P. aeruginosa (PSA) and A. baumannii-calcoaceticus complex (ACB) as part of the SENTRY Antimicrobial Surveillance Program.Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin with activity against Gram-negative bacteria. CFDC and comparator activities were analyzed against resistant and molecularly characterized 2,241 PSA and 682 ACB were consecutively collected from 63 US sites in 2020-2021. Susceptibility testing was performed by broth microdilution and CFDC testing used iron-depleted media. FDA and CLSI breakpoints were used for CFDC. CLSI criteria were applied to comparators, except for imipenem-relebactam (IMR) that used FDA breakpoints. Isolates with a resistance phenotype to ≥ 3 classes were defined as multidrug resistant (MDR). ACB and PSA with imipenem and/or meropenem (MER) MIC ≥4 μg/mL or ceftazidime (CAZ) and/or cefepime MIC ≥ 16 μg/mL were subjected to next-generation genome sequencing for screening of acquired carbapenemase genes.50/90, 0.06/0.25 μg/mL) and did  meet the MIC screening criteria. CFDC also had similar MIC50 values (0.12 μg/mL) against the MDR and carbapenemase-positive PSA populations, whereas other agents had compromised activity. 42.5% (290/682) of ACB met the MIC screening criteria, while 35.9% (245/682) had an MDR phenotype, and 27.3% carried carbapenemase genes . In general, CFDC, IMR, meropenem-vaborbactam, MER, ceftazidime-avibactam, and CAZ had activity against ACB that did not meet the MIC screening criteria, but only CFDC  was active against the resistant ACB subsets.31.6%  and 16.9%  of PSA met the MIC screening criteria and showed an MDR phenotype, respectively (Table). Carbapenemase genes were detected in 6 (< 1%) PSA. CFDC had similar MIC against PSA that did not , Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support.


Synovial inflammation in knee osteoarthritis (OA) causes disorganized synovial angiogenesis and complement activation in synovial fluid, but links between complement and synovial microvascular pathology have not been established. Since complement causes vascular pathology in other diseases and since sex-differences exist in complement activation and in OA, we investigated sex differences in synovial fluid complement factors, synovial tissue vascular pathology, and associations between complement and synovial vascular pathology in patients with late-stage knee OA.Patients with symptomatic, late-stage radiographic knee OA undergoing total knee arthroplasty or high tibial osteotomy provided matched synovial fluid and tissue biopsies during surgery. Complement factors  and terminal complement complex (sC5b-C9) were measured in synovial fluid by multiplex or enzyme-linked immunosorbent assay, respectively. Features of synovial vascular pathology  were assessed by histopathology. Multivariate linear regression models were used to assess associations between synovial fluid complement factors and histopathological features of vascular pathology, with adjustment for age, sex, body mass index, and sex interaction. Sex-disaggregated comparisons were completed.p=0.05). Median vasculopathy scores  were higher in males. In the full cohort, C5 concentration was associated with lower vascularization scores  while accounting for sex*C5 interaction. In sex-disaggregated analyses, increased C5 concentration was associated with lower vascularization scores  in male patients, but not in female patients. Males had higher sC5b-C9 compared to females. Additionally, males with high C5 had a higher synovial fluid concentration of sC5b-C9 compared to males with low C5. No differences were found in females.Synovial fluid biomarker and histopathology data were included from 97 patients. Most synovial fluid complement factors and synovial tissue histopathological features were similar between sexes. Synovial fluid C5 trended to lower levels in males (-20.93 ng/mL  Higher synovial fluid C5 levels were associated with increased complement activation and decreased synovial vascularization in males but not in females with OA. Future studies should test whether synovial fluid complement activation suppresses synovial angiogenesis and identify mechanisms accounting for C5-related sex-differences in synovial fluid complement activation in patients with knee OA. Synovial inflammation in knee osteoarthritis (OA) causes disorganized synovial angiogenesis and complement activation in synovial fluid, but links between complement and synovial microvascular pathology have not been established. The formation of new blood vessels by angiogenesis is stimulated during wound healing and dysregulated by chronic inflammation . In kneeInflammation in OA primarily involves components of the innate immune system, including activation of the complement cascade , 12. ComWang et al. reported that the synovium and synovial fluid of knee OA patients have higher gene expression of complement effectors and lower complement inhibitors compared to healthy individuals . KnockouFemale sex is a major OA risk factor . Females2f of 0.15 (moderate effect size) with 80% power and an α = 0.05, indicating we were adequately powered for our primary analysis with the total cohort (n=97) (Participants were recruited from the Western Ontario Registry for Early Osteoarthritis (WOREO) Knee Study, a prospective cohort designed to investigate clinical, biomechanical, and pathophysiological features of inflammation in patients with knee OA. The present study included 97 consecutively recruited patients with symptomatic, late-stage knee OA. Eligibility criteria included patients with Kellgren-Lawrence (KL) grades of 3 or 4 , frequent (n=97) . Knee rain vivo samples as we did not use protease inhibitors at the time of synovial fluid processing and this assay does not measure the more stable glycosylated (des Arg) metabolite.At the time of surgery, prior to knee joint arthrotomy, synovial fluid was aspirated to avoid contamination by blood. Synovial fluid samples were then centrifuged at 2800g and at 4°C for 15 minutes. Supernatants were aliquoted into cryovials for storage at -80°C until use. Synovial fluid samples with sufficient volume and minimal blood contamination were selected for multiplex analysis. Samples were treated with hyaluronidase (1 µg/mL) prior to assay with the Human Complement-2, Milliplex Multiplex Assay using Luminex , performed commercially at Eve Technologies, Calgary, AB, Canada, to measure the concentration of complement factors C5, C2, CFI, adipsin (factor D), and MBL (mannose-binding lectin) (ng/mL). We excluded the short-lived, unstable intermediates C4b and C5a in these To assess levels of a stable complement activation product and ensure that lower C5 levels were not due to consumption due to rapid C5 cleavage/activation, synovial fluid samples from a subset of patients that had adequate synovial fluid volume were treated with hyaluronidase prior to measuring terminal complement complex (sC5b-C9) using an enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer’s protocol . We included samples from 16 male and 16 females patients, with n = 8 per quartile (highest/lowest) of C5 concentration. Absorbance reading at 620 nm (reference) was subtracted from the absorbance reading at 450 nm. Amount of sC5b-C9 (ng/mL) present in each sample was interpolated using a standard curve.To standardize the location of tissue collection, all synovial tissue biopsies were obtained from the lateral suprapatellar recess at the time of surgery. After overnight fixation in 4% paraformaldehyde, synovial tissue samples were processed, and paraffin embedded. Serial sections (5 µm thickness) were mounted on glass slides, stained with hematoxylin and eosin, and 5 high powered fields (hpf) per patient at least 100µm apart were assessed. Six histopathological features were graded as described previously : perivasTo assess classical pathway activation in synovial tissue, we performed immunofluorescent detection for C1q. Slides were heated at 62°C for 30 minutes, cooled to room temperature, and then deparaffinized and rehydrated. For antigen retrieval, sections were immersed in 70°C Tris buffer  and allowed to cool to below 30°C. Sections were permeabilized by incubation in 0.2% Triton-X in 1x PBS for 10 minutes. Slides were rinsed with 1X phosphate buffered saline (PBS) and blocked with 5% bovine serum albumin blocking solution . Samples were incubated overnight at 4°C with anti-human C1q . We included