated substrate at an optimized temperature (growth). In this article, we focused on how the morphology of the perovskite thin film can be tuned by playing with the process of nucleation of the perovskite crystals. The process of nucleation can be controlled either by changing the temperature of the precursor solution before spin coating or preheating the substrate. The technique of preheating the substrate before coating the perovskite layer has been explored recently. It is one of the latest advanced strategies used to modulate the mechanism of crystal growth and, hence, the crystal design. There have been few studies on the effect of substrate temperature on the morphology of the perovskite film. Among some of the earlier studies is a report by te films . In thiste films . Figure m. Above Tm, \u0394G > 0 is not favorable for spontaneous transformation, whereas below Tm, \u0394G < 0 favors spontaneous phase transformation. In this study, the perovskite film was coated on the substrates preheated to five temperatures below Tm, (Tm for MAPbI3 = 120\u00b0C).In 2018, Srivastava et al. described the underlying theory of crystallization thermodynamics altered by the preheating temperature which leads to a significant change in the film morphology . In a siI) is given by the product of the number of critical sized particles (N*) and the frequency at which they become supercritical, i.e., the frequency at which they cross the interface to join the product phase (\u03bd\u2032), as given by the equation  according to this equation is shown in m, which agrees with the observed results. It was found that the perovskite film coated on the substrate with a preheating temperature of 100\u00b0C has an optimum morphology for the high open-circuit voltage and uniform charge and ion diffusion across the interface. Moreover, the dependence of the growth rate (U) with the temperature reveals that the growth of the nucleated particles is maximum at a temperature just below the maximum nucleation temperature. Hence, in this study, the films were annealed at the temperature of 100\u00b0C for 10\u00a0min. This study suggests that the mechanism of fast nucleation followed by delayed growth is the best method to obtain high-quality perovskite films  and organic methylammonium iodide (MAI) precursors , the conversion of MAI with PbI2 is inhibited, leading to the formation of the unreacted amorphous film. Therefore, tailoring of the optimum substrate temperature for both spin coating  can work as an antisolvent, given that it is miscible with the solvent of the perovskite ink; at the same time, perovskite precursors are expected to show poor miscibility with the antisolvent. The antisolvent treatment method helps the perovskite ink to reach the supersaturation state and form the nucleus sites. Formation of nuclei is governed by the variation in Gibbs free energy (G). For homogeneous nucleation, change in the Gibbs free energy is given by the following equation \u0394G=\u0394GS+\u0394mated as \u0394G\u2217=16\u03a0\u03b3To achieve the system with stable nuclei for further growth, the critical Gibbs free energy and critical radius of the nuclei need to be reduced. This requires a decrease in the surface energy between the supersaturated solution and the crystalline surface. The addition of additives such as benzylamine (PMA) in a/an solvent/antisolvent is known to decrease the surface energy (\u03b3) and help in achieving uniform, compact, and small grain-sized perovskite films suitable for the high-performance light-emitting diodes (LEDs) .max corresponds to the equalization of chemical potentials of liquid and nucleus sites. Now, the role of the antisolvent treatment is to increase the chemical potential of the nucleus site and cause a positive gradient in the \u00b5-n curve, which is the characteristic signature of Frank\u2013van der Merwe (FM) growth. Thus, by tuning between the Volmer\u2013Weber (VW) and FM growth, we controlled the crystallization dynamics to achieve the required film morphology. The volume of the antisolvent, dripping delay in antisolvent treatment, and the rate of pouring of the antisolvent can tune the perovskite film morphology by controlling the chemical potential of the nucleus sites. Kumar et el. used antisolvent dripping delay to achieve textured film morphology  or dissolving FAI. The strong coordination of tBuOH:EA makes this possible  . Althougpossible . Antisolpossible .via bypassing the intermediate phase (CH3NH3I\u2013PbI2\u2013DMSO)  or o-dichlorobenzene (o-CB) with a larger dipole moment, as compared to toluene, can induce the liquid to a crystalline phase transformation instantaneously I2\u2013DMSO)  (Jung etI2\u2013DMSO) . The int2. The role of IA formation is investigated in MASnI3-based perovskite films, and it has been observed that by using DMSO (strongly coordinating solvent) as the solvent forms more stable IAs as compared to DMF. Since DMF has a lower coordination affinity, lower boiling point, and higher vapor pressure as compared to DMSO, using DMF in the perovskite film preparation immediately forms the perovskite structure resulting in a black-colored film, whereas in case of DMSO, a gradual yellow to black color change occurs while annealing the films, which indicates that DMSO shows a higher stability of IAs as compared to IAs formed by DMF solvent. Formation of the new IAs from different solvents  has been confirmed, using the XRD and FTIR measurements. The formation of IAs is helpful in controlling the crystal size, crystallization rate, and ultimately the morphology of the perovskite films. Stable IA formation leads to a much compact, uniform, and large grain-sized perovskite films. Cai et al. investigated the effect of the solvent ratio in a binary solvent system. They found that 20\u201340% of DMF or GBL mixture in DMSO results in an optimum morphology (DMF (Dimethylformamide), DMSO (Dimethylsulfoxide), DMA , GBL (\u03b3-butyrolactone), and NMP (N-methyl-2-pyrrolidone) are the most commonly used solvents in making good quality perovskite films for the high-performance perovskite solar cells and light-emitting diode. All of them are aprotic, high boiling point, and low vapor pressure solvents, and they are only useful in making full coverage, pinhole-free compact film when used along with some additional treatments such as antisolvent treatment or gas-assisted crystallization . The chorphology   of 19.2\u00b0, 22.4\u00b0, 21.6\u00b0, and 47.4\u00b0, respectively. The surface coverage for the controlled samples (without any solvent additive) was found to be around 20\u201330% only. Surface coverage for EA and CB additives was improved to more than 95%  stoichiometry.Solvent additives can be used to optimize the surface energy of the solvent, and thus, according to than 95% . They fovia solvent extraction by an antisolvent treatment. Preheating the substrate can affect surface-induced nucleation and control the crystal growth. It has been observed that the formation of uniform pinhole-free perovskite films can be realized by elevating the substrate temperature close to the equilibrium melting point (Tm). However, only those substrate temperatures below Tm are thermodynamically favorable. The temperature-dependent growth rate reveals that the growth of the nucleated particles is maximum when the substrate temperature is kept closer to Tm. The chemical potential of the nucleic site can be tuned in order to tune between the FM and VW growth mode with the help of tuning the delay of the antisolvent treatment. This helped us to tune the perovskite film morphology for the application in the high-performance devices. Also, the rate of antisolvent dripping plays a significant role in improving the crystallization process; a slow rate of antisolvent dripping is found to result in better quality perovskite films and solar cells. Apart from this, the solvent boiling point, coordination affinity, and dipole strength also play a significant role in deciding the ultimate film morphology, defect density, and charge transfer resistance. Solvents with a higher coordination affinity such as DMSO help to slow down the crystal growth rate and achieve the larger sized grains suitable for the perovskite solar cells. An optimized ratio of the low and high coordination affinity solvent can help to control the grain size and film morphology in accordance with the application.In summary, we have reviewed the latest, advanced strategies to control the perovskite crystal growth in fabricating the high-quality hybrid halide perovskite thin films, suitable for optoelectronic devices. It has been found that the key parameter deciding the nucleation and subsequent growth of the nucleated particles is the Gibbs free energy. We mainly focused on the three most popular strategies, namely, substrate temperature treatment, antisolvent treatment, and cosolvent engineering. We found that these techniques are found to be highly effective in controlling the film morphology and obtaining the high-performance perovskite solar cells and perovskite light-emitting diodes. The condition for the nucleus formation is to achieve the supersaturation of the perovskite precursor solution, either by heat treatment or"}
+{"text": "This paper estimates the impact of public guarantees on crisis predictive indicators among small and mid-size enterprises (SMEs). We use a confidential database provided by the Italian Ministry of Economic Development on the universe of guarantees granted by the Central Guarantee Fund. We apply difference-in-difference regressions and propensity-score matching estimators to a sample of approximately 40,000 SMEs over the 2010\u20132018 period. We find that obtaining a public guarantee improves profitability both in the short- and medium-term. On the other hand, SMEs\u2019 financial health worsens in the short run, but financial burdens are alleviated 2\u00a0years after the issuance of a guarantee. The economic and financial effects of government-backed loans are amplified for micro-sized firms, companies operating in the service sector and direct guarantees. Our results can thus support public authorities in designing credit guarantee schemes capable of preventing SMEs\u2019 zombification and protecting them from the risk of debt overhang. Access to public credit guarantee schemes negatively impact SMEs\u2019 financial equilibrium, but their recovery occurs 2\u00a0years after guarantee issue. How does one select eligible firms to prevent zombification? Using a confidential dataset provided by the Italian Ministry of Economic Development on guarantees issued by the Central Guarantee Fund, we investigate this topic with an unprecedented level of salience. Our findings reveal the need for cautious interventions on firms in financial distress and for the introduction of stress tests to select beneficiaries. Our results show that specific guarantee lines could be applied for direct guarantees granted to micro-sized enterprises and companies operating in the service sector to maximize the additionality of public resources. This study has practical, policy and societal implications, guiding SMEs in their assessment of the overall medium-term effects of guarantees and policy-makers in their rethinking of guarantee schemes to resolve trade-offs between effectiveness and sustainability. Throughout European countries, plans for recovery and resilience following the coronavirus disease (COVID-19) pandemic have included the strengthening of public guarantee programmes. In addition to moratoria on loan repayments, tax deferrals, grants, equity facilities and wage subsidies, public guarantee schemes represent a fundamental instrument within the countercyclical public policy toolkit deployed by governments to support the liquidity needs of firms, particularly small and mid-size enterprises (SMEs) ?This study aims to evaluate the effects of government-backed finance on SMEs\u2019 crisis indicators 1\u00a0year and 2\u00a0years after guarantees are issued. We attempt to understand whether public guarantees are able to impact the business continuity prospects of guaranteed SMEs by comparing them to non-guaranteed SMEs. We specifically address the following research questions:To answer these questions, we use a confidential database provided by the Italian Ministry of Economic Development that includes all guarantees issued by the Italian Central Guarantee Fund (CGF) from 2012 to 2016, which we integrate with data downloaded from the Aida Bureau van Dijk database for a period ending in 2018. We use difference-in-difference regressions (DiD or diff-in-diff) and propensity-score matching estimators to compare a proprietary sample of 17,810 guaranteed firms with a sample of approximately 21,000 comparable SMEs.Papers that analyse the impacts of government-sponsored credits typically focus on a specific country given the need to compare transactions regulated by the same legislation  to obtain predictive signals of corporate crises. Public guarantee funds pursue economic policy objectives but must simultaneously ensure sustainability in the use of public funds for the benefit of the taxpayers. Therefore, the selection of eligible companies while considering business continuity prospects over time from a forward-looking perspective is essential. Then, the study measures the impacts of new guaranteed loans on the overall health of the company as measured by predictive indicators of corporate crises. The overall package of these indicators was not previously investigated by the international literature and represents one of the first empirical tests applying the recent Code of the Business Crisis and Insolvency . The Code aims to create functional instruments to facilitate the early identification of the debtor\u2019s financial crisis to prevent insolvency and, when such efforts fail, manage the insolvency with the aim of overcoming the crisis and restoring the company to profitability. Therefore, the warning signals to prevent a crisis investigated in this study include not only economic and capital adequacy indicators but also debt sustainability indices, which are particularly relevant in evaluating the effectiveness of public support for access to new financing. Regarding this issue, the literature has paid limited attention to the impact of public programmes on firms\u2019 liquidity and the overall structure of corporate debts. We attempt to contribute to filling this gap by studying the impact of guarantees on the overall cost of corporate debt and debt sustainability with cash flows.The effects of public guarantees on SMEs\u2019 going concern were investigated in the context of economic and financial crises  and the repayment periods of guaranteed loans and reducing guarantee fees and documentation requirements AECM, .Public guarantee programmes are among the main tools used to support access to finance during and after a recession period. As a strategic response to a financial and economic crisis, these schemes seem to be more effective than other direct public interventions  a direct guarantee can be activated to cover a loan granted by a bank, a leasing company or other financial intermediaries without directly intervening in the lender-borrower relationship; or (2) a counterguaranteeSMEs that exceed a predefined probability of default cannot access the Fund. Eligible firms, on the other hand, have different conditions for accessing the Fund based on their obtained score. For example, national regulations establish the maximum coverage percentages of the guarantee to the total amount of the loan and the maximum guaranteed amount, which can vary based on the obtained score. The guarantee never covers 100% of the loan, but the coverage rate can reach 80% of the loan for direct guarantees and 90% for a counter-guarantee to an MGI whose guarantee cannot, however, exceed 80% of the loan. Such conditions also differ according to the amount and type of operation guaranteed, in the short- or medium\u2013long-term, for liquidity or investments.During and after relevant economic or financial shocks, when firms\u2019 survival is exposed to growing risks, the CGF has experienced significantly increasing rates in both the applications for admission received and accepted. In particular, the role of the CGF was strengthened during the international financial crisis started in 2008 and after the European sovereign debt crisis started in 2010. It was refinanced with approximately two billion euros between 2008 and 2012 and then again between 2013 and 2014 with 1.2 billion euros. Afterwards, during the COVID-19 pandemic, the Italian government adopted extraordinary measures that reinforced and significantly expanded the programme by broadening its coverage, relaxing its eligibility criteria and increasing its endowment.During the period covered in this study, the number of requests forwarded to the CGF increased by approximately 60%, the number of requests accepted has risen by almost 70%, while the number of companies guaranteed grew by approximately 60%.During the COVID-19 emergency, the functioning of the CGF has been temporarily modified. Currently, it could be necessary to rethink the structure of the programme during the post-pandemic era.This paper is related to the literature that studies the effects of public interventions in terms of supporting the access to finance of micro-sized enterprises and SMEs. This focus on smaller companies is particularly relevant since such companies are a crucial element of most European countries: in 2020, they accounted for more than 99.8% of all the businesses in the EU-27 non-financial business sector, contributed more than half of the value added of the Union and accounted for approximately two thirds of the total employment of the private sector with over 90 million employees  economic additionality and (2) financial additionality.Employment by considering increases in the number of employees or the employment growth rate , the accessed loan and the sector and geographical area of the guaranteed firm. In the dataset, we include the balance sheet information of the guaranteed firms, which is sourced from the Aida Bureau van Dijk database. To cover the periods before and after each guarantee was granted, we collect data from 2010 to 2018. The values are downloaded from the 2\u00a0years before the issuance of each guarantee for bias correction; financial and economic data from 2017 and 2018 are used to measure the impacts of the public guarantees. Table We develop a consistent control sample of comparable firms that did not receive public guarantees during the same period Rubin, . We downTo identify the determinants of access to the CGF, we refer to variables used in the literature to evaluate firms\u2019 creditworthiness Altman, . AccordiPrevious studies  and SMEs\u2019 ongoing concern indicators (measured by crisis indicators). To evaluate economic additionality, we choose the ROI because it can be used to measure, in terms of profitability, the effect of new investments financed by guaranteed loans , we choose the covariates by conducting a probit regression. The formula is as follows:We use a matching estimator, ithm Li, . The ATTi observed in the year before the guarantee is granted (t-1). We use the k-nearest matching algorithm and identify k-matched (control) observations from the sample of non-guaranteed firms, namely the \u201cuntreated\u201d firms, for each observation. The control observations are the non-guaranteed firms that are closest to the treated observations in terms of propensity score. The number of control observations, k, is arbitrarily determined. We estimate the average treatment of the sample with four equally weighted matches, controlling for heteroskedasticity . Indeed, Abadie & Imbens  and industry  of each firm. We proxy the size of each firm based on its number of employees: micro-sized enterprises have up to 10 employees; small firms have between 11 and 50 employees; and medium firms have between 51 and 250 employees. Using the aforementioned k-nearest matching estimator, for each subsample, we estimate the effect of guarantees on the changes in profitability and the indicators of business crisis 1 and 2\u00a0years after the issuance of the guarantees. Then, we apply propensity score matching to each subsample.i at time t; n is equal to 1 and 2; To overcome the potential limitations of propensity score matching, we also apply the DiD methodology. Equation\u00a0 represenFirst, a probit regression is conducted to verify the relevance of the covariates. Table Table The guarantees help increase the difference between guaranteed and non-guaranteed firms. First, the guarantees have a strong effect on the ROI growth rate during the year after the intervention of the CGF. The impact on this variable is even greater 2\u00a0years after the issuance of the guarantee. The loans obtained due to the credit-support programme contribute, with a high level of statistical significance, to the improvement of ROI for guaranteed firms, both in the short- and medium-term. These results confirm the main findings of the accredited literature on economic additionality /total assets growth rate.The consideration of further important aspects related to the ongoing concern of SMEs is the main contribution of our study. Therefore, first, our analysis reveals a relevant effect on the liquidity position of the examined guaranteed companies. The growth rate of the current ratio among the firms that have been granted public support is higher than that of companies without this support. No direct impact is recorded on the ratio of cash flow to total assets. According to our results, guarantees are not able to directly impact the ability of companies to generate cash flow. On the other hand, a significant effect is produced on the ratios related to the firms\u2019 financial health:The relation between interest expenses and total sales seems to worsen in the short- and medium-term. In particular, for micro-sized enterprises, obtaining a guarantee involves a significant increase in the growth rate of this ratio.Firms that use state-backed credit incur costs. Usually, guarantees are not provided free of charge, but their costs in the focal period range from 0.25 to 1% of the guaranteed amount.On the other hand, the interest rates of guaranteed loans should reflect the presence of a public guarantee as follows: a guaranteed loan should be more inexpensive than it would have been without the guarantee. This is also related to the inclusion of all the main public guarantee programmes on the list of credit risk mitigators of the EU Capital Requirement Regulation. Nevertheless, in addition to the guarantee fees passed onto borrowers, companies can face an increase in the interest rates applied to other unsecured loans due to the potential worsening of their financial structure and creditworthiness. Indeed, once they are accepted for public guarantees, supported companies change their financial structures and increase their debt-to-equity ratios, as shown by our main results. Growth is particularly exhibited by micro- and medium-sized enterprises in the short-term and by small firms in the medium run. There is not only an increase in the leverage of guaranteed firms compared to that of unsecured companies but also an improvement in the composition of corporate debts. The growth in a company\u2019s \u201c/total assets\u201d ratio decreases when it obtains a guarantee, improving its financial structure. This may be related to an increase in assets due to the new investments made possible by the guaranteed loan and/or to a partial reimbursement of old tax debts and social security debts that occurs once the new liquidity has been obtained.Such a firm\u2019s financial health is thus partly threatened by this new access to finance but with some positive aspects. Moreover, this deterioration in financial health is not persistent in the medium-term. The effects on the ratios \u201cfinancial interest/sales\u201d and \u201cdebt/equity\u201d lose significance, and the coefficients decrease in the second year after the issuance of guarantees. For medium-sized companies, there is no significant relationship in the medium-term.In summary, obtaining a guarantee helps increase the differences between guaranteed and unsecured firms, except in relation to the capacity to produce cash flow. However, while the ROI growth rate continues to grow significantly 2\u00a0years after the issuance of a guarantee, the negative effects on financial health lessen over time. Regarding the overall impact on business ongoing concerns, guarantees do not lead to an increase in business continuity risks in the medium-term. Table The signs of the relationships are the same for every industry. Nevertheless, relevant differences in terms of magnitude are detected. In the service industry, the effects of guarantees are more evident, both in terms of profitability and in terms of crisis indicators. In particular, positive and significant increases in the growth rates of the \u201cfinancial interest/sales\u201d growth rate ratio and the \u201cshort-term assets/short-term liabilities\u201d growth rate ratio are recorded , and a relevant decrease in the growth rate of the \u201c/total assets\u201d ratio is highlighted. On the other hand, a higher growth rate in the debt-to-equity ratio is exhibited by the trade sector.In general, services are usually characterized by higher long-term barriers to investment due to financial constraints. This was confirmed by a recent survey conducted by the European Investment Bank (EIB), according to which obstacles to investment activities related to the availability of finance are more significant in the services sector than other industries EIB, . This maTable The results are similar for the two forms of guarantees, but as assumed, the effects have different magnitudes. Both the impact on profitability and that on the crisis indicators are more pronounced in the case of direct guarantees. The bank directly requests public guarantees mainly for companies able to achieve more substantial profit improvements in the short- and medium-term. Nevertheless, the impact of direct guarantees, on the whole, put the financial health of companies at greater risk. Higher profitability can be achieved in the face of greater negative impacts on firms\u2019 financial structures and burdens of financial charges. However, the intervention of an MGI is able to reduce the overall risk borne by the bank and the risks to companies\u2019 business continuity prospects simultaneously.t-test to examine differences in observable characteristics between the treatment firms and the control firms before the matching  in addition to the type of intervention (direct guarantee or counterguarantee/reinsurance). Therefore, specific portions of public funds could be applied where the effects produced by such guarantees are stronger as is the case of direct guarantees granted to micro-sized enterprises and companies operating in the service sector to maximize the additionality of public resources in the context of curtailed budgets.Currently, the CGF considers the differences among business sectors only for better assessing the probability of default among eligible firms. The kinds of ratios and thresholds used to evaluate each variable vary according to the industry in which the focal company applying for the fund operates. Considering the impact of guarantees on different sectors, to improve its economic additionality and its effects on business continuity, the CGF could consider firms\u2019 industries also as a variable in determining the specific amounts to be allocated to establish defined economic policy objectives.Nevertheless, during recovery phases, it is essential that public guarantee schemes do not interfere with efficient downsizing or consolidation within specific sectors to prevent dangerous zombification effects. Hence, the search for maximizing the effectiveness of public intervention must be limited to the subset of illiquid companies with viable business models in the context of targeted credit-support programmes. The use of crisis prevention indicators, which combine the analysis of economic and financial profiles, seems to be a useful reference for solving the trade-offs in the design and implementation of public guarantee funds. Our main findings thus have relevant implications for policy-makers, contributing to the public debate on rethinking credit guarantee schemes after the pandemic to find an equilibrium between effectiveness and sustainability.Moreover, our results may have important practical implications for SMEs that are potentially eligible for a public guarantee. When applying for a public guarantee, SMEs should assess not only the impact on access to a specific financing type but should also properly estimate the medium-term effect on the overall economic and financial conditions. In this regard, our study points out that firms characterized by high levels of debt and financial distress should carefully consider their application for a public guarantee to avoid raising warning signs of a potential corporate crisis.Despite the relevance of our results and the robust methodologies we have applied, our empirical analysis has some limitations that may be overcome by future research. First, our analysis explores the Italian context. Although this choice is useful in comparing guarantees issued in the same regulatory context and granted to eligible firms selected according to uniform criteria, a cross-country analysis could effectively verify the impact of macroeconomic and financial country-specific conditions on our main results. Such variables could help justify regulatory differences and a different level of public support for SMEs at an international level.Second, we carried out this analysis over the period 2010\u20132018. It could be interesting to explore a broader period, including the most recent years, to determine if our results vary according to changed economic context. In particular, future research could test the medium-term effects of the easing of eligibility criteria and the temporary changes to the functioning of the CGF introduced during the pandemic emergency period, applying the methodology based on crisis predictive indicators presented in this paper. This is particularly important in a context of global inflation and energy crisis, where the debate on the design and implementation of support measures for SMEs is highly topical."}
+{"text": "Apus apus) flies over a wide geographic range in Eurasia and Africa during migration. Although some studies have revealed the migration routes and phenology of European populations, A. a. apus (from hereon the nominate apus), the route used by its East Asian counterpart A. a. pekinensis (from hereon pekinensis) remained a mystery.As a widely distributed and aerial migratory bird, the Common Swift (pekinensis breeding in Beijing from 2014 to 2018, and analysed full annual tracks obtained from 25 individuals. In addition, we used the mean monthly precipitation to assess the seasonal variations in humidity for the distribution ranges of the nominate apus and pekinensis. This environmental variable is considered to be critically relevant to their migratory phenology and food resource abundance.Using light level geolocators, we studied the migration of adult apus, pekinensis experiences drier conditions for longer periods of time. Remarkably, individuals from our study population tracked arid habitat along the entire migration corridor leading from a breeding site in Beijing to at least central Africa. In Africa, they explored more arid habitats during non-breeding than the nominate apus.Our results show that the swifts perform a round-trip journey of ca 30,000\u00a0km each year, representing a detour of 26% in autumn and 15% in spring compared to the shortest route between the breeding site in Beijing and wintering areas in semi-arid south-western Africa. Compared to the nominate pekinensis breeding in Beijing might suggest an adaptation to semi-arid habitat and dry climatic zones during non-breeding periods, and provides a piece of correlative evidence indicating the historical range expansion of the subspecies. This study highlights that the Common Swift may prove invaluable as a model species for studies of migration route formation and population divergence.The migration route followed by The online version contains supplementary material available at 10.1186/s40462-022-00329-2. Hirundo rustica) [Bird migration, connecting remote places, has captured the attention of humans for thousands of years, yet systematic scientific research only started about a hundred years ago . It is grustica) , 5. Glacrustica) , thus plrustica) , 8, and rustica) . Modern rustica) , 11, of rustica) , 13.Apus apus) is widely distributed in the Palearctic and often nests in cavities in buildings [apus which breeds more northerly, from Europe and north-western Africa through to northern Asia, and the eastern subspecies pekinensis  breeds from western/central Asia to eastern Asia [pekinensis overlaps with two other migratory swifts, i.e. the Pacific Swift (Apus pacificus), and the White-throated Needletail (Hirundapus caudacutus), whose migratory paths have been recently elucidated by light-level geolocators [As a typical long-distance migratory and aerial insectivore, the Common Swift . The study population is located almost at the easternmost part of the geographical range, and we expect that they might travel the longest migration distance between its potential breeding and nonbreeding areas as compared to the nominate apus. In turn, we expected that pekinensis exhibit a different migration strategy in terms of route and penology in comparison to the nominate subspecies. Because long-distance bird migrants commonly track seasonal resources between breeding and wintering grounds [pekinensis and apus experience very different environmental conditions throughout their migration. To this end, we tested the differences in precipitation along the migratory flyways between pekinensis and the nominate\u00a0apus, since the amount of rainfall has been regarded as one key climatic factor that negatively correlate with swift\u2019s survival [In this study, we characterized the migration route and phenology of  grounds , we furtsurvival , 32. TogWe carried out fieldwork at the Kuoru Pavilion  situated in the Summer Palace in Beijing, China. Once per breeding season from 2014 to 2018, we captured breeding swifts using mist nets arranged around the pavilion between 03:45 and 06:00 on a single day around May 22th. Upon first capture, randomly selected individuals were fitted with light-level geolocators  using a full body harness made of soft braided flat nylon string  around the neck and both wings , the totIn this study, a total of 66 swifts were outfitted with geolocators of which 22 were retrieved the following year. We also banded 250 birds without attaching geolocators and recaptured 131 after one year. The average recovery rate with geolocator across four years was 30.0% (range 20.0\u201341.9%), which was lower than that in Sweden (ca 50%), but approximately close to those from other colonies (ca 30%) , and lowFinally, we successfully recaptured a total of 25 geolocators by 2018 including five birds with two-years of tracking data (three retrieved in the third year and two retrieved twice in the following two years). To allow consistent comparisons , we retaGeoLight 2.0.0 [R 4.0.2 [QGIS 3.14 for further analysis.The light-level data were analysed using ht 2.0.0 , an easy[R 4.0.2 . More im[R 4.0.2 , 21, 37.[R 4.0.2 , 20, 22.[R 4.0.2  to deter[R 4.0.2 , 20, whichangeLight function  [QGIS. Since the reported travel speed of Common Swift was above 250\u00a0km/day, and the stopovers below 2\u00a0days were considered indistinguishable from slow movement due to the inaccurate positioning [Given the error margin of the light-level geolocators , 40, espays\u2009=\u20093)  to deteritioning , 21, 37,pekinensis swifts might move northeast in the later part of winter, often causing the central position of wintering area and median time of wintering period to be out of sync. Therefore, we chose the first position after the swifts entering the wintering area to define the end of autumn migration, and the last position before leaving to define the start of spring migration. The dates of arrival and departure from the wintering area were simultaneously determined in this step.During the wintering period, the swifts were constantly moving around covering short distances per day, forming restricted \u201cresidence areas\u201d with distinct arrival and departure paths, which were defined as wintering areas. The Fast east\u2013west movements change the perceived day and night lengths the swifts experienced, interfering with accurate positioning, particularly latitude, which was more obvious during spring migration, so the end of the spring migration was determined by the first point less than 500\u00a0km away from Beijing, or at 116\u00b0E (longitude of Beijing).In order to reduce the effects of uncertainty associated with location estimates using geolocator data, we used the distances connecting 3-day average positions to calculate the total migration distance . We alsoapus. In addition, we also compared our data with some general information on movement patterns from several European populations of apus in published studies which also used farthest sites as migration endpoints [We compared the sexual and seasonal variations of migration parameters using t-tests. In addition, we compared the migration phenology of two groups of individuals from different subspecies, using a detailed set of published data from Sweden (N\u2009=\u200925) , 37,reprndpoints .apus were known to have different migration patterns in the northern and southern European populations, we compared them with pekinensis from Beijing respectively. Firstly, we extracted the precipitation data of four breeding sites of pekinensis with phenological records [apus in central and western Europe  [pekinensis and May to August for nominate apus, to compare the mean monthly precipitation experienced by the two subspecies in breeding areas. Secondly, since the positions of Common Swifts are not fixed during the wintering period, we used the precipitation of the corresponding month at each position [apus is incomplete during the wintering period, we only take the precipitation experienced by the three populations in November and December for comparison. Similarly, we used the random point method to measure the precipitation in the whole wintering areas, and determined the wintering periods and ranges of the two subspecies: November to February for pekinensis, October to April for northern and November to February for southern populations of nominate apus [apus and\u00a0pekinensis as well as the annual precipitation at \u201crandom sites\u201d, and compared\u00a0the mean monthly precipitation during the wintering period of the three populations\u00a0between the \u201creported sites\u201d and \u201crandom sites\u201d. The historical precipitation was obtained from WorldClim 2.1 [https://www.worldclim.org), with a resolution of 2.5\u00a0min.We assessed the condition of aridity in both reported and randomly selected positions throughout the distribution ranges using monthly precipitation from 2014 to 2018. Since the populations of the nominate  records \u201343, inclouthern)  (Additioouthern) , 44 (Addouthern) , 22, we ate apus , 21, 22.Clim 2.1   . Thereafter they moved to central Africa, reaching the approximate longitude of the eastern Congo Basin in early September, where they remained for around 39\u2009\u00b1\u200916\u00a0days (mean\u2009\u00b1\u2009SD) before slowly moving south. Due to the Autumnal Equinox, we were not able determine how the locations of most individuals during this period relate to the Congo Basin. The swifts reached the Southern African Plateau (on average 1000\u00a0m asl) around November 5th. There, the swifts stayed for 100\u2009\u00b1\u200916\u00a0days, roaming the area before moving northeast around February 13th . Then they crossed the Red Sea, leaving Africa in early April, and flew back to the breeding area almost nonstop, arriving in Beijing on average at April 18th   (Table p\u2009<\u20090.001).The migration distance in autumn was 14,733\u2009\u00b1\u2009775\u00a0km, which was significantly longer than in spring (13.572\u2009\u00b1\u2009999\u00a0km) . Therefore, the average total migration speed in autumn, 134\u2009\u00b1\u200917\u00a0km/day, was significantly slower than 217\u2009\u00b1\u200934\u00a0km/day in spring . Excluding the stopover periods, the average travel speed in autumn was 423\u2009\u00b1\u2009171\u00a0km/day, which was slightly slower than 528\u2009\u00b1\u2009171\u00a0km/day in spring    Tables . Moreovepekinensis vs. northern nominate apus: t\u2009=\u2009\u22120.43, df\u2009=\u200926.25, p\u2009=\u20090.67, and pekinensis vs. southern nominate apus: t\u2009=\u2009\u22121.45 df\u2009=\u200926.61 p\u2009=\u20090.16). Based on random sampling throughout the whole breeding areas, pekinensis experienced significantly lower rainfall , as compared to ranges explored by nominate apus  and when the complete wintering ranges were considered   in areas with less than 600\u00a0mm of annual precipitation. This figure is much higher than that in the nominate apus from Sweden, which spent only 25\u00a0days mainly in the Sahara Desert (10.5% of non-breeding period) [pekinensis is also significantly lower than that for the nominate apus throughout its breeding range  and wintering area , as calculated from random positions. When comparing the mean monthly precipitation between \"reported sites\" and \"random sites\" during the wintering period, we found that the precipitation experienced by these known individuals was different from that of the whole wintering area, with higher precipitation for northern nominate apus , and lower for pekinensis , but similar for southern nominate apus .The subspecies  period) , 20. In apus breeding in Sweden for which migration routes and phenology have been well described [pekinensis from Beijing had a significantly longer breeding period, stopover time, migration distance and duration, shorter wintering period and lower movement speed, except not significantly for travel speed in spring  [pekinensis eastward along this same route, which has been hypothesised but will need more evidence to be verified [apus in an arid refugium during the glacial period before the eastward population expansion. In the case of the nominate apus, we speculate that its ancestral populations colonized Europe via the west coast of Africa, from a relatively wet refugium, bypassing their greatest barrier \u2013 the Sahara Desert. However, these scenarios must be inferred by coupling genomic-based historical demographic analysis and paleoclimate reconstruction[pekinensis along this route and prior isolation from\u00a0the nominate apus in an arid refugium during the evolution of the two subspecies.Taking a direct route from the wintering grounds meant that ountains \u201356, ther canorus  and Falcmurensis , with abqueenii) . This deverified . On thisstruction. Such anpekinensis has been studied. We suggest that future work should be directed to investigate migration patterns in multiple populations of pekinensis throughout its geographical range in central and western Asia. While exploring these populations, parallel studies allow us to compare whether chain migration is also present in pekinensis [pekinensis populations would geographically overlap with\u00a0the nominate apus or the Beijing pekinensis population studied here. Moreover, it would be extremely intriguing to delineate migration routes of populations at the contact zones , in order to determine whether a migratory divide can act as barrier to maintain population divergence between\u00a0the nominate apus and pekinensis [At present, only a single population of kinensis ,and to wkinensis .pekinensis of the Common Swift, also advances our knowledge of other migratory swift species in East Asia. Only recently, migration patterns of two East Asian breeding swifts,i.e. Pacific Swift Apus pacificus [Hirundapus caudacutus [pekinensis, it is worth noting that their non-stop flight involves crossing the airspace over oceans. The formation of migratory patterns (e.g. the choice of migratory routes and wintering grounds) of Pacific Swift and White-throated Needletail may reflect an adaptation to explore forest habitats en route in a tropical climate [Using light level geolocation data not only sheds new lights on the migration of East Asian acificus  and Whitudacutus  have bee climate , 17.pekinensis tracked from Beijing, which travelled the 14,000\u00a0km one-way route through eastern and central Asia to south-western Africa each year. Compared with the nominate apus, the tracked pekinensis experienced more arid environmental conditions and habitats during the non-breeding periods. This pattern might indicate that the subspecies pekinensis of the Common Swift has adapted to explore arid regions at certain stages of their annual cycle, and might be correlative with the historical colonisation route by tracking preferred habitats from sub-Saharan Africa into Asia. However, whether this specialization in habitat preference, together with other possible environmental variables, could be part of the explanation as to why pekinensis diverged from nominate apus in their evolutionary history remains to be tested. The two subspecies may thus prove to be good models for further comparative studies of the specialized intrinsic genetic, behavioural and physiological mechanisms that allow swifts to manage a highly mobile life-style spending substantial part of the year constantly on the wing [In conclusion, our results painted a picture of the poor-known migration progress of the wing .Additional file 1. Table S1. Distribution of data over years. Table S2. Key phenological parameters of migration of different sex individuals of A. a. pekinensis breeding in Beijing. Table S3. Results of comparative analysis of characteristic parameters between female and male pekinensis using t-test. Table S4. Results of comparative analysis of characteristic parameters between pekinensis from Beijing (N=25) and nominate apus from Sweden (N=25) using t-test. Table S5. Monthly and annual precipitation (mm) at several sites reported in the breeding areas of both subspecies.Additional file 2. Table S6. Migration phenology of each individual in this study.Additional file 3. Figure S1. The breeding and wintering areas of two subspecies of Common Swift and the random sample points used in this study.Additional file 4. Table S7. List of random samples and monthly precipitation (mm) during the breeding season.Additional file 5. Table S8. Monthly precipitation (mm) at several sites reported within the wintering areas of both subspecies.Additional file 6. Table S9. List of random samples and monthly precipitation (mm) during the wintering season.Additional file 7. Figure S2. Map showing the tracks away from breeding site and across the Junggar Basin.Additional file 8. Figure S3. Maps showing the migration routes of five individuals in two years. The pentagram represents the bird breeding site and fieldwork location -Beijing. The dotted lines indicate the lack of data in the two weeks before/after the autumn/spring equinoxes. The yellow lines represent the autumn routes, the blue line represent the spring routes."}
+{"text": "Staphylococcus aureus is an opportunistic pathogen causing osteomyelitis through hematogenous seeding or contamination of implants and open wounds following orthopedic surgeries. The severity of S. aureus-mediated osteomyelitis is enhanced in obesity-related type 2 diabetes (obesity/T2D) due to chronic inflammation impairing both adaptive and innate immunity. Obesity-induced inflammation is linked to gut dysbiosis, with modification of the gut microbiota by high-fiber diets leading to a reduction in the symptoms and complications of obesity/T2D. However, our understanding of the mechanisms by which modifications of the gut microbiota alter host infection responses is limited. To address this gap, we monitored tibial S. aureus infections in obese/T2D mice treated with the inulin-like fructan fiber oligofructose. Treatment with oligofructose significantly decreased S. aureus colonization and lowered proinflammatory signaling postinfection in obese/T2D mice, as observed by decreased circulating inflammatory cytokines (tumor necrosis factor-\u03b1 [TNF-\u03b1]) and chemokines , indicating partial reduction in inflammation. Oligofructose markedly shifted diversity in the gut microbiota of obese/T2D mice, with notable increases in the anti-inflammatory bacterium Bifidobacterium pseudolongum. Analysis of the cecum and plasma metabolome suggested that polyamine production was increased, specifically spermine and spermidine. Oral administration of these polyamines to obese/T2D mice resulted in reduced infection severity similar to oligofructose supplementation, suggesting that polyamines can mediate the beneficial effects of fiber on osteomyelitis severity. These results demonstrate the contribution of gut microbiota metabolites to the control of bacterial infections distal to the gut and polyamines as an adjunct therapeutic for osteomyelitis in obesity/T2D.IMPORTANCE Individuals with obesity-related type 2 diabetes (obesity/T2D) are at a five times increased risk for invasive Staphylococcus aureus osteomyelitis (bone infection) following orthopedic surgeries. With increasing antibiotic resistance and limited discoveries of novel antibiotics, it is imperative that we explore other avenues for therapeutics. In this study, we demonstrated that the dietary fiber oligofructose markedly reduced osteomyelitis severity and hyperinflammation following acute prosthetic joint infections in obese/T2D mice. Reduced infection severity was associated with changes in gut microbiota composition and metabolism, as indicated by increased production of natural polyamines in the gut and circulating plasma. This work identifies a novel role for the gut microbiome in mediating control of bacterial infections and polyamines as beneficial metabolites involved in improving the obesity/T2D host response to osteomyelitis. Understanding the impact of polyamines on host immunity and mechanisms behind decreasing susceptibility to severe implant-associated osteomyelitis is crucial to improving treatment strategies for this patient population. Staphylococcus aureus infections as a result of obesity-induced chronic inflammation, immune cell dysfunction, and impaired immune defenses (\u20138\u2013Staphylococcus aureus (MRSA)  are at increased risk for invasive defenses \u20134. Osteoefenses \u2013. Obese/Tefenses \u2013, 7. In gefenses \u2013, 8\u201310. Fs (MRSA) \u201314. In aS. aureus osteomyelitis  as beneficial metabolites that directly reduced bone infection severity similar to oligofructose.Obesity-induced gut dysbiosis is reversed by changes in the host diet, a primary driver of gut microbiota composition and metabolism . One way39\u201344\u2013S. aureus USA300 infection, infections were monitored for 14\u2009days, a time frame within which acute osteomyelitis can develop. In agreement with previous research suggesting that obese/T2D mice are at increased risk for more severe invasive osteomyelitis than healthy counterparts, the obese/T2D + CL control mice exhibited more severe infections than lean/control mice regardless of supplement, with 5% greater weight loss (S. aureus implant-associated infections by \u223c1 log fold compared to the obese/T2D + CL control mice (Oligofructose (OF) is a soluble fiber that has been reported to alleviate some of the secondary complications of obesity and type 2 diabetes in humans and rodents \u201343. To d\u2013ght loss  and incrght loss  to E  Brown-Brenn Gram stain to visualize S. aureus communities in crystal violet, (ii) Alcian blue hematoxylin-orange G (ABHO) to visualize mature calcified bone in orange to red and soft tissues in pink to red for abscesses, and (iii) tartrate-resistant alkaline phosphatase (TRAP) for osteoclast activation in pink to indicate areas of osteolysis  from fiber fermentation, and an unknown species of the S24-7 family and Allobaculum genus . Oligofructose supplementation at week 14 rapidly increased Bifidobacteria abundance to 34% in lean/control + OF mice and 18% in obese/T2D + OF mice . This expansion in Bifidobacteria continued while mice were given oligofructose 14\u2009days after infection at week 16, where Bifidobacteria abundance was found to be at 44% and 39% in lean/control + OF and obese/T2D + OF mice, respectively . We next quantified absolute abundance of B. pseudolongum for weeks 12, 14, and 16 by quantitative PCR  discriminative of each group, we performed linear discriminant analysis effect size (LEfSe) on the gut microbiota at 14\u2009days postinfection (week 16). Several taxa were differentially abundant between lean/control + CL mice and obese/T2D + CL control mice . The gutlus spp. . Multiplved diet \u201360. In cum genus  61, 62), 62AkkerCR qPCR;  to G. Wion limit . After orol mice . FollowiTo determine which gut-microbial derived metabolite(s) were altered following oligofructose supplementation, we performed targeted metabolomics on plasma and cecal material isolated from mice after 2 weeks on supplement but before infection . SamplesSimilar trends were observed in analysis of metabolites from the cecum  and B. BS. aureus bone and soft tissue burden in obese mice by approximately 1-log fold while having no effect in lean/control mice. These results demonstrate that the effects of polyamines on S. aureus osteomyelitis in obese/T2D mice were similar to those of oligofructose . Therefore, we conclude that polyamines contribute to the reduced infection severity observed in obese/T2D mice when supplemented with oligofructose through immune modulation and not direct killing of the pathogen  and controls given phosphate-buffered saline (PBS) was similar . Weight fructose  64, 65), 65S. aupathogen .S. aureus osteomyelitis. One modifier of gut dysbiosis is dietary fiber. To explore this approach, we supplemented diet-induced obese/T2D mice with an inulin derivative, oligofructose, and examined infection outcomes, including bacterial burden, immune signaling, and metabolic profiles. To our knowledge, this is the first study to show efficacy of oligofructose in reducing infection severity during tibial implant-associated osteomyelitis, a site distal to the gut. Our results further showed that polyamines were involved in mediating improved infection severity.A major component of host response to infections and systemic inflammation is the gut microbiota , 67. In S. aureus in a biofilm state, which are physiologically different than abscesses in tissue and bone  mice demonstrated increased neutrophil infiltration in infections of the hind paw and heightened production of chemokines CXCL1 and CXCL2 12\u2009h after infection  family and Allobaculum genus (B. pseudolongum as an example of the distinct shifts induced by oligofructose because (i) A. muciniphila was not significantly altered in obese/T2D mice given oligofructose and (ii) taxa from the S24-7 family and Allobaculum genus are currently uncharacterized. Both S24-7 and Allobaculum have been associated with fermentable fiber supplementation and improved glucose tolerance  experiments with various Bifidobacteria species demonstrated their capability to produce spermidine in vitro, (ii) shotgun metagenomic sequencing of gut microbiota from obese mice fed resistant starch indicated that polyamine synthesis was associated with increases in B. pseudolongum, and (iii) treatment of mice with arginine and Bifidobacterium animalis led to increased levels of circulating and colonic polyamines, which corresponded with decreased levels of TNF-\u03b1 and IL-6  due to previously characterized resistance conferred by speG, a gene in the arginine catabolic mobile element , 62Bifiddolongum  and 6. Adolongum , 61, 72.dolongum . Consistdolongum . Polyamidolongum  30, 63), 63Bifiddolongum . Therefodolongum . We suggand IL-6 \u201378. The nd IL-6 \u2013, and thend IL-6 \u2013. However element , 65. We,S. aureus community colonization in the bone and ameliorated the hyperinflammatory response in obese/T2D mice, indicating that the fiber reduced infection severity. The observations corresponded with changes in the gut microbiota and overall metabolism that suggested that polyamines, and not short-chain fatty acids, were involved in improving infection response. Increased B. pseudolongum abundance and other bacteria affected by oligofructose likely contribute to heightened production of polyamines, as their increased abundance was associated with increased acetyl-ornithine, an intermediate of polyamine synthesis produced solely by bacteria. Treating obese/T2D with polyamines in drinking water provided direct evidence for the potential of spermine and spermidine in reducing infection severity. Overall, this study uncovered a novel role for oligofructose and polyamines in the context of bone infections, warranting further investigation into their role in immunoregulation and potential as adjunct therapeutics for obese/T2D hosts against invasive S. aureus-mediated osteomyelitis.In conclusion, we successfully demonstrated the therapeutic potential of the dietary fiber oligofructose on osteomyelitis in obese/T2D hosts . OligofrS. aureus at week 14 using a pin model described below and monitored for an additional 2 weeks following infection. At harvest, mice were sacrificed with CO2 and cardiac puncture as a secondary method unless otherwise noted.All handling of mice and associated experimental procedures were reviewed and approved by the University Committee on Animal Resources at the University of Rochester Medical Center. Male C57BL/6J mice from The Jackson Laboratory  were housed five per microisolator cage in a two-way housing room on a 12-h light/dark schedule. Male mice were used, as they gain weight more consistently than female mice . At 6 weS. aureus USA300 LAC::lux, a kind gift from Tammy Kielian , was used to infect tibias of mice. Bacteria were added to 10\u2009mL of tryptic soy broth (TSB) and cultured overnight at 37\u00b0C with shaking at 250 rpm. Stainless steel wire (0.02\u2009\u00d7\u20090.5\u2009mm) was used to make L-shaped pin implants (4\u2009\u00d7\u20091\u2009mm), which were submerged in overnight culture for 20 min prior to implantation (\u223c5\u2009\u00d7\u2009105 CFU). Mice were anesthetized with 60\u2009mg/kg ketamine and 4\u2009mg/kg xylazine and received preoperative extended-release buprenorphine. The right tibial area was shaved and disinfected with 70% ethanol and betadine. An \u223c5-mm incision was used to uncover the tibia, and 30- and 26-gauge needles were used to drill a hole through the bone to fit the pin. Pins were inserted into the drilled holes, and the surgical site was closed with 5-0 nylon sutures.At week 14 (2 weeks after supplementation but prior to infection), mice were fasted for 6\u2009h during early morning, and a glucose tolerance test was performed. Mice were injected intraperitoneally with 300\u2009mg/kg glucose in 0.9% NaCl using U-100 insulin syringes . Tail vein blood glucose levels were then monitored before injection and at 15, 30, 90, 120 min postinjection using a OneTouch Verio glucometer and test strips .At harvest, an incision was made at the site of infection to expose soft tissue abscesses. The shortest and longest diameters of the abscess were measured using a digital caliper , and an average of the two diameters was used as the final measurement for abscess size. For bacterial burden quantification, soft tissue abscesses and pin implants were homogenized in lysing matrix A tubes at 6.0 m/s for 40\u2009s with an MP FastPrep-24 bead-beating system. Tibias were homogenized for an extra 40\u2009s. Homogenized tissue and bone were then serially diluted and plated on tryptic soy agar to determine CFU.Infected tibias were fixed in 10% neutral buffered formalin at room temperature for 3\u2009days. Samples were then rinsed in PBS and distilled water. Soft tissue and pin implants were removed prior to imaging. Three-dimensional images of infected tibias were acquired by high-resolution microcomputed tomography . Tibias were scanned with the following parameters: 145-kV energy setting, 300-ms integration time, voxel size of 10.5 mm, slice increment of 10\u2009mm, and a threshold of 210. Osteolysis was determined by quantification of the void area both from medial and lateral views.S. aureus communities and TRAP areas were performed using analysis protocol packages (APPs) in Visiopharm v.2021.07 .Following \u03bcCT imaging, fixed tibias were decalcified with 14% EDTA for 7\u2009days. Following decalcification, samples were stored in 70% ethanol until they were processed and embedded in paraffin. Transverse sections 5\u2009\u03bcm in size were cut and mounted on glass slides. Images were scanned with a VS120 virtual slide microscope  and visualized using Olympia OlyVIA for abscess counting . Quantification of Taq polymerase  using dual indexed primers . Amplicons were then normalized and pooled using SequalPrep normalization plates . Pooled library was paired-end sequenced on an Illumina MiSeq  at the University of Rochester Genomics Research Center. Each sequencing run included: (i) positive controls consisting of a 1:5 mixture of Staphylococcus aureus, Lactococcus lactis, Porphyromonas gingivalis, Streptococcus mutans, and Escherichia coli; (ii) negative controls consisting of sterile saline; and (iii) extraction controls of saline. The background microbiota was monitored at multiple stages of sample collection and processing. All sterile saline, buffers, reagents, and plasticware used for sample collection, extraction and amplification of nucleic acid were UV irradiated to eliminate possible DNA background contamination. Elimination of potential background from the irradiated buffers, reagents, plasticware, and swabs was confirmed by 16S rRNA amplification. Data from these background negative-control samples are deposited in the SRA along with positive controls (BioProject ID PRJNA786881).Samples were processed and sequenced as previously described . Fresh fRaw reads from the Illumina MiSeq basecalls were demultiplexed using bcl2fastq version 2.19.1 requiring exact barcode matches . QIIME 2Bifidobacterium pseudolongum   . Briefly2EDTA anticoagulant tubes . To isolate plasma, blood was centrifuged at 2,000\u2009\u00d7\u2009g for 10 min at 4\u00b0C. Plasma was transferred to clean microcentrifuge tubes and stored at \u221280\u00b0C until analysis. Plasma samples were analyzed by Eve Technologies Corporation  using a multiplex assay for levels of 31 different cytokines and inflammatory mediators. Cytokines/chemokines with more than one undetectable sample were omitted in the heat map.Blood samples were collected at sacrifice via cardiac puncture and stored in KA separate cohort of mice was dedicated to targeted metabolomic profiling. Mice were fed on the same feeding scheme as before but were harvested 2 weeks after supplementation without infection to prevent confounding effects of infection on metabolism. Mice were fasted for 6 h prior to intraperitoneal injection with 100\u2009mg/kg Euthasol  and cervical dislocation. Plasma was isolated as previously described from blood collected via cardiac punctures. Cecal material was isolated using sterile forceps and placed directly into clean, preweighed microcentrifuge tubes. Both plasma and cecal samples were snap-frozen in liquid nitrogen and stored at \u221280\u00b0C prior to metabolomic analysis. The Metabolomics Innovation Centre  was contracted to identify and quantify up to 150 metabolites commonly found in samples.g. Ten microliters of each sample was loaded onto the center of the filter on the upper 96-well plate and dried in a stream of nitrogen. Subsequently, phenyl-isothiocyanate was added for derivatization. After incubation, the filter spots were dried again using an evaporator. Extraction of the metabolites was then achieved by adding 300 \u03bcL of extraction solvent. The extracts were obtained by centrifugation into the lower 96-deep-well plate, followed by a dilution step with MS running solvent. For organic acid analysis, 150 \u03bcL of ice-cold methanol and 10 \u03bcL of isotope-labeled internal standard mixture was added to 50 \u03bcL of sample for overnight protein precipitation. Then, it was centrifuged at 13,000\u2009\u00d7\u2009g for 20\u2009min. Fifty microliters of supernatant was loaded into the center of the wells of a 96-deep-well plate, followed by the addition of 3-nitrophenylhydrazine (NPH) reagent. After incubation for 2 h, butylated hydroxytoluene (BHT) stabilizer and water were added before LC-MS injection. Mass spectrometric analysis was performed on an ABSciex 4000 QTrap tandem mass spectrometry instrument  equipped with an Agilent 1260 series ultra-high-performance liquid chromatography (UHPLC) system . The samples were delivered to the mass spectrometer by an LC method followed by a DI method. Data analysis was done using Analyst 1.6.2.A targeted quantitative metabolomics approach was used to analyze the samples using a combination of direct injection (DI) mass spectrometry (MS) with a reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) custom assay. This custom assay, in combination with an ABSciex 4000 QTrap (Applied Biosystems/MDS Sciex) mass spectrometer, can be used for the targeted identification and quantification of up to 150 different endogenous metabolites, including amino acids, acylcarnitines, biogenic amines and derivatives, uremic toxins, glycerophospholipids, sphingolipids, and sugars , 88. TheS. aureus was grown overnight in TSB at 37\u00b0C with shaking at 250\u2009rpm. Overnight culture was washed twice in PBS and normalized to an optical density (OD) of\u20090.5 in TSB. The normalized bacteria solution was further diluted 1:200 with a total volume of 200 \u03bcL in TSB alone or TSB with supplemented polyamines (3 mM spermine and/or spermidine) in a flat-bottom 96-well plate. Absorbance readings at 600\u2009nm were then monitored every 30 min using a kinetic reading with a Biotek ELx808 microplate reader  set at 37\u00b0C with medium shaking. Growth curves were performed in triplicates.B. pseudolongum copy number quantitation, and individual metabolite analysis, a one-way analysis of variance (ANOVA) with a Tukey\u2019s multiple comparisons post hoc test was performed. All statistics were analyzed using GraphPad Prism 9.3.1 except for 16S rRNA sequencing and metabolomics data, which were analyzed in RStudio 4.0.3. Differences between groups were considered significant with a P value of <0.05. P values less than 0.1 are included in figures to denote trending changes.Statistical analyses for 16S rRNA sequencing and metabolomics were performed as described above. For weight assessment, the glucose tolerance test, bacterial burden analysis, histology, \u03bcCT, individual plasma cytokine assessment, PRJNA786881, including positive and negative controls on each plate.Illumina 16S rRNA V3-V4 amplicons were deposited in the SRA under BioProject ID number"}
+{"text": "S. aureus pathogens. The interaction of nano-antibiotics with the bacterial cells was investigated via fluorescent confocal microscopy. Conventional tests (MIC and MBC) were used to examine the antibacterial properties of nano-antibiotic formulations. Simultaneously, a bioluminescence assay model was employed, revealing the rapid and efficient assessment of the antibacterial potency of colloidal systems. In comparison to the free-form antibiotic, the targeted nano-antibiotic exhibited enhanced antimicrobial activity against both the planktonic and biofilm forms of S. aureus. Furthermore, our data suggested that the efficacy of a targeted nano-antibiotic treatment can be influenced by its antibiotic release profile.The poor bioavailability of antibiotics at infection sites is one of the leading causes of treatment failure and increased bacterial resistance. Therefore, developing novel, non-conventional antibiotic delivery strategies to deal with bacterial pathogens is essential. Here, we investigated the encapsulation of two fluoroquinolones, ciprofloxacin and levofloxacin, into polymer-based nano-carriers (nano-antibiotics), with the goal of increasing their local bioavailability at bacterial infection sites. The formulations were optimized to achieve maximal drug loading. The surfaces of nano-antibiotics were modified with anti-staphylococcal antibodies as ligand molecules to target  Antibiotic resistance presents one of the biggest threats to global health today. Despite continuous efforts to develop new antibacterial drugs, including those with novel mechanisms of action, the occurrence and dissemination of antibiotic resistance seem to be inevitable . Under sOne suggested approach to tackling antibiotic resistance is to optimize the accessibility of antibiotics at their designated sites of action using effective antibiotic dosage forms . This coStaphylococcus aureus, a leading cause of a wide range of clinical infections, was selected as the representative model of the bacterial pathogen. To target staphylococcal infection sites, the surfaces of nano-particles were conjugated with anti-protein A antibodies, which have a high affinity for bacterial surfaces. The conjugation process was optimized to ensure the proper orientation of antibodies on the nano-particle surface, preserving their antigen-recognition ability -3,5-diphenyl tetrazolium bromide (MTT) assay and Lactate dehydrogenase (LDH) test against the alveolar cell line A549. Cells were grown in DMEM supplemented with 10% . Statistical analysis was performed using the Mann\u2013Whitney non-parametric test, with In the present study, we successfully encapsulated two fluoroquinolone antibiotics, ciprofloxacin and levofloxacin, within PLGA-based nano-carriers to develop nano-antibiotics. The surfaces of these nano-antibiotics were modified by conjugating anti-staphylococcal antibodies, which significantly improved their ability to recognize and bind to targeted bacteria. Our findings demonstrated that both untargeted and targeted nano-antibiotics increased the anti-biofilm efficacy more compared to that of the free-form antibiotics. The advantages of targeted delivery were particularly evident in the short-time killing assay of bacteria (dynamic environment). These targeted formulations may minimize the off-target effects on healthy tissues or the microbiome, while enhancing the bioavailability of antibiotics at the site of infection. However, it is crucial to note that the drug release kinetics of the formulation could influence the targeting effect. In future experiments, the consideration of incorporating controlled release strategies, such as stimuli-responsive drug release systems triggered by bacterial pathogens, should be taken into account to maximize the efficacy of antibiotic therapy."}
+{"text": "During the COVID-19 lockdown, a distortion of time passage has been widely reported in association with a change in daily rhythm. However, several variables related to these changes have not been considered. The purpose of the present study was to assess the changes in dispositional mindfulness, time experience, sleep timing and subjective memory functioning. A longitudinal study was conducted on 39 Italian adults  assessing mindfulness, ad hoc questions of sleep habits during workdays and free days, chronotypes, subjective time experience, and memory functioning before (December 2019\u2013March 2020) and during (April 2020\u2013May 2020) the first Italian COVID-19 lockdown. Participants reported delayed sleep timing, a slowdown in the perception of the present time, a decrease of time pressure, and an increase in the feeling of time expansion/boredom. In addition to correlations between mindfulness, memory functioning, and subjective sleep duration during workdays, a mediation model showed that changes in the dispositional mindfulness determined a delay of bedtime during workdays through the mediation effect of increased feeling of time expansion/boredom. This finding highlighted the role of mindfulness in reducing the feeling of time expansion/boredom for regulating the sleep timing. The theoretical and practical implications of the findings are discussed. In December 2019, from the Wuhan province of China, the coronavirus disease 2019 (COVID-19) spread rapidly worldwide and was caused by severe acute respiratory syndrome\u2014or coronavirus2 (SARS-CoV2) .Several national governments in almost all countries around the world imposed a strict social distancing measure, with the closure of most business and recreation activities , as well as the shutdown of school and group meetings . This loIn addition, a change in the experience with time has been reported during the home confinement. Generally, the studies have reported a slowing down of time during the home confinement ,28,29,30The proposed idea could be grounded on the evidence, during the COVID-19 lockdown, of a change in life rhythm ,19,21, wHowever, to better address the aim of the present study, the circadian typology  should In the present study, the dispositional mindfulness  was considered as an additional individual difference. Trait or dispositional mindfulness could be defined as a personality trait and refers to the innate capacity of paying and maintaining attention to the present moment with a nonjudgmental attitude ,50. The Finally, in the present study, the memory functioning was considered because memory involves a retrospective component  and a prospective component  ,70, and In the present research, the general aim was to assess the relationships between the subjective passage of time, sleep timing and regulation, chronotype, dispositional mindfulness and self-reported memories slips in a longitudinal study (from December 2019 to May 2020) in Italy. Contrarily to the majority of the studies, which required participants to compare, for example, their sleep quality during the COVID-19 lockdown with that usually exhibited in the pre-lockdown months, the present study could directly assess the changes experienced from pre-lockdown to first Italian lockdown in the same individuals for all variables considered. Thus, first of all, it was expected in all participants a slowdown of the subjective passage of time, longer sleep duration , delayed bedtimes, reduced social jetlag, lower dispositional mindfulness and a reduction of self-reported memory slips. In addition, a slower passage of time, more irregularity in the sleep\u2013wake habits and lower dispositional mindfulness should be expected in the evening-types, with respect to the other two chronotypes, while no chronotype differences should be expected for subjective memory functioning. Second, negative associations between reduction of the dispositional mindfulness and increase of dilatation in the passage of time, later bedtime, increase of sleep irregularity, and better memory functioning were expected. At the same time, a slowdown in the passage of time should be associated with a later bedtime, reported sleep irregularity and better memory functioning. Finally, the following mediation model was expected: the changes in the dispositional mindfulness from pre-lockdown to during COVID-19 pandemic outbreak should predict the changes in the feeling of time expansion/boredom, which, in turn, should predict the bedtimes/social jetlag of participants. Specifically, it was expected that people with a reduction of their dispositional mindfulness during home confinement should report a greater slowdown of their passage of time, and then a delayed sleep timing and less regularity.t(38) = 0.0001, p = 1.00). In the sample, there were 10 evening-types, 21 intermediate-types and 8 morning-types. These chronotypes did not differ for gender (2\u03c7(2) = 1.10, p = 0.58), and the rMEQ score did not correlate with age  or education level .The comparison between the mean rMEQ score at pre-lockdown  and that at COVID-19 lockdown period  was not significant .During the COVID-19 lockdown, participants shifted in advance their bedtimes and wake-up time during workdays, and their MPoS was significantly advanced . Additiot(38) = 3.78, p = 0.001), WTW  = 3.85, p = 0.0001), MPoS  = \u22123.41, p = 0.002), FTP  = 4.16, p = 0.0001), and FTE/B  = \u22124.75, p = 0.0001) were significantly different from zero. These results confirmed that during the COVID-19 lockdown, participants experienced a change of these variables, respectfully, to the pre-lockdown period.In order to deeply address the significant results, the difference between the pre-lockdown period and the COVID-19 lockdown for each variable was calculated for every participant, as done by . Thus, aTo further examine the relationships between the individual changes over these two periods, a difference between values reported in pre-lockdown period and those obtained during the COVID-19 lockdown for each variable was calculated . For all bedtimes and wake-up times, the difference was calculated between COVID-19 lockdown times and pre-lockdown times . rs ranged from \u22120.25 to +0.21 and ps > 0.12; correlations not reported in Beyond the fact that the rMEQ score did not correlate with any difference in values of every variable  = 3.31, p = 0.015). Although the dMASS did not directly predict dBTW (p = 0.32), dMAAS was negatively associated with dFE/B (p = 0.007), which, in turn, was positively associated with dSTW (p = 0.008), suggesting an indirect effect . In other words, during the COVID-19 lockdown, less dispositional mindfulness induced a greater feeling of boredom, and this increased feeling of time expansion induced a late shift of bedtime during workdays.Finally, the mediation model, hypothesizing an indirect effect of dispositional mindfulness on sleep timing during workdays, through the feeling of time expansion/boredom, was tested . The modThe general aim was to assess the relationships between the subjective passage of time, sleep timing/regulation, chronotype, dispositional mindfulness, and self-reported memories slips in a longitudinal study from the pre-lockdown period (December 2019) to the first Italian COVID-19 lockdown . To address this general aim, in the present study the changes from pre-lockdown to home confinement were assessed for all variables. Then, the relationships between individual changes in these variables were addressed through correlational analysis. Finally, a possible mediation model explaining how changes in the dispositional mindfulness could predict the changes in the feeling of time extension/boredom, which, in turn, could be related to delayed bedtime during workdays during the COVID-19 pandemic, was tested.Considering that this study could directly evaluate the impact of the home confinement in the passage of time, sleep\u2013wake habits, morningness\u2013eveningness preference, dispositional mindfulness and subjective memory functioning in the same participants, the present study showed that from December 2019 to March\u2013May 2020 participants modified their sleep\u2013wake  timing during the work/school days and in their feeling of time pressure and expansion. Although these findings partially confirmed the expectations, participants longitudinally reported an advanced shift for bed and wake-up times compared to those reported previously of social isolation ,46,47,48Contrarily from the expected correlational relationships, in the present study the negative associations between the reduction of the dispositional mindfulness and better retrospective and prospective memory, as well as the increase of the feeling of time expansion/boredom were only found. The former association described that a reduction of present-centered attention\u2013awareness in everyday experience was related to a better memory functioning during social isolation. Although this relationship seemed to be counterintuitive, not only the present finding could be in line with mixed results on other cognitive tests ,13,16,17The main novelty of this longitudinal study was related to the mediation model tested: the dispositional mindfulness exerted an effect on the feeling of time expansion/boredom, which, in turn, predicted the delayed bedtime during the workdays. Bearing in mind the strict relationship between the sleep timing  and sleep health, it is possible to advance the idea that the feeling of slowing down of time affected the sleep timing and sleep quality due to an increased boredom ,31,66,67https://eurohealthobservatory.who.int/monitors/hsrm/Accessed on 7 May 2023), it is not possible to exclude that a new variant of COVID-19  will determine a new widely contagion diffusion, requiring to re-adopt a severe lockdown. Indeed, in Europe, for example, there are different countries with a high rate of contagion , and the mean number of cases in the last 7 days (at the moment of the paper has been written) was equal to 122,146, suggesting that COVID-19 remains an important topic for the health of the population . Thus, the present results could suggest that online training in mindfulness and home-based mindfulness practice could decrease individual boredom, increase the attention to the passage of present time, and reduce bedtime procrastination with a direct impact on sleep quality [Altogether, the present findings indicated a possible way of the impact of home confinement\u2014which was a government measure to limit the coronavirus spread\u2014on the sleep disorders reported. Indeed, a reduction of the dispositional mindfulness could determine an increase of the feeling of time expansion/boredom  to May  2020, and it was difficult to take into account that social desirability and/or response bias could affect the data, given that no one could know how to cope with this exceptional phenomenon. In addition, the second data collection was performed at least two months after the first administration of questionnaires. Future studies should adopt more objective and/or behavioral measures of the assessed variables , reducing the possible impact of subjective biases in the responses. Related to this point, an additional limit could be related to the MAAS, which focused on the absence of attention to and awareness of present experience, for the assessment of the dispositional mindfulness. The Five Facet Mindfulness Questionnaire (FFMQ) or the Philadelphia Mindfulness Scale (PHLMS)  could bet(37) = 0.80, p = 0.43). In this sample, 5.10% of participants obtained an eighth-grade diploma, 41.00% declared a high school diploma, 23.10% obtained a bachelor\u2019s degree, and 28.20% reported a master\u2019s degree. The remaining 2.60% of participants obtained a PhD title. There was no gender difference for the educational level . All participants lived in the south of Italy, and the occupational status covered unemployment, students, and workers in both private and public fields. When the demographic characteristics of the original sample were compared with those of the final sample, no significant differences were found for age (t(80) = \u22120.38, p = 0.70), gender distribution (2\u03c7(1) = 0.001, p = 0.97) and educational level , suggesting that the drop-out of participants did not influence the sample characteristics.As described in the study by Mirolli et al. , a conveI rush through activities without being really attentive to them\u201d). Specifically, participants are requested to report how often they believed they had an experience referenced by each item on a scale from 1  to 6 . The total score of the MAAS involves calculating mean score across the 15 items, with higher averaged total score indicating greater mindfulness. In the present study, the Cronbach\u2019s alpha of the MAAS was equal to 0.85 and 0.90 for pre- and during lockdown, respectively.Dispositional mindfulness was assessed by the Mindful Awareness Attentional Scale (MAAS) \u2014for the The sleep\u2013wake habits were assessed through four ad hoc questions  relatingThe reduced version of the Morningness\u2013Eveningness Questionnaire (rMEQ) \u2014 for thehttps://www.ed.ac.uk/ppls/psychology/research/facilities/philosophy-and-psychology-library/psychological-tests/prmq). The PRMQ was selected because it is a reliable tool for assessing memory functioning in daily life [Do you fail to recognize a place you have visited before?\u201d) and prospective  memory mistakes, ranging on a 5-point scale from 1 (\u201cnever\u201d) to 5 (\u201cvery often\u201d). The total score can range from 16 to 80 and was obtained by summing the scores of each question. Considering that 8 items are used to assess retrospective memory errors and 8 items are used to assess prospective memory mistakes, both subscales can range from 8 to 16 as the result of the sum of score at each relative question. In line with Crawford et al. [www.psyc.abdn.ac.uk/homedir/jcrawford/prmq.htm. In this way, for total PRMQ score, as well as Pro and Retro subscales scores, higher scores indicated better memory functioning. The internal consistency of the PRMQ was equal to 0.85 and 0.90 for pre- and during COVID-19 lockdown period, respectively.For cognitive performance, the Prospective and Retrospective Memory Questionnaire (PRMQ)  was usedily life . In addiily life , and thud et al. , the rawvery slowly\u201d) to +2 (\u201cvery fast\u201d). Specifically, the first two questions were related to the perception of present time, in the form of trait-like and  state-like (\u201cHow fast do you expect the next hour to pass?\u201d) perception of passage of present time (PT). In the present study, the sum of subjective assessment was calculated, and thus, positive values reflected a perception of a fast passage of present time, while negative values reflected a slow passage of present time. Then, four questions covering the retrospective judgments of the passage of past time intervals were proposed. Specifically, these questions asked how fast last week, last month, last year, and the past 10 years had passed, using the previous 5-point scale. In a similar way, participants had to indicate how fast their childhood (defined as the period before 12 years), adolescence (between 13 and 19 years), young adulthood (between 20 and 29 years) and adulthood (between 30 and 39 years) had gone by. For the purpose of the present study, the mean scores of subjective judgments of time intervals (TI) and those for life periods (LP) were separately calculated. As before, positive values indicated a fast passage of time whereas negative values indicated a slow passage of time. The second part of the questionnaire was used to assess the Subjective Time Experience, given that participants were requested to indicate their feeling of time pressure  or their feeling of time expansion/boredom . To judge their feelings, for these 10 statements, participants used a 5-point scale from 0 (\u201cstrong rejection\u201d) to 4 . For each feeling, a mean score was computed. In the last part of this questionnaire, participants judged how their time experience could be explained by three temporal metaphors of speed  or by three temporal metaphors of slowness . As before, a mean score for each metaphor category was calculated.Finally, we adopted the Time Awareness and Subjective Time questionnaires , a different gender distribution among chronotypes was assessed. In addition, Pearson and Spearman correlation analyses were performed to assess association, respectively, between age and educational level with an rMEQ score.Statistical analyses were performed using SPSS software (IBM). First of all, I assessed the presence of possible changes in rMEQ scores between the two selected survey periods. In case of a null result, I decided to categorize participants as evening-, intermediate- and morning-types on the basis of their score in rMEQ obtained in the COVID-19 outbreak. Then, using a chi-squared test , as a between-subjects factor, and Time (pre-lockdown vs. during lockdown) as a within-subjects factor, was run on each variable score. To further examine the relationships between the individual changes over time for all variables, I calculated the differences in scores between pre-lockdown and during COVID-19 shutdown and analyzed their correlations with Pearson correlation analysis. When significant Time effect was found in previous ANOVAs, a t-test against zero was performed in order to assess whether the changes in the score were significantly different from zero.Finally, a mediation analysis assessed whether the changes in the feeling of time expansion/boredom mediated the relationship between the changes in the dispositional mindfulness and in the bedtime habit during the workdays, controlling for gender, age and educational level. A mediation analysis could use bootstrap analysis to evaluate the significance of indirect effects by using macro Process from SPSS developed by Hayes (Model 4) . The booThe alpha level was equal to 0.01 in order to be more conservative in the statistical choice (see  for a siThis longitudinal study assessed the changes from pre-pandemic to COVID-19 lockdown for dispositional mindfulness, sleep timing, subjective passage of time and self-reported memory functioning. The present study showed a significantly delayed bedtime, wake-up time, and delayed MPoS, a decrease of time pressure and an increase in the feeling of time expansion/boredom. In addition, the changes in the dispositional mindfulness correlated negatively with the changes in scores of PRMQ, as well as the changes in the feeling of time expansion/boredom, suggesting that less attention to and awareness of the present moment induced better subjective memory functioning and a slowing down of the passage of time. More importantly, the present study reported that during the home confinement, the changes in the dispositional mindfulness predicted the increase of the feeling of time expansion/boredom which, in turn, predicted the delayed bedtime during workdays. Thus, this study showed how the presence component of the mindfulness was related to the subjective passage of time during social isolation, and this relationship could delay the bedtime, which has been associated with several health outcomes . Extendi"}
+{"text": "The placenta is an extraembryonic organ, which is essential to maintain a normal pregnancy. However, placental development in humans is poorly understood because of technical and ethical reasons.We analyzed the anatomical localization of each trophoblastic subtype in the cynomolgus monkey placenta by immunohistochemistry in the early second trimester. Histological differences among the mouse, cynomolgus monkey, and human placenta were compared. The PubMed database was used to search for studies on placentation in rodents and primates.The anatomical structures and subtypes of the placenta in cynomolgus monkeys are highly similar to those in humans, with the exception of fewer interstitial extravillous trophoblasts in cynomolgus monkeys.The cynomolgus monkey appears to be a good animal model to investigate human placentation. The placenta forms a maternal\u2013fetal junction and has several essential physiological functions, such as facilitating the supply of nutrition to the fetus, exchange of gases, and removal of wastes from the fetus.Eutherian mammals share similar fetal organ structures, but they show substantial diversity in the gross appearance of the placenta as follows. The representative classification includes discoid (rodents and primates), diffuse (horses and pigs), multicotyledonary (ruminants), and zonary (carnivores) .In this review, we examine the utility of the cynomolgus monkey, which is a nonhuman primate (NHP), as an animal model for human placentation. Comparative studies among mice, cynomolgus monkeys, and humans have shown that the anatomical structures and trophoblast subtypes of the placenta in cynomolgus monkeys are highly similar to those in humans, with the exception of fewer interstitial extravillous trophoblasts in cynomolgus monkeys. The study of the cynomolgus monkey placenta with shallower extravillous trophoblast (EVT) invasion than that in humans may lead to the understanding of the molecular mechanism of placentation, by focusing on this difference in placental structure between the two species.2Human placental research has been conducted using clinical samples, including the term placenta and aborted conceptus. In vitro models, such as primary trophoblasts or choriocarcinoma cell lines, were the only option to investigate mechanistic insight into human placentation for many years because of the lack of adequate placental models. Nevertheless, many findings have been made in the human placenta.2.1After fertilization, human oocytes develop into blastocysts composed of two cell lineages called the inner cell mass and trophectoderm (TE), which are considered to form the fetus and placenta, respectively (Carnegie stage 4).2.2GCM1.GCM1 enhances human endogenous retrovirus envelope gene expression of ERVW\u20101 and ERVFRD\u20101, which are known as Syncytin\u20101 and \u20102, leading to cell fusion.Proliferative vCTBs differentiate toward multinucleated STBs, which cover the entire surface of placental villi through cell fusion.HIF1\u03b1 upregulation under hypoxia.Intervillous oxygen tension rises from 2% to 3% oxygen at 8\u201310\u2009weeks of gestation to more than 6% oxygen after 12\u2009weeks.WNT5A and IL33 secreted from decidual macrophages enhance vCTB and CCT proliferation in first trimester villous explant culture.EGF and HB\u2010EGF, which are expressed by decidual fibroblasts, are also associated with an increase in CCT proliferation.TEAD4 and TP63 contribute to vCTB cell proliferation, and the NOTCH1 intracellular domain prevents EVT differentiation by maintaining CCT proliferation.TP63 inhibits EVT migration and represses EVT marker genes, such as ITGA1, ITGA5, ITGB1, and MMP2.Not only oxygen tension, but also maternal cell\u2010derived soluble factors, function in the maintenance and controlling the differentiation of trophoblasts. 33.1Mice are widely used as an animal model for understanding human placentation because mice show similar physiological features of the placenta to those in humans, and manipulating gene function is relatively easy in mice. Innovative genome engineering techniques and the existence of useful stem cell lines have provided considerable knowledges for early embryonic development and placentation. However, there are substantial differences in the developmental mechanism and anatomical features between mice and humans.3.2Research on determining gene regulatory networks related to placentation using early mouse embryos and trophoblast cell lines has been conducted.Cdx2 is progressively restricted to the outer cells in the morula.CDX2 expression is detectable only after the early blastocyst stage.Cdx2 functions as a master regulator by enhancing a set of key trophoblast genes, such as Eomes, Tfap2c, Gata3, Elf5, and Ets2, to maintain the proliferation and inhibition of differentiation of trophoblast stem cells (TSCs),CDX2 is not expressed in human TSCs (hTSCs).However, there are clear differences related to gene regulation networks between mice and humans as follows. First, These different gene regulatory networks and signaling pathways might provide cues to solving the evolutionary differences between mice and humans. Taking into consideration that there are similarities and differences in epigenetic regulation between the mouse and human placenta, other animal models that are closer to humans are required to understand human placentation.44.1NHPs including New World monkeys  and Old World monkeys , which belong to macaques, are considered one of the most useful animal models to understand the mechanism of human embryogenesis and diseases. They are useful models because they show highly similar anatomical, physiological, and genomic features to those in humans. In particular, cynomolgus monkeys are considered a useful model because they can be bred throughout the year, and human disease models can be created by the lentivirus and CRISPR\u2010Cas9 systems.4.2While approximately 280\u2009days are required for human gestation, the gestation time in cynomolgus monkeys is approximately 160\u2009days.Human and macaque placentae are the discoid type, but both single and bidiscoid placentae are observed in cynomolgus monkeys during their pregnancy Figure\u00a0. The sec+/ITGA6+ in the inner layer of the vCTB and KRT7+/ITGA6\u2212 in the outer layer of the STB, similar to in humans. VIM+ mesenchymal and endothelial cells were localized inside of the placental villi. In CTBs at anchoring villi around the distal part of the placenta, ITGA6 expression was gradually decreased from the fetal to maternal side, suggesting that integrin switching also occurs during EVT differentiation in the cynomolgus monkey. Although deep EVT invasion into the decidual stroma is shared with humans and great apes, Old World monkeys, including cynomolgus macaques, show much shallower trophoblast invasion.+ endovascular EVTs in cynomolgus monkeys  is classified into class I, comprising 55.1To investigate gene functions and molecular mechanisms for placental tissue, the in vitro culture system is used to understand biological progress. TSCs were first established under FGF4\u2010treated culture condition as an in vitro model of placental cells in mice.5.2KRT7, TFAP2C, and GATA3, and primate placenta\u2010specific nonprotein\u2010coding microRNAs that are located on chromosome 19.ELF5 promoter region, and a lack of HLA class I molecule expression are also markers of first trimester mononuclear trophoblasts.In contrast to rodents, establishing TSC lines in primates took a long time. Human primary mononuclear CTBs were successfully isolated from the term placenta using the Percoll gradient method, but trophoblasts could not be maintained because they immediately differentiated toward STBs.CG and OCT4, which are STB and inner cell mass markers, respectively, are also expressed under the undifferentiated condition.As an NHP placental model, trophoblast cells are isolated from rhesus monkey blastocysts. These cells can be maintained for more than 23 passages. 5.3Recently, a new three\u2010dimensional culture system called organoid culture has been developed using TSCs in humans. Placental organoids contain both CTB and STB populations, such as placental villi.Although placental organoids are expected to be a useful tool to examine postimplantation trophoblast features in vitro, a preimplantation model called the blastoid model has been newly generated. Blastoids, which have a blastocyst\u2010like structure and features, are generated by the aggregation of embryonic stem cells and TSCs.6Cynomolgus monkeys are a useful animal model to provide novel insight into embryonic and placental development in primates because of ethical and technical obstacles in humans. While these two species show high similarities, there are some differences in early development between cynomolgus monkeys and humans . Therefore, identifying the mechanism underlying these differences may answer the fundamental question of what is involved in the inherent anatomical structure of the placenta in humans.In this article, we summarize multispecies comparisons mainly in trophoblast cells in placental development. A recent single\u2010cell RNA sequencing analysis of the human placenta comprehensively showed the cell population, gene expression profile, and cell\u2013cell interaction of all subtypes, highlighting the mechanism of immunotolerance that plays an important role in normal pregnancy.This work was supported by JSPS KAKENHI Grant Numbers 22K15028 (to S.M.), 23H03860 (to E.O.), 20K15699 (to M.M.), 20H05359, and 22H04669 (to M.E.), and the Naito Foundation (to M.E.). The funding source had no role in the design, practice, or analysis of this study.Authors declare no conflict of interests for this article.All animal experiments were conducted by following the guideline approved by the Institutional Animal Care and Use Committee of Shiga University of Medical Science (21\u2010002 and 22\u2010025)."}
+{"text": "We further observed a loss of physiologic age-dependent iron accumulation among people with schizophrenia, in that the iron level among cases was already high in young adulthood. Ferritin, which stores iron in a redox-inactive form, was paradoxically decreased in individuals with the disorder. Such iron-ferritin uncoupling could alter free, chemically reactive, tissue iron in key reasoning and planning areas of the young-adult schizophrenia cortex. Using a prediction model based on iron and ferritin, our data provide a pathophysiologic link between perturbed cortical iron biology and schizophrenia and indicate that achievement of optimal cortical iron homeostasis could offer a new therapeutic target.Despite loss of grey matter volume and emergence of distinct cognitive deficits in young adults diagnosed with schizophrenia, current treatments for schizophrenia do not target disruptions in late maturational reshaping of the prefrontal cortex. Iron, the most abundant transition metal in the brain, is essential to brain development and function, but in excess, it can impair major neurotransmission systems and lead to lipid peroxidation, neuroinflammation and accelerated aging. However, analysis of cortical iron biology in schizophrenia has not been reported in modern literature. Using a combination of inductively coupled plasma-mass spectrometry and western blots, we quantified iron and its major-storage protein, ferritin, in post-mortem prefrontal cortex specimens obtained from three independent, well-characterised brain tissue resources. Compared to matched controls ( Current treatments for schizophrenia do not address its most debilitating symptoms such as blunting of affect and volition and impairment in executive and social functioning . MoreoveDistinct from heme-iron, most tissue iron is bound to the intracellular protein ferritin and stored in neurons and glia . When reFor yet-to-be-identified purposes, most brain regions continue to accumulate iron, albeit at a slower rate, throughout adult life , 39. GivIn the present study, we focused on the PFC, whose dysfunction is a hallmark of schizophrenia . In healBased on specimens from three independent brain tissue resources, we show here that prefrontal iron content in individuals with schizophrenia is elevated compared to matched controls. We demonstrate that this iron elevation, which likely reflects the disorder itself rather than confounders such as demographic variables, lifestyle, or antipsychotic medications, confers the highest disease risk in individuals who died as young adults (<35). Next, we show that protein levels of ferritin, which stores iron in a redox-inactive form, are decreased in schizophrenia patients, and this decrease can predict disease more accurately in individuals with otherwise low iron levels. Finally, we show that a classification based on tissue levels of iron and ferritin and their ratio is superior to any one of these measures alone in discriminating cases from controls, providing a rationale for implicating perturbed cortical iron biology in schizophrenia.Post-mortem human brain samples were derived from three independent brain tissue resources: New South Wales Brain Tissue Resource Center (NSW-BTRC), Victorian Brain Bank Network (VBBN) and National Institute of Mental Health Human Brain Collection Core (NIMH-HBCC). Individuals with a diagnosis of schizophrenia or schizoaffective disorder and healthy controls were matched for age, sex, brain pH and post-mortem interval (PMI). Ascertainment and exclusions are described in the Supplementary Methods, with clinical and post-mortem characteristics summarized in Tables\u00a0N-ethylmaleimide (NEM), Roche cOmplete\u2122, EDTA-free Protease Inhibitor Cocktail  and phosphatase inhibitors cocktail, Roche PhosSTOP . The homogenization buffer for VBBN samples also contained Roche cOmplete\u2122, EDTA-free Protease Inhibitor Cocktail  and aprotinin (0.1\u2009mg/mL). Homogenized samples were centrifuged at 10,000\u2009g for 10\u2009min at 4\u2009\u00b0C. Supernatants were collected and protein concentrations determined using BCA Protein Assay Kit . Supernatants were aliquoted and stored at \u221280\u2009\u00b0C until use.Brains were obtained at autopsy (see Supplementary Methods for details relating to specific brain banks), stored frozen (\u221280\u00b0\u2009C) and PFC tissue was dissected and processed as previously described . Briefly2O2)  was added to each sample. Samples were allowed to stop effervescing, for 30\u2009minutes, before heating again for a further 15\u2009minutes at 70\u2009\u00b0C. Then cooled to ~22\u2009\u00b0C. Samples were further diluted with 1% HNO3 diluent up to 500 \u03bcL (dilution factor 1:10) and iron and copper were measured by inductively coupled plasma-mass spectrometry (ICP-MS), using an Agilent Technologies 7700\u00d7 ICP-MS system  under routine multi-element operating conditions. As previously described , a, aTAX1BPia Table\u00a0 142] an anTAX1BPia Table\u00a0 126] in inTAX1BPConsistent with ferritin\u2019s neuroprotection against iron-mediated build-up of toxic lipid peroxides , increashypodopaminergia in schizophrenia [During adolescence, excitatory synapses in the PFC may be pruned to generate a proper excitatory/inhibitory balance , and an ophrenia , 160. Moophrenia . Effectsophrenia .An additional mechanism linking our findings to schizophrenia reflects iron\u2019s emerging roles in neuronal signalling. Cross-talk between N-methyl-D-aspartate receptors (NMDARs) and iron , wherebyLack of data addressing iron dyshomeostasis in specific cell populations represents a limitation of the current study. For instance, proteomics analyses of the schizophrenia corpus callosum unravelled lower levels of both heavy- and light-chain ferritin , 170, inC. elegans model of aging [We note that of the total tissue iron that we assayed upon acid extraction, only a minor fraction that remains unbound to proteins is implicated in oxidative damage. Unfortunately, this labile iron pool is not easily measured in post-mortem tissue, leaving as analytical markers of cell iron status the total iron (associated with the minor labile iron pool as well as the predominant soluble protein fraction including ferritin) and ferritin itself. The concentration of the labile iron pool is kept steady by mechanisms such as ferritin expression , and theof aging , 174, anDespite decades-long research, major culprits driving prefrontal deficits in individuals with schizophrenia remain elusive. Focusing on iron biology, in the current study we showed that compared to matched controls, prefrontal tissue from schizophrenia cases was characterised by elevated iron, diminished ferritin, and most notably, an elevated iron-to-ferritin ratio. Using a prediction model based on iron and ferritin, our data are consistent with a potential pathophysiologic link between perturbed cortical iron biology and schizophrenia. As a prognostic biomarker, imaging cortical iron in-vivo could shed light on a potential relationship between iron, clinical course and treatment response, as was recently reported with early psychosis glutathione imaging . TherapeSUPPLEMENTAL MATERIALDataset and code"}
+{"text": "Iron is known to accumulate in neurological disorders, so a careful balance of the iron concentration is essential for healthy brain functioning. An imbalance in iron homeostasis could arise due to the dysfunction of proteins involved in iron homeostasis. Here, we focus on ferritin\u2014the primary iron storage protein of the brain. In this study, we aimed to improve a method to measure ferritin-bound iron in the human post-mortem brain, and to discern its distribution in particular cell types and brain regions. Though it is known that glial cells and neurons differ in their ferritin concentration, the change in the number and distribution of iron-filled ferritin cores between different cell types during autolysis has not been revealed yet. Here, we show the cellular and region-wide distribution of ferritin in the human brain using state-of-the-art analytical electron microscopy. We validated the concentration of iron-filled ferritin cores to the absolute iron concentration measured by quantitative MRI and inductively coupled plasma mass spectrometry. We show that ferritins lose iron from their cores with the progression of autolysis whereas the overall iron concentrations were unaffected. Although the highest concentration of ferritin was found in glial cells, as the total ferritin concentration increased in a patient, ferritin accumulated more in neurons than in glial cells. Summed up, our findings point out the unique behaviour of neurons in storing iron during autolysis and explain the differences between the absolute iron concentrations and iron-filled ferritin in a cell-type-dependent manner in the human brain.The rate of loss of the iron-filled ferritin cores during autolysis is higher in neurons than in glial cells. Non-heme iron accumulates in the normal ageing brain until the fourth decade of life , sets off immediately after death, causing a breakdown of cells, changing structural components, and hindering cell identification , e.g. in Krebs et al. . Howeverpost-mortem at the region and cellular level. We observed that autolysis influences the loss of iron-filled ferritin cores in a time-dependent manner. Moreover, our study reveals different rates of loss of iron-filled cores between neurons and glial cells during autolysis.For this, we investigated the concentrations of iron and ferritin cores filled with iron in the human brain samples post-mortem intervals (PMI) are given in Table The samples used here were collected from six deceased human subjects who had to undergo routine brain autopsy at the Diagnostic and Research Institute of Pathology of the University Hospital Graz . The subjects had not suffered from a known neurological disorder. The sex of the subjects was not considered for this study. To ensure that the analyses were not confounded by age, the specimens were collected from patients with ages ranging from 61 to 86. At this age saturation in iron accumulation has already been reached  were cut out of the samples and immersed in chemical fixative containing 2% formaldehyde, 2% glutaraldehyde in 0.1\u00a0M sodium cacodylate buffer. After fixation, the samples were rinsed in the same buffer, postfixed in 1% osmium tetroxide solution in the same buffer, dehydrated in a graded series of alcohol, immersed in propylene oxide, and embedded in TAAB embedding resin . After curing for 3\u00a0days at 60\u00a0\u00b0C, thin sections were cut using a Leica UC6 or a Leica UC7 ultramicrotome at a thickness of either 60 or 70\u00a0nm. The sections were contrasted with platinum blue and lead citrate solutions. One block was randomly selected from each brain area for imaging and elemental mapping.Following the autopsy, 6\u201310 sub-samples of approximate wet weight 0.1\u20130.2\u00a0g  using respectively the three-window method and the two-window method. The iron cores inside the ferritins are visible as bright spots using EFTEM.An unbiased sampling protocol described earlier  each quantified the number of iron-loaded ferritins and assessed the cell types. Ferritins were only counted if they were clearly visible as a bright spot on both the iron L\u2014jump-ratio and the iron L\u2014elemental maps and if at least three of the four researchers agreed on their number. In case of disagreement, the images were analysed again simultaneously. If the number of ferritins exceeded 20 in any map, the mean value of the counts from four researchers was considered as the final count. Cell types were identified on the bright field images and only noted if three of the four researchers agreed. For cell identification, the criteria described by Aten et al. , Hermel R2* relaxation data, a two-dimensional radiofrequency-spoiled multi-echo gradient-echo sequence with 6 equally spaced echoes was applied . Generalized auto-calibrating partially parallel acquisition (GRAPPA) was performed with an acceleration factor of 2.MR imaging of the extracted brain hemisphere was performed using a 3.0 Tesla scanner . A phased array head coil with 20 elements was used. For acquiring R2* maps were calculated with the relaxometry toolbox  were used. Brain tissue was freeze-dried to constant mass with a Gamma 1\u201316 LSC freeze-dryer  and the wet/dry mass ratio was determined. Freeze-dried samples were weighed to 0.1\u00a0mg into the 12\u00a0mL quartz vessels of a microwave-heated autoclave, UltraCLAVE IV . After the addition of 3\u00a0mL subboiled nitric acid and 2\u00a0mL of water the quartz vessels were placed in the 40 positions rack of the autoclave and the autoclave was pressurized with Argon to a pressure of 4\u00a0\u00d7\u00a0106\u00a0Pa. The following microwave heating program was applied: from room temperature to 80\u00a0\u00b0C in 5\u00a0min, from 80 to 150\u00a0\u00b0C in 15\u00a0min, from 150 to 250\u00a0\u00b0C in 20\u00a0min, and 250\u00a0\u00b0C for 30\u00a0min. After cooling to 80\u00a0\u00b0C, the pressure was released and the samples were transferred to 50\u00a0mL polypropylene tubes  and filled to the mark.All solutions were prepared with ultrapure water . Nitric acid (>\u200965% p.a.) was further purified via sub-boiling. For quantification, single elements standards .For the quantification of iron  at a concentration of 200\u00a0\u00b5g/L was added as an internal standard online before the nebulizer. The samples were pumped through a peristaltic pump tubing with an i.d. of 1.05\u00a0mm and the internal standard with a tubing of 0.19\u00a0mm. The elemental iron was quantified with an inductively coupled plasma mass spectrometer  at a mass-to-charge ratio of Fe p\u2009<\u20090.05; ***p\u2009<\u20090.001; ****p\u2009<\u20090.0001 for Figs.\u00a0Statistical analysis was performed using GraphPad Prism 9.2.0. One-way ANOVA with a Tukey\u2019s multiple comparisons test was used for statistical analysis: *Ferritins consist of a shell and an iron-filled core, and the core is visible as an electron-dense body in negative contrast electron micrographs of isolated ferritins Fig.\u00a0b. The saTo visualize the ferritin cores in electron micrographs, we generated iron L\u2014elemental maps and iron L\u2014jump-ratios of brain samples for four different brain areas in each of six different deceased subjects. The maps consistently exhibited ferritins as bright spots of 5\u201310\u00a0nm in diameter in all four examined brain regions Fig.\u00a0a\u2013d. The 2 dimensions and showed that the ferritins were not evenly distributed but clustered in certain micrographs  of 24\u00a0h, but in this sample, autolysis had progressed considerably, preventing us from faithfully identifying cell types. Accordingly, this sample was excluded from cell type identification experiments.We next counted the number of ferritin cores in samples taken from FGM, FWM, putamen, and GP of each patient. The counts were made within 60 micrographs of 556\u2009\u00d7\u2009556\u00a0nmphs Fig.\u00a0. We obse3 from the micrographs\u2019 3D dimensions. Plotting ferritin concentration in each brain region for each patient revealed that either the putamen, GP, or both, had a significantly higher concentration of ferritins than either the FGM or FWM in five out of six patients  in the putamen and GP than in FGM and FWM Fig.\u00a0a\u2013c). qMRFWM Fig.\u00a0b. SignifFWM Fig.\u00a0c.Fig. 6qR2* value, the higher the iron concentration  ferritins than the FGM and FWM. The basal ganglia as the storage site for iron have long been known  and the absolute iron concentration (ICP-MS), confirming an earlier, comparative study of both methods\u00a0, as this is non-invasive, but this does not enable to assign the ferritin to specific cell types. Organotypic brain slice cultures could be the closest option to understanding cellular ferritin distribution and human iron regulation in-vivo, which however are accompanied by both ethical and practical limits of obtaining suitable samples.Though it is not possible to extrapolate from post-mortem tissue to living humans, it would be possible to determine their iron distributions using quantitative MRI  that contained most (86%) of the ferritins we had detected in five out of the six patients. We found that most of the iron in the human brain is stored in oligodendrocytes, but iron is also found in astrocytes or unidentifiable glial cells and neurons. Their presence in astrocytes is in line with previous light microscopic results demonstrating an increasing number of ferritin-positive astrocytes in the ageing human basal ganglia  expressed on the brain capillary endothelial cells (Moos et al. Second, neurons also express iron exporter ferroportin, which allows them to excrete any excess iron. Additionally, neurons express both TfR and divalent metal transporter 1, enabling the import of transferrin-bound iron (Burdo et al. In summary, we found high involvement of neurons in the storage of ferritins at high iron load. Moreover, iron-loaded ferritins are lost post-mortem, in a yet unknown process, and loss of iron from ferritin cores occurs at a higher rate in neurons than in glial cells."}
+{"text": "The increasing popularity of robotic assisted surgery (RAS) as it is implemented in to sub specialities poses many challenges to ensuring standards in quality and safety. The area of Reconstructive and Functional Urology (RFU) has a wide range and largely complex heterogeneous procedures. In recent years RFU has started to incorporate RAS as the primary method to undertake these procedures due to improved vision, dexterity, and access to deep cavities. To ensure patient safety majority of institutions maintain minimal requirements to operate using RAS however across specialities and institutions these greatly vary.A narrative review of all the relevant papers known to the author was conducted.Specific challenges facing RFU is the inability to rely on case numbers as a surrogate means to measure competency as well the ongoing consideration of how to differentiate between surgeons with robotic training and those with the clinical experience specific to RFU.This review explores current models of training and credentialling and assess how it can be adapted to suggest a standardised guideline for RFU to ensure the highest standards of patient care. These policies focus on uro\u2010oncological procedures such as RARP and RAPN . The Society of European Robotic Gynaecological Surgery (SERGS) has recently addressed the important aspects of credentialling of robotic gynaecology procedures.In addition to heterogeneity, RFU is challenging to credential because many of the procedures are complex and technically challenging and the case volume for each procedure to be credentialed is low compared to RARP or RAPN. Further the conditions themselves require specialist training to ensure surgical judgement regarding appropriate choice of procedure.This review explores the training and credentialling process that is described for RAS in other subspecialities to determine aspects that can be adapted to creation of a training and credentialling guideline for reconstructive and functional urology to ensure safe patient care and good outcomes are achieved with \u2018onboarding\u2019. What is wrong with the SERGS draft that we are not just adopting it?22.1Meaningful advantages of RAS for intra\u2010pelvic vaginal wall surgery, such as sacrocolpopexy, is the achievable range of motion, easily targeted magnification and abolition of tremor compared to laparoscope that has resulted in surgeons resorting to the use of tacks (such as ProTack\u2122) and other maneuverers to overcome the challenges of suturing. Robotic surgery for apical vaginal prolapse was first reported in 2006.Colposuspension has seen a revival in popularity following the transvaginal mesh controversy. There is very limited literature on the RAS Burch Colposuspension. A small pilot study comparing robotic hysterectomy and robotic colposuspension with open hysterectomy and colposuspension demonstrated no difference in continence rates, however there were advantages when comparing blood loss and hospitalisation time.2.2Bladder neck contracture, though uncommon, is a challenging complication of prostate surgery and pelvic trauma. A robotic\u2010assisted approach to V\u2010Y\u2010plasty of the bladder neck has recently been described by two small case series by Granieri and Musch et al. The results are largely comparable to open surgery with respect to median surgical time and estimated blood loss.Artificial urinary sphincters have been placed in both males and females using RAS. While there are no randomised comparisons, in females blood loss (17\u2009ml vs. 275\u2009ml), shorter length of stay (3.5 vs. 9.3\u2009days) and intraoperative complication rates (37.5% vs. 62.5%) appear to favour the use of RAS.2.3Multiple studies2.4There are several other RAS reconstructive urological procedures described by expert pioneers, generally with case series describing feasibility but in most series with relatively small numbers including: bladder reconstruction and urinary diversion,33.1Lessons from urologic and non\u2010urologic literature demonstrate the learning curves involved in robotic surgery and progress over time from self\u2010taught pioneers, who then mentored their trainees to structured modular curricula, which are still evolving.3.2Due to the high volumes of cases, RARP have been established as the entry point of onco\u2010urology modular training curriculums described by leading organisations such as the European Association of Urology (EAU). An international study by Lovegrove et al reported on a modular pathway for RARP using the ERUS RARP Assessment Score.3.3Rice et al describe the vastly improved learning curves with progression from non\u2010structured toward structured training from robotic pancreatico\u2010duodenectomy. From self\u2010taught pioneers (1st generation) through mentorship alone (2nd generation) to mentorship and modular curriculum, they demonstrated significant reduction in operating times accompanied by a fall in complication rates and blood loss between 1st versus 2nd/3rd generations.4Case selection requires advanced non\u2010technical skills: history and physical examination relevant to functional urology problems, interpretation of appropriate urodynamic testing, medical imaging and knowledge of the surgical procedures that have been performed previously. For each procedure, adequate knowledge of the other options and their relative risks and benefits are required.4.1Specific knowledge is required of the risks of a RAS in any given individual due to the need for the extended head down position and the risks of operating in the abdominal cavity. The position of steep Trendelenburg at 25\u201345 degrees can lead to brain and upper airway oedema in addition to increase in intracranial pressure and cerebral blood flow.Trainee (novice) surgeon (resident/registrar/fellow)Experienced surgeons who have laparoscopic skillsExperienced surgeons with extensive previous experience of open but minimal laparoscopic experiencePrior to independent operating a stepwise approach is generally recommended including: \u2010 completion of a recognised robotic surgical system training course, observership of cases with an experienced surgeon, simulation and wet\u2010lab training to develop familiarity with the system and cultivate skillsThe training requirements are tailored to the surgeon's existing skill level, which can be categorised as follows:This process should be done with the demonstration of proficiency and safety in robotic procedural skills as opposed to high numbers alone. In general, a surgeon's proficiency with patients should be complemented by ongoing simulation practice and supervision with the full range of procedures to be undertaken until fully proficient.4.2The guidelines recommended by BAUS, NYU and SERGS4.3There are several simulation services available: the Robotic Surgical Simulator (RoSS), Mimic \u2122 Simulation Software and the Simsurgery Educational Platform (SEP), which have proven face, content and construct validity. The simulation aspect should be considered as a step with its own curriculum with scores of >80% in three consecutive tests to be achieved, that is, a competency\u2010based evaluation. This is juxtaposed with the number of hours on the console, which is less relevant. To ensure simulation practice is effective it should be supervised by an expert surgeon or educationalist. Virtual reality (VR) may also become an increasingly prevalent tool to enhance simulation. A study from Ebbing et al reported their experience of implantation and validation of VR RobotiX Mentor\u00ae simulator in RARP modular training. Ebbing's group demonstrating face and content validity for a robotic surgical training programme while focusing on key steps of the surgery such as non\u2010guided bladder neck dissection (ngBND) and non\u2010guided neurovascular bundle dissection (ngNVBD).So called \u2018wet labs\u2019 where surgical simulation is practices on live animal or cadavers are important to the training progression. These provide a standardised demonstration of a complex range of skills involving placement of ports, troubleshooting of complications in addition to specific operative procedures. Due to cost and access, a wet lab is generally used once full proficiency is achieved with simulation and other training and before independent operating. These \u2018wet lab\u2019 training is costly requiring standards of good laboratory practice and complex governance of ethical animal care and donor body systems ongoing work and development would need to be undertaken to ensure the wet\u2010lab teachings would be of a high standard. To date simulation for RFU procedures is in its early stages. In the future the development of RFU specific simulations requiring virtual or \u2018wet lab\u2019 training would be required to demonstrate proficiency.4.4This requires the establishment of standardised criteria to credential or judge a surgeon's competency. The development and subsequent validation of The Global Evaluative Assessment of Robotic Skills (GEARS) tool was created by deconstructing the fundamental elements of robotic surgical procedures. The six domains evaluated include depth perception, bimanual dexterity, efficiency, autonomy, force\u2010sensitivity and robotic control. GEARS is the first and currently only such tool, its domains being applicable to any speciality. The tool is applied to a step of the operation that reflects a pivotal point/complex task, which in turn can be applied to various operations\u2010index or complex to assess competency of a surgeon.4.5The final aspect of training is applying the theoretical knowledge and skillset to procedures. Modular training breaks down a procedure into key steps/components and entails advancing through the steps of increasing difficulty.Recently, there has been publications that works to address establishing performance metrics for the RARP procedure. A study by Mottrie et al developed proficient based progression metrics for RARP that reliably and objectively distinguish experienced and novice robotic surgeons.Specifically, to address the problem of relatively low case numbers in reconstructive and functional urology, one operation can be distributed between trainees (component operating) permitting impactful improvement of technical skills while preserving patient safetyA key aspect of this model is determining what is a basic or index procedure. Basic operations tend to involve fewer steps with simple dissection while establishing spatial awareness and fine\u2010tuning purposeful movement. RAS in Urogynaecology/RFU has index operations that are commonly performed laparoscopically such as Burch colposuspension and the authors with robotic experience would propose robotic retropubic sling removal as an index robotic procedure. Both procedures are favourable for their relative predictability permitting standardisation of steps. They are also not rare procedures potentially offering the trainee robotic surgeon the opportunity for repetition to cement and develop skills. Current urology trainees and some surgeons may have some or significant exposure to onco\u2010urological procedures, such as RARP, but condition specific training in RFU is still required.Prior to the development of the RAS skills, a model of advanced training, which teaches and evaluates the appropriate clinical evaluation and investigation leading to a decision to treat, as well as a pathway for standardised (or suggested) post\u2010operative evaluation to ensure that the robotic surgery sits within a whole package of delivering the best possible patient journey. This would give the appropriate balance between technical robotic skills, which are the focus of most current training guidelines and non\u2010surgical technical skills around clinical history, examination and decision making, which are underrepresented but arguably even more contributory to poor outcomes in reconstructive surgery.This basic training outline is applicable to any expertise level though would need adjustments based on proficiency. Experienced surgeons in either laparoscopic or open surgery wanting to add RAS to their repertoire should be expected to complete the same curriculum and assessments. For instance, a surgeon may take the equivalent amount of time in E\u2010knowledge as a resident but progress quickly to Modular training/Proctorship due to their already established technical and procedural skills. Irrespective of experience, each stage requires proof of competency through simulation and proctorship prior to full credentials being granted. Irrespective of a surgeon's robotic skills, it would be expected that a surgeon would have the adequate expertise in either laparoscopic or open surgery if the robot malfunctioned or in a similar practise to laparoscopic surgery, if the conditions became unsafe such as access or life\u2010threatening bleeding that the operation would be converted to open. It would be an expectation that in addition to robotic training, a surgeon maintains their additional open skillset or have an open surgeon on standby to assist if necessary.The learning curve is still substantial for an experienced laparoscopic surgeon to transition to robotic surgery. A CUSUM learning curve study of sacrocolpopexy found a learning curve of 78 cases for two surgeons who had each already completed 300 laparoscopic sacrocolpopexies, based on intraoperative bladder/bowel complications.5General requirements*Appropriate local licence with no practice restrictions*Current membership of board or college for appropriate continuing educationPre\u2010requisites*The physician must be independently privileged to perform the open/laparoscopic or minimally invasive procedures as applicable to the procedure for which he or she is requesting robotic assisted privileges.*Demonstrate performance of at least 20 open and laparoscopic surgeries with acceptable results and complications rates, reflective of the scope of privileges requested, in the past 12\u2009months.Provisional versus full credentialingTo achieve credentialing there needs to be \u2018sign\u2010off\u2019, which can be viewed as two stages*Sign\u2010off by the mentor (for surgeon in practice) or*Fellowship/programme director (for fellow/resident) and*Sign\u2010off by a proctor.Surgeons in practice, without robotic fellowship training, should be directed to follow the modular curriculum to acquire competency in a structured manner. They would need to identify a mentor to supervise this via component and then whole case completion prior to seeking a proctor to \u2018sign\u2010off\u2019.Procedure specific requirements:Most mature credentialing frameworks require procedure specific credentialing. The NYU credentialing document requires each procedure  to be assessed and signed off individually.Maintaining proficiency. To continue with RAS, either basic or advanced procedures, the surgeon must perform a minimum 20 cases each calendar year using the robotic surgical system though it is unclear where the evidence for this number has been determined.Documenting Competency and Proficiency. Surgeons undertaking RAS should keep a record of total operative times, estimated blood loss and complications, with regular review by credentialling committee and/or individual maintaining simulation proficiency is also appropriate.Transferrable Credentialling. A surgeon with experience who is new to a given institution but has provided evidence of basic requirements should be provided independent credentials however the first 5 cases should be assisted by a robotic surgeon and undergo focused review by the credentialling committee or individual.Generally, the numbers required to be deemed competent are arbitrary. If a given trainee has competency in RARP with >20 supervised cases, it would follow that specific RFU credentialling may require fewer cases than where such prior credentialing exists.For credentialing, several committees or associations6Once credentialling is achieved, the next issues faced by institutions is how are they maintained. Generally, it is expected that a surgeon will continue to operate using RAS.Though training has shifted towards competency\u2010based models, one of the shortcomings remains low case numbers/low volume centres. It is generally accepted that high case numbers do not necessarily equate to competency. A high volume of cases does however provide the opportunity to develop and apply skills and in turn demonstrate competency. Most credentialling requires completing a certain number of procedures, making it difficult to undertake at low volume centres. Given the uncommon nature of some operations, considerations need to be given to grouping procedures with similar skillsets to allow this to be achievable without significant impact on patient outcome. Proposed methods to maintain standards are annual simulation and regular undertaking of operative assistance to remain familiar with the docking and undocking of the robots and its equipment. Another means to assess surgical skill is surgeons should include audit of operative times, blood loss and complications based on Clavien\u2013Dindo Classification.7modular curriculum with \u2018components\u2019, which could encompass skills across several different procedures to address volume constraintsemphasis on index procedures to establish transferable skills, where possible, such as Burch Colposuspension or sacrocolpopexy or Burch takedown?develop the use of mixed reality/simulation models to support the opportunities to perform component skills in a safe environment, especially for complex procedures.The variety and heterogeneity of cases, combined with low numbers of some types of cases presents a challenge that differs significantly from existing robotic uro\u2010oncologic and other specialty procedures. Our group would like to identify a RFU curriculum with:8Our group would like to use the above experiences to develop a guideline on training and credentialing of robotics in reconstruction and functional urology through the use of a modified Delphi study engaging specific societies and international expertise. The challenge in developing such a framework Figure\u00a0 in RFU iEF, HY, HH and HO created the initial concept of the work. FH wrote the initial manuscript. All authors refined the final manuscript, and agree to be accountable for all aspects of the work.The authors declare that they have no disclosure of interest."
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