negative (no primary antibody) control sections. Goat anti-mouse conjugated to Alexa Fluor-647  in 1X PBS was added to the slides for an hour at room temperature. Slides were mounted with Molecular Probes Prolong Gold Antifade Mountant with DAPI .Slides were imaged at 40X using a Leica Aperio VERSA 8 microscope scanner located at Molecular Pathology Core Facility, Robart’s Research Institute. Immunofluorescence images were analyzed quantitatively using QuPath (v0.3.1)  pixel clGraphpad Prism 8  or Stata IC/15.1 .All analyses were done using Descriptive statistics for patient’s demographics and clinical characteristics were calculated as mean +/- standard deviation (range) and frequency  for continuous and categorical variables, respectively. Two-tailed independent student’s t-tests were used to compare age, BMI, and KOOS pain between sexes. A Fisher’s Exact test was used to compare proportions of KL grades between sexes. Statistical significance was set at p<0.05.Unpaired, two-tailed t-tests  were conducted to assess differences in synovial fluid complement concentrations between sexes. Measures were reported as the difference in mean concentrations +/- 95% confidence intervals. To assess for relationships between synovial fluid levels of C5 and other measured complement components, we fitted a series of sex-disaggregated multivariate linear regression models with adjustment for age and BMI. Results were reported as unstandardized beta coefficients +/- 95% confidence intervals.To assess sex-differences in complement activation, we used an unpaired, two-tailed t-test to test if terminal complement complex (sC5b-C9) was different between males and females. To determine if C5 concentration was indicative of complement activation, a two-way ANOVA with Tukey’s post-hoc test was used to assess differences in synovial fluid sC5b-C9 (complement activation product) between patients from the highest and lowest quartiles of synovial fluid C5 levels for both sexes. Results were reported as mean difference +/- 95% confidence intervals.To assess sex-differences in classical pathway activation, we used an unpaired, two-tailed t-test to measure any differences in percent positive synovial lining cells for C1q in males and females. To identify whether synovial lining C1q was indicative of complement activation, a two-way ANOVA with Tukey’s post-hoc test was used to assess differences in percent positivity of synovial lining cells for C1q between patients from the highest and lowest quartiles of synovial fluid C5 levels for both sexes. Results were reported as mean difference +/- 95% confidence intervals.A series of multivariate linear regression models were fitted to test the association between median synovial vascular pathology scores (predictor) and synovial fluid C5 concentration (outcome) while adjusting for age, sex, and BMI. To test whether the relationship between C5 and vascular pathology depended on sex, we included sex by C5 concentration interaction terms. As recommended by SAGER guidelines , sex-disA total of 126 patients with late-stage knee OA were screened for eligibility. Four (3.17%) were excluded because they did not have synovial biopsies available. Twenty-four (19.05%) were excluded due to having inadequate synovial fluid volumes  or blood contamination . One (0.79%) patient was excluded due to low bead counts during the multiplex assay. Therefore, a total of 97 patients with complete synovial fluid biomarker and histopathology data were included in this analysis. Demographics and clinical characteristics for the cohort are presented in Synovial fluid concentrations of complement factors are reported for the total cohort and separated by sex in In males, there was no evidence of associations between C5 and other complement factors C2, MBL, CFI, and adipsin Table 3.The average of the median synovial histopathology scores of the total cohort, as well as the averages separated by sex, are shown in Multivariate linear regression showed that synovial fluid C5 concentrations were inversely associated with median synovial vascularization, while adjusting for age, sex, and BMI  compared to females . However, we cannot rule out complement-related changes in inflammatory mediators such as cytokines or inflammatory cell function, as these were not measured in this study. Future studies investigating how complement activation directly affects synovial vascular pathology and how this may impact clinical outcomes in knee OA patients is warranted, and such studies should investigate sex differences.Numerous studies have reported pro- or anti-angiogenic roles of complement signaling. Importantly, disease context and the pathological cell types involved may influence the divergent roles of complement . If we iOur sex-specific analyses suggest that complement activation may play more of a role in synovial vascular pathology mechanisms in males than it does in females with knee OA Figure 4Upstream complement pathway activation was not assessed in this study but may influence downstream events. Synovial fluid levels of C5 trended to be higher in females than in males and were associated with C2 levels in females only. This suggested a potential sex-specific link between C5 levels and classical pathway activation in females, but we found no sex differences in C1q deposition in synovial tissues regardless of synovial fluid C5 levels. However, we cannot rule out a link between C1q and synovial vascularization since C1q has also been linked with angiogenesis through mechanisms that bypass downstream complement activation , and oure.g., 70-80 per group) to be appropriately powered for the sex-disaggregated analyses. Synovial angiogenesis is a pathological feature in knee OA and associated with worse joint pain yet, it is not known whether disease-related or therapeutic reduction of synovial vascularization would be protective or lead to worse disease outcomes. Given the strong relationship between synovial vascularization and OA symptoms, the effects of reducing synovial angiogenesis on OA outcomes should be investigated.Our study is limited to cross-sectional relationships. Although we cannot conclude that causal relationships exist between complement and synovial vascular pathology, our findings raise the novel hypothesis that complement signaling inhibits synovial angiogenesis in knee OA and that sex differences modify these effects. The focus on late-stage knee OA also limits the generalizability of our findings to the context of early knee OA. Future studies should also assess longitudinal relationships since increased vascularization has been shown to be associated with increased KL grade at follow up . Nearly This is the first study to describe an association between synovial fluid complement C5 levels and synovial tissue microvascular pathology in patients with knee OA. Importantly, we also identified sex differences in this association and in synovial fluid complement activation. Higher synovial fluid C5 levels were associated with greater terminal complement activation and decreased synovial vascularization in males, but neither occurred in female patients. These findings raise the hypothesis that sex differences in synovial fluid complement activation in knee OA may lead to differential effects on synovial microvascular pathology. Prospective studies are required to confirm whether complement directly or indirectly inhibits synovial angiogenesis or is merely an indicator of the inflammatory milieu within the joint. Our findings also serve as a compelling reminder to include sex-disaggregated analyses in OA research.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Western University Health Sciences Research Ethics Board. The patients/participants provided their written informed consent to participate in this study.ES and HP contributed to study design, data collection and analysis, and drafted the manuscript. MC contributed to data analysis. TB and TA contributed to study design, data interpretation and critically revised the manuscript. All authors read and approved final manuscript.This work was funded in part by grants from the Academic Medical Organization of Southwestern Ontario , the Canada Research Chairs program, and Western University’s Bone and Joint Institute. HP is supported by Frederick Banting and Charles Best Doctoral Award from CIHR.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.


Fluid overload in patients in the intensive care unit (ICU) is associated with higher mortality. There are few randomized controlled trials to guide physicians in treating patients with fluid overload in the ICU, and no guidelines exist. We aimed to elucidate how ICU physicians from Nordic countries define, assess, and treat fluid overload in the ICU.We developed an online questionnaire with 18 questions. The questions were pre-tested and revised by specialists in intensive care medicine. Through a network of national coordinators. The survey was distributed to a wide range of Nordic ICU physicians. The distribution started on January 5th, 2022 and ended on May 6th, 2022.We received a total of 1,066 responses from Denmark, Norway, Finland, Sweden, and Iceland. When assessing fluid status, respondents applied clinical parameters such as clinical examination findings, cumulative fluid balance, body weight, and urine output more frequently than cardiac/lung ultrasound, radiological appearances, and cardiac output monitoring. A large proportion of the respondents agreed that a 5% increase or more in body weight from baseline supported the diagnosis of fluid overload. The preferred de-resuscitation strategy was diuretics (91%), followed by minimization of maintenance (76%) and resuscitation fluids (71%). The majority declared that despite mild hypotension, mild hypernatremia, and ongoing vasopressor, they would not withhold treatment of fluid overload and would continue diuretics. The respondents were divided when it came to treating fluid overload with loop diuretics in patients receiving noradrenaline. Around 1% would not administer noradrenaline and diuretics simultaneously and 35% did not have a fixed upper limit for the dosage. The remaining respondents 63% reported different upper limits of noradrenaline infusion (0.05–0.50 mcg/kg/min) when administering loop diuretics.Self-reported practices among Nordic ICU physicians when assessing, diagnosing, and treating fluid overload reveals variability in the practice. A 5% increase in body weight was considered a minimum to support the diagnosis of fluid overload. Clinical examination findings were preferred for assessing, diagnosing and treating fluid overload, and diuretics were the preferred treatment modality. Fluid overload is common in intensive care unit (ICU) patients and is associated with mortality in patients with sepsis , acute kFluid overload during critical illness is hypothesized to be caused by a pathological increase in global vaso-permeability in combination with excessive intravenous administration of fluids . When diLoop diuretics are commonly applied to treat fluid overload in the ICU , but no Criticalcarereviews.com  reported fluid overload to be a common occurrence in their ICU. Most of the physicians  agreed/strongly agreed that fluid overload is a modifiable consequence of fluid administration and 86% (706/827) found it to be a modifiable source of morbidity. However, a majority also thought of fluid overload as an inevitable consequence of appropriate fluid resuscitation 58%, (482/829) agreed/strongly agreed and a manifestation of sodium and water retention due to endocrine factors and AKI . The physicians were divided regarding the subject of fluid overload being an issue that resolves spontaneously, 31% (251/823) agreed/strongly agreed and 41% (335/823) disagree/strongly disagreed. The study demonstrated a broad consensus against fluid overload being a finding without clinical consequence  .When assessing fluid status, physicians preferred clinical parameters. The physicians often or very often used clinical examinations such as the daily and cumulative fluid balance , edema , urinary output , body weight , and the patient’s oxygen requirements . Physicians used other modalities such as ultrasound and radiological appearances more sparsely .The physicians considered findings from clinical examination supportive of the diagnosis of fluid overload . Additionally, when diagnosing fluid overload, 85% (658/770) found that the potential complications of fluid overload , and radiological findings  were more supportive of the diagnosis of fluid overload than a positive cumulative fluid balance of 3 liters . Many of the respondents  agreed/strongly agreed to a 10% or more increase in body weight from baseline supported the diagnosis of fluid overload while 59% (454/770) of the physicians agreed to a minimum of 5% increase to support the diagnosis .Most physicians agreed/strongly agreed to clinical findings suggestive of fluid overload , radiological features suggestive of fluid overload  and increasing body weight  as indications for de-resuscitation. High inspired oxygen concentration  and a positive fluid balance  were additionally seen as indications. A total of 45% (340/751) agreed that AKI was an indication for commencing de-resuscitation .The ICU physicians preferred fluid removal targeted to daily net fluid balance  followed by clinical examination findings  and baseline body weight . Other modalities such as ultrasound findings or physiological parameters to target fluid removal were rarely used .Diuretics were the preferred de-resuscitation strategy  followed by minimization of maintenance fluids and drug diluents  and minimization of resuscitation fluids . Administration of albumin was used sometimes/often or very often by 66% (480/732) of the physicians in case of in case of plasma albumin < 20 mM. Commencement of RRT was less likely, but 83% (598/718) of physicians would rarely/infrequently leave an ICU patient with fluid overload untreated . When chA majority agreed/strongly agreed that they would review their fluid removal plan in all the listed clinical situations in The physicians were questioned about how they would react to different complications and contraindications when having commenced loop diuretics in a stable ICU patient with fluid overload. In the case of mild hypotension (MAP 55–65 mmHg) the physicians were most likely to commence low-dose vasopressors, continue loop diuretics, and monitor closely  . In caseWe have conducted a Nordic survey on self-reported practices used in the ICU concerning patients with fluid overload. Our survey reveals variability in the practice, but some tendencies emerge. Clinical examination findings were the most preferred modality when assessing, diagnosing, and targeting fluid strategy. This is in line with previous surveys in the field that demonstrated how clinical examination findings, changes in body weight, and fluid balance were the most frequent assessment modalities in the ICU , 16. ThePrecise determination of fluid balance is known to be complicated. The Nordic physicians most commonly used clinical examination findings to assess fluid overload, this is potentially problematic for several reasons. The formation of peripheral and pulmonary edema is complex and formed due to many contributing factors in addition to fluid overload . Body weRadiological findings, echocardiograms, and lung ultrasound were used to a certain extent by the Nordic physicians when assessing fluid status. The survey from NZ/A/UK demonstrated different preferences concerning these modalities. Nordic physicians used ultrasound more frequently, especially lung ultrasound (27% of Nordic physicians vs. 8% of physicians from NZ/A/UK used it often/very often). Conversely, UK/A/NZ physicians used radiological findings more frequently (69% of NZ/A/UK physicians vs. 44% of Nordic physicians).Chest X-ray findings can be difficult to interpret in ICU patients and the diagnostic performance for fluid overload is at best mediocre . UltrasoMultiple retrospective studies have shown that an increase above both 5 and 10% weight-based cumulative fluid balance is significantly correlated with increased mortality in AKI  and surgAcute kidney injury was considered an indication for de-resuscitation amongst a large portion of the Nordic physicians (45% out of 751). There might be a divide in the understanding of this question. Some respondents might interpret it the question as AKI either with or without fluid overload. Our intention was to inquire the indication for commencing de-resuscitation in the case of AKI and fluid overload. International guidelines from the Kidney disease: Improving global outcomes (KDIGO) suggests not using loop diuretics in the case of AKI except in the case of fluid overload . Given tThe Nordic physicians were inclined to intervene in the case of fluid overload in their patients. Diuretics were the most preferred method for de-resuscitation followed by minimization of maintenance fluids, resuscitation fluids, and drug diluents. A previous international survey distributed among three intensive care societies found similar results . RRT wasMost studies in the field of fluid therapy focus on resuscitation fluids but also maintenance fluids and fluid creep  are substantial sources of fluid accumulation . In one The evidence for using a combination of different types of diuretics is sparce for the general ICU patient . The exiA large portion of the respondents recognize increasing oxygen requirements and radiological findings as indications for commencing de-resuscitation, presumably because these are surrogate markers for ARDS . Current2/FiO2 ratio at 24 h  of the Nordic physicians were inclined to administrate intravenous albumin in the case of p-albumin less than 20 mM. Albumin is recommended in the case of ARDS in newer English guidelines  but not  at 24 h .The respondents were divided about treating fluid overload with loop diuretics in patients receiving noradrenaline. Few physicians would not administrate noradrenaline and loop diuretics simultaneously. The majority of respondents 63% had a fixed upper limit of noradrenaline dose between 0.05 and 0.5 mcg/kg/min and 35% of respondents found it patient dependent and did not have an upper limit. Another survey in the field found that 50% of the physicians had no fixed upper, the physicians from that survey were thus more liberal than the Nordic physicians . This prThis study has considerable strengths. The questions in our survey have previously been applied and pretested for validity and have been conducted on similar study populations. We performed our own pretesting, a clinical sensibility test, and an item reduction, and remodified the survey before distribution. Another strength is the number of respondents we received. The size of the participant population indicates a proportionate representation of the ICU physicians in Nordic countries and of their general views.The limitations of this study are the absence of a reliability test. Missing data were handled by case-wise deletion, and imputation might have given more precise results. Respondents of the survey might have stronger opinions or greater interest in the subject compared to the non-respondents, which could increase the non-response bias. The survey had 412 item non-responders which are respondents that neglected to answer one or more questions. All in all, there is a risk of significant non-response and potentially non-response bias in our study. We applied wave analysis to assess non-response bias by comparing how initial respondents answer the questionnaire compared to late respondents (proxies to non-respondents). The difference is interpreted on a five-point Likert scale, where two adjacent points  represent adjacent statements on the Likert scale e.g., “Strongly disagree” and “Disagree.” Non-response bias in the survey was estimated to be small between −0.06 and 0.26, which demonstrates similar attitudes in the two groups. It shows a low possibility of bias in the survey .The response rate in the current study was 28%. The distribution of the survey varied in the participating countries which is visible when comparing the percentage of physicians from each Nordic country. Our sampling strategy was relatively broad which could have resulted in distribution to physicians who might not work in the ICU and therefore were less likely to open the survey link. There can also be physicians who are employed in more than one ICU and potentially have received the survey invitation more than once. This demonstrates that the response rate might be skewed and would be more precise if the distribution had been more selective.Self-reported practices among Nordic ICU physicians when assessing, diagnosing, and treating fluid overload reveals variability in the practice. A 5% increase in body weight was considered a minimum to support the diagnosis of fluid overload. Clinical examination findings were preferred for assessing, diagnosing, and treating fluid overload, and diuretics were the preferred treatment modality.The raw data supporting the conclusions of this article will be made available by the authors, at a reasonable request.SW and EZ designed the study. EZ and MS-L conducted the statistical analyses. All authors made a contribution to the acquisition of the data, manuscript, and approved the final version for publication.


An emulsion template method was an effective way to prepare oleogels. However, there were few reports on how hydroxypropyl methylcellulose-pectin (HPMC-PC) mixtures affected the physicochemical properties of the obtained oleogels. In this study, the oleogels were prepared by an emulsion template method. The influences of HPMC and PC concentrations on the formation and physical properties of the emulsions and oleogels were investigated, by analyzing particle size distribution, microstructure, rheological test, oil loss, and crystallinity. The results of particle sizes and microstructure showed that a high concentration of HPMC and PC exhibited a better emulsification performance. The rheological tests indicated that a high concentration of HPMC and PC contributed to an increase in the mechanical strength of emulsions and oleogels. Moreover, an increase in an HPMC and PC concentration was beneficial to reduce the oil loss of oleogels. However, the change of HPMC and PC concentrations had no significant effect on the X-ray diffraction pattern of oleogels. This study could provide a theoretical basis for the construction of polysaccharide-based oleogels. Obesity caused by a high intake of fat increased the risk of diabetes and cardiovascular disease . TherefoAn oleogel could be regarded as an organic liquid trapped in a three-dimensional network, which had the features of solid fat . Lots of small molecule surfactants, such as waxes , monoglyHPMC is an amphiphilic polysaccharide with both hydrophobic methoxy groups and hydrophilic hydroxypropyl groups. HPMC exhibits good surface activity and can be used to prepare oleogels through the network structure. The oleogels constructed by HPMC in combination with thickening agents exhibited better gel properties, compared with those using a single component HPMC . Jiang ePectin (PC) is an acidic heteropolysaccharide extracted from plant cell walls and is commonly used for thickening, gelling, and emulsification . Most ofIn this work, the microstructure, rheological properties, oil retention, and X-ray diffraction of emulsions and oleogels were characterized to investigate the effects of polysaccharide concentrations on the formation and physical properties of oleogels. It could provide a theoretical basis for the practical application of oleogels in the food industry.The small droplets often aggregated with each other to form large oil droplets during the storage process of the emulsion system, resulting in the instability of emulsions. The effect of HPMC or PC concentrations on emulsions was investigated by analyzing microstructure and particle size distributions in this work. For emulsions where the concentration of HPMC was varied, the concentration of PC was kept constant at 0.1 wt%. For emulsions where the concentration of PC was varied, the concentration of HPMC was kept constant at 0.8 wt%. When an HPMC concentration was lower than 0.4 wt%, the phase separation phenomenon occurred during the drying procedure; when the concentration was higher than 1.0 wt%, HPMC particles were difficult to fully hydrate in the aqueous solution. The HPMC concentration in this experiment was selected from 0.4 wt% to 1.0 wt%. As shown in −1).The rheological properties of emulsions were further measured using a small-amplitude oscillatory shear (SAOS) rheometer. As shown in The process of preparing oleogels by the emulsion template method was shown in −1), proving that the oleogels were pseudoplastic fluids [As shown in c fluids . Moreovec fluids ,28. Furtc fluids ,30,31.−1 to 10 s−1, which meant that the connections between the oleogel particles could be broken with sufficient force, resulting in a decrease in flow resistance [In previous reports, the rheological properties of oleogels could vary greatly with the deformation of the structure ,33. Thersistance . Mert etsistance . Moreovesistance . A lower oil loss rate (OLR) is a desirable property of oleogels, which means a high ability to retain oil in its structure and suitable physical stability. The OLR of different oleogels were shown in In order to understand the structure of oleogels, the internal structures of polysaccharide powder and oleogels were determined and analyzed by X-ray diffractometer (XRD). The XRD patterns of HPMC powder, PC powder, and different oleogels were shown in w/w, %).The hydroxypropyl methylcellulose (HPMC) , with a viscosity of 400 mPa s at 2% aqueous solution at 20 °C was purchased from Shanghai Titan Scientific Co., Ltd. . Pectin from citrus peel was purchased from Sigma-Aldrich , its galacturonic acid content was more than 74.0% (in dry basis) according to the manufacturer’s instruction. The degree of methylation (DM) was 70.2 ± 0.4%. Medium-chain triglycerides (MCT) oil  were provided by Hainan Dabai Health Medicine Co., Ltd. All sample concentrations were expressed as a mass percentage  for 30 min. Then, 60 wt% MCT was added to the polysaccharide mixtures with different HPMC concentrations and the mixtures were sheared at 12,000 rpm/min for 2 min using a high-speed shearing homogenizer  to obtain the emulsions. The emulsions were placed in a vacuum drying oven  at 70 °C for about 36 h to obtain dried products with constant weight. Then, the dried material was sheared using a pulverizer  for 6 cycles of 5 s each to obtain oleogels.Polysaccharide mixtures with different concentrations of PC were obtained by mixing 0.8 wt% HPMC with different concentrations of PC  for 30 min to obtain polysaccharide mixtures with different PC concentrations. The following steps were the same as above.An appropriate amount of the emulsions was diluted in distilled water, and the particle size distribution and volume-average diameter  of the emulsions were measured using a laser particle size distribution analyzer . The refractive index of water, refractive index and light shielding ratio of the samples were set to 1.33, 1.44 and 8–12%, respectively. The reported results were the average of three parallel experiments at room temperature.Optical microscopy  was used to observe the microstructure of the emulsions. A total of 10 μL of the emulsion was placed on an optical microscope slide and the oil droplets were observed and recorded by a computer-built camera with a 40× magnifying glass.−1 and 10 s−1 for the oleogel samples. All measurements above were performed 25 °C. The reported results were the average of three parallel experiments.The rheological properties of the emulsions and oleogels were measured using a rheometer  with 35 mm plate geometry. The test gap between the plates was fixed at 1 mm for the emulsion and oleogel samples. During strain sweep testing, the elastic and viscous moduli were recorded at a constant frequency of 1.0 Hz with strain amplitudes ranging from 0.1–100% and 0.01–100% for emulsions and oleogels, respectively. The frequency was set from 0.1 to 10 Hz and the fixed strain was 0.5% in the frequency sweep experiment. The range of shear rate in flow measurements was set from 0.1% to 100%. Alternating shear rates of time sweep were 0.1 s1) and were centrifuged at 10,000 rpm for 15 min (4 °C) using a high-speed centrifuge . After centrifugation, free oil on the upper layer of the centrifuge tube was removed completely, and the oleogel samples after the removal of liquid oil were measured (m2). The oil loss was calculated by the following Equation (1).Oil loss was measured according to a centrifuge method . The oleThe diffraction spectrum of the sample was measured using an X-ray diffractometer  at room temperature. The range of diffraction angles was 5°–60° (2θ), and the scanning speed of the sample was 10 deg/min. The wavelength of the X-ray radiation was 1.540593.p < 0.05.The data were analyzed using SPSS. All experimental data were the average of three parallel experiments and the statistical significance was determined at In this work, oleogels were prepared by an emulsion template method and the effects of HPMC and PC concentrations on the formation and physicochemical properties of oleogels were investigated. The results showed that the higher concentrations of HPMC and PC exhibited better emulsifying capabilities. An increase in HPMC or PC concentrations improved the mechanical strengths of oleogels and was beneficial to reducing the oil loss of oleogels. The change of HPMC and PC concentrations had no significant effect on the X-ray diffraction pattern of oleogels. Furthermore, the release rules of free fatty acids and nutrients carried by oleogel during in vitro digestion and their controlled release capabilities of nutrients could be explored in the future.


Prompt appropriate antimicrobial therapy is crucial for suspected or confirmed invasive infections. The S. aureus (≤0.12 mg/L) was used for in vitro comparison only.587 CoNS isolates (1/patient) were consecutively collected in 30 US centers in 2017-2019. Bacterial identification was performed by MALDI-TOF, and susceptibility testing using CLSI broth microdilution methodology in a central laboratory. CLSI breakpoints were applied for comparators and the ORI susceptible (S) breakpoint for S. epidermidis , followed by S. hominis , S. capitis , S. lugdunensis , and S. haemolyticus . 12 other species represented < 10 (1.5%) isolates each. Overall, 59.1% of isolates were methicillin-resistant (MR), with the highest rate in Sepi (73.2%), followed by Shae (68.0%), Shom (46.8%), and Scap (30.2%). No MR isolates were detected in Slug. ORI  inhibited 96.1% of CoNS at ≤0.12 mg/L. Linezolid , daptomycin , and vancomycin  were also active against CoNS. ORI displayed similar MIC50 (0.03-0.06 mg/L) and MIC90 (0.12-0.25 mg/L) values against Sepi, Shom, Scap, and Shae, and inhibited 96.0%, 96.1%, 97.7%, and 84.0% of these isolates at ≤0.12 mg/L, respectively. All Slug isolates were inhibited by ORI at ≤0.015 mg/L. ORI inhibited 94.8% of all MRCoNS at ≤0.12 mg/L, and 95.5%, 94.4%, 92.3%, and 82.4% of MR Sepi, Shom, Scap, and Shae species, respectively. VAN, DAP, and LZD inhibited 100.0%, 100.0%, and 93.9% of MRCoNS isolates at their susceptible breakpoints, respectively.The most common species were ORI was highly active and inhibited ≥96% of all CoNS and individual species ( >10 isolates) at ≤0.12 mg/L, regardless of methicillin profile, except for Shae. VAN, DAP, and LZD were also active against CoNS causing BSI in US medical centres.Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support Jennifer M. Streit, BS, MT(ASCP), Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Office for Assistant Secretary of Defense for Health Affairs: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Spero Therapeutics: Grant/Research Support.


The aim of the study is to assess netarsudil's intraocular pressure (IOP)-lowering potential when prescribed as an adjunctive agent, to examine the effect of baseline IOP on patients' response to netarsudil, and to explore patients' characteristics predictive of pronounced responses to netarsudil.  This is a single-center, multiprovider retrospective cohort study set at Massachusetts Eye and Ear. Patients with a diagnosis of glaucoma or ocular hypertension on netarsudil and at least one other hypotensive agent for glaucoma who had at least one month of follow-up were included. Patients with additional procedures or glaucoma medication changes were excluded. The main outcome measures were IOP reduction, Kaplan–Meier survival analyses, netarsudil responder type, and complication rates.  236 eyes of 236 patients were included. The mean baseline IOP was 19.06 ± 4.6 mmHg on an average of 4 ocular hypotensive medications. 196 (83.1%) patients experienced IOP reduction at the first follow-up visit of 2.84 ± 0.30 mmHg at 55.66 ± 51.89 days. IOP reduction at the second visit among these patients was 3.01 ± 0.44 mmHg at 133.24 ± 77.63 days. After starting netarsudil, 59% had a sustained response (median duration of 315 days), 25% had a robust response (>20% IOP reduction for at least 80% of visits), and 10% had a super response (>20% and >10 mmHg IOP reduction). Netarsudil was effective as an adjunctive therapy across all baseline IOP categories with greater relative IOP reduction in higher baseline IOP groups.  Netarsudil is an effective adjunctive glaucoma therapy. IOP reductions between 2 and 3 mmHg are typical, but a minority had more pronounced and sustained effects (>10 mmHg). Further analysis is needed to assess specific demographic and clinical factors predictive of these robust responses. Netarsudil ophthalmic solution 0.02% , a Rho-associated protein kinase and norepinephrine transporter inhibitor, is a novel class of topical ocular hypotensive medical therapy introduced in 2017. It lowers intraocular pressure (IOP) by simultaneously decreasing trabecular meshwork resistance, aqueous humor formation, and episcleral venous pressure [When used as monotherapy, netarsudil's average IOP-lowering effect was 5.2–6.6 mmHg .p < 0.001) in patients on two or more ocular hypotensive medications, including netarsudil with 16.2% of eyes showing a sustained ≥20% IOP reduction during the first two follow-up visits. However, the magnitude of response to netarsudil addition remains unexplored. If known, these data may help providers anticipate the degree of IOP drop according to the patient's demographic and clinical data.The efficacy of netarsudil as adjunctive therapy, however, has only been recently explored in the literature. The MERCURY trials demonstrated that the fixed-dose combination (FDC) of netarsudil (0.01% and 0.02%) and latanoprost 0.005%) lowered IOP more effectively than its individual components , 5. Simi% loweredIn this study, we characterized netarsudil's IOP-lowering effect when used as a second, third, fourth, fifth, and sixth agent and characterized the response duration in a survival analysis. We also uniquely identified patient cohorts with a pronounced and sustained response to netarsudil, whom we designated as “Robust long-term responders” (>20% IOP reduction for at least 80% of visits) and “Super responders” (>20% and >10 mmHg IOP reduction). Such a significant IOP reduction has not been extensively reported to our knowledge in any clinical trials or case reports. Finally, we explored the characteristics associated with a favorable IOP-lowering response as well as reasons for netarsudil's discontinuation.This is a retrospective cohort study of patients with a diagnosis of glaucoma or ocular hypertension on netarsudil and at least one other hypotensive agent for glaucoma at Massachusetts Eye and Ear (MEE). Approval was obtained from the Mass General Brigham Institutional Review Board, and data collection abided by the Declaration of Helsinki and the Health and Portability and Accountability Act. Medical records of patients who were prescribed netarsudil by a provider at MEE between April, 2018, and August, 2020, were identified and reviewed. Records were included for analysis if they met the following criteria: (1) the presence of at least 2 IOP recordings prior to netarsudil initiation and (2) at least 1 month of follow-up without additional procedures or glaucoma medication changes. For patients treated with netarsudil in both eyes, the right eye was included in our study. Patients were included in the analysis until they experienced one of the following censoring events: netarsudil discontinuation, a change in the number of adjunctive hypotensive medications, intraocular surgery, or laser procedure. Self-reported medication adherence was recorded.The primary outcome measures were intraocular pressure (IOP) reduction (mmHg) from baseline and percent IOP reduction from baseline. Baseline IOP was calculated by averaging the 2 most recent IOP measurements prior to starting netarsudil. IOP measurements were performed with Goldmann applanation tonometry. IOP reduction following netarsudil initiation was assessed at various time intervals which were selected based on a statistical function that evenly distributes office visits across the five chosen time intervals: 1 to 39 days, 40 to 74 days, 75 to 124 days, 125 to 299 days, and 300+ days.The patients' response to netarsudil was defined according to the following designations Figures  and 2. “In addition, patients were further divided into different groups based on baseline IOP relative to the mean IOP, which was 19.06 mmHg. “Low” IOP was defined as IOP < 15, “Medium” IOP was 15 ≤ IOP < 20, “High” IOP was 20 ≤ IOP < 25, and “Very high” was defined as IOP ≥ 25 .t-tests on IOP reduction from baseline at specific time intervals or office visits. Secondary analyses included Kaplan–Meier survival analysis for failure to preserve >20% IOP reduction from baseline for two consecutive visits. Tertiary analyses were logistic regression model outcomes for the odds of a subject being classified as a given responder type. Logistic regression models were fitted using baseline data to model the log odds of subjects belonging to various responder groups of interest. Stepwise selection in both directions utilizing Akaike's information criterion was performed on each fully saturated model to determine a final, “best” model for each outcome. Statistical analysis was performed in R software.Primary analyses were unpaired p < 0.001), resulting in a significantly greater representation in the “Very High” IOP baseline category, 9 subjects , compared to the other responder groups (p < 0.001)  with 0.85% of patients falling in the 10–18 years age range, 22.88% in the 19–64 years, and 76.27% in the 65–98 years; 53% were female, and 70% were Caucasian Tables . The med< 0.001) .p < 0.0001) (p < 0.0001). Interestingly, nonresponders demonstrated a significant change as well after this visit, but with IOP increasing by an average of 22.44% ± 17.13% (p < 0.0001) . The IOP reduction at the first visit among all patients was 2.84 ± 0.30 mmHg, 14.34% ± 22.19% ( 0.0001) . When an 0.0001) .p < 0.0001). At this visit, long-term, robust long-term, and super responders all had a significant IOP reduction with long-term and robust long-term responders having an average reduction of 3.53 and 5.66 mmHg, respectively, and super responders having an average reduction of 12.30 mmHg  . IOP reduction at the second visit among all patients was 3.01 ± 0.44 mmHg, 14.94% ± 28.04% ( groups) .p < 0.0001 for each comparison), and the overall ANOVA was significant (p < 0.0001). Figures A Tukey test was also conducted to determine if the mean IOP reduction from baseline significantly differed between each responder type. Each of the pairwise Tukey comparisons was significant ) maintained >10 mmHg reduction for an average of 218.82 ± 285.59 days (7.19 ± 9.39 months). Failure to preserve the super responder status occurred when patients had an IOP reduction from a baseline of less than 10 mmHg at two consecutive office visits.p = 0.39), indicating that age was neither protective nor harmful to survival. We also stratified age into three groups 10–18 years, 19–64, and 65–98 and recalculated the Cox PH coefficient for age as a noncontinuous variable and got a similar result  model analysis was conducted to investigate the effect of age under the same Kaplan–Meier survival criteria. The Cox PH coefficient for age is 0.007 , and the overall ANOVA was not significant (p = 0.785). A stacked bar chart was generated to visually compare differences in IOP reduction for the number of glaucoma agents ; this indicates that those subjects with a “Very High” baseline IOP had a significantly greater mean IOP reduction than any of the other baseline IOP groups. The overall ANOVA was significant (p < 0.0001). Likewise, a Tukey test was conducted to determine whether the mean IOP reduction depended on the patient's baseline IOP. When comparing means, the “Low,” “Medium,” and “High” baseline IOP categories were each significantly different from the “Very High” baseline IOP category. The mean difference in reduction is −5.29 mmHg when comparing “Low” to “Very High,” −4.42 mmHg for “Medium” to “Very High,” and −3.38 mmHg for “High to “Very High” . It is important to note, however, the small sample size of patients in this responder group (n = 17) with only 1 patient having a “Medium” baseline IOP which reduces the certainty of the true effect size of this association. Finally, the odds of being a super responder was 3.127 (95% CI 0.984–11.078) for patients with a history of prior surgery compared to those without previous eye surgery with our results trending towards significance (p = 0.061).Secondary analyses for the odds of a patient being classified as a responder yielded a final, parsimonious model post-stepwise selection using AIC. The findings from five different multiple logistic regression models, representing each responder type, are seen in p = 0.034). In addition, the odds of being a robust long-term responder was 2.03 (95% CI: 0.922–4.386) times for patients with a glaucoma type other than POAG compared to those with POAG with our results approaching significance (p = 0.074).The odds of being a robust long-term responder was 9.353 (95% CI: 1.790–172.813) times that for subjects with a “Medium” baseline IOP categorization as compared to those with a “Very High” baseline IOP categorization, maintaining the other parameters as constant (p = 0.003), which was the highest in long-term responders.The adverse effects experienced across all patients and by responder type are summarized in In our retrospective cohort study, we reported netarsudil's IOP-lowering effect as an adjunctive agent. Our study found an IOP-lowering effect when prescribed as adjunctive therapy with an average reduction from baseline of 2.84 ± 0.30 mmHg (14.34% ± 22.19%) at the first follow-up visit and 3.01 ± 0.44 mmHg (14.94% ± 28.04%) at the second follow-up visit. Our results are consistent with those of previous studies exploring netarsudil's efficacy as a monotherapy and adjuvant therapy. The ROCKET-1 monotherapy clinical trial reported IOP lowering of 3.3–5.0 mmHg (17–22%) at 3 months . That stOur study included a longer follow-up period compared to the aforementioned studies and thereby offered important insights when assessing the durability of netarsudil's effect. We found that patients experienced a sustained hypotensive effect lasting well beyond one year, with survival analysis demonstrating a 50% probability of preserving >20% IOP reduction at 400 days. Moreover, 59% (144/244) of all patients in the study experienced a sustained response (IOP reduction from baseline at >80% of office visits) to netarsudil. Explanations for the durability of netarsudil's IOP-lowering effect include potential trabecular meshwork remodeling –12 and ip = 0.061) , referred to in this study as “super responders,” who demonstrated ≥10 mmHg IOP reduction from baseline as well as a sustained ≥20% IOP reduction at >80% of follow-up visits. Such a pronounced IOP reduction has not been previously reported to our knowledge in clinical trials or case reports. Mounting anecdotal accounts from providers with standout cases of pronounced netarsudil hypotensive effect warrant further exploration of baseline characteristics predictive of such a response. Our logistic regression analysis identified that among the characteristics contributing to the super responders' 12.1 mmHg (45.7%) IOP reduction from baseline was having a “Very High” baseline IOP (IOP ≥ 25) and a history of prior eye surgery (= 0.061) . The logWe also noted that the degree of IOP reduction was not affected by the baseline number of hypotensive agents . This isIOP inclusion criteria for this study were broad (mean baseline IOP: 19.06 ± 4.6). Netarsudil's adjunctive efficacy was apparent across all baseline IOP categories with greater relative IOP reduction in the “High” and “Very High” baseline IOP groups (38.3% and 53.84%) relative to the low and medium groups (24.53% and 28.3%). There are few prior studies that have explored the role of baseline IOP and its effect on netarsudil efficacy. In the FDA clinical trials for netarsudil and netarsudil/latanoprost FDC, Brubaker et al. found a greater proportion of patients with ≥20% IOP reduction in patients with baseline IOP ≥ 25 mmHg when compared to those with baseline IOP < 25 mmHg . Some ofSimilar to prior studies, we found that netarsudil was well tolerated in most patients , 22. In Our study has several limitations, including the absence of a control group. In addition, given that this study was not a clinical trial and instead was aimed at investigating netarsudil's efficacy in a real-world glaucoma practice, there was no wash-out period; therefore, the hypotensive effect cannot be entirely attributed to netarsudil. Furthermore, while the study did control for changes in the glaucoma regimen with its stringent inclusion criteria, it did not account for changes in the glaucoma regimen coinciding with netarsudil's initiation, such as the concurrent discontinuation of other glaucoma medications. Finally, there was no practical way to distinguish normal diurnal fluctuations of IOP from the IOP measured in the clinic as this study focused on the real-world use of netarsudil as an adjunctive medication and was thus subjected to the constraints of regular daily clinic hours which start at 7:00 am and end at 7:00 pm EST.Our results support the use of netarsudil as an adjunctive treatment that can provide significant and sustained IOP lowering with once-daily dosing for patients across a broad baseline IOP range and number of baseline medications. Future studies should aim to create more explicit terminology that considers criteria related to netarsudil response over a longer follow-up period as well as further assesses specific demographic and clinical factors predictive of these robust responses.The research was conducted as part of the employment of the authors.


NF2. Previous mathematical models of schwannoma incidence have not attempted to account for alterations in specific genes, and could not distinguish between nonsense mutations and loss of heterozygosity (LOH).A vestibular schwannoma (VS) is a relatively rare, benign tumour of the eighth cranial nerve, often involving alterations to the gene Here, we present a mechanistic approach to modelling initiation and malignant transformation in schwannoma. Each parameter is associated with a specific gene or mechanism operative in Schwann cells, and can be determined by combining incidence data with empirical frequencies of pathogenic variants and LOH.u = 4.48 × 10−10 and the rate of LOH = 2.03 × 10−6/yr in Schwann cells. In addition to new parameter estimates, we extend the approach to estimate the risk of both spontaneous and radiation-induced malignant transformation.This results in new estimates for the base-pair mutation rate We conclude that radiotherapy is likely to have a negligible excess risk of malignancy for sporadic VS, with a possible exception of rapidly growing tumours. NF2 and at least one other genetic hit , 74pLOH possible . Such stntiation .Fig. 7ReTP53 in glioblastoma multiforme (GBM). Copy number losses on 17p and coding mutations in TP53 occur with a similar frequency, fLOH (17p) ≈27%  (on the same loci) must at least have similar orders of magnitude for alterations to occur with such similar prevalence. Furthermore, the same study observed copy number losses on 22q in 44% of cases, which is consistent with a role for NF2 in GBM [TP53 are unheard of in vestibular schwannoma [As a result of the efforts of the Pan-Cancer Analysis of Whole Genomes Consortium (PCAWGC), a wealth of gene- and tumour-specific data on the prevalence of LOH is now available , 78. Whip) ≈27%  Fig. 3g)TP53 in g2 in GBM . Convershwannoma .TP53 must occur “early” rather than “late”, and LOH on 22q must occur “late” in GBM [NF2 in GBM, it is therefore likely to be a relatively “late” driver. In contrast, our model for malignant transformation in vestibular schwannoma suggests that the last alterations before malignancy should be in an unidentified tumour suppressor (or suppressors) TSX, with NF2 already being inactivated or lost in the benign tumour. If there is any role for TP53 in malignant schwannoma, it can therefore be expected to be “late”, with NF2 being “early”. This is the reverse of the case in GBM [TP53 has been speculated to be involved in the malignant transformation of VS [This same work of the PCAWGC found that these hits to ” in GBM . If there in GBM . Loss-ofon of VS .GFX, nGFX, was found to be 2002. This high value may indicate that there are several oncogenes that contribute to sporadic incidence, representing a high level of genetic diversity. This is also likely to be a slight overestimate of the real figure, and may be revised once LZTR1 is taken into account.The number of sensitive sites on the third hit nGFX was found to be bimodal, with two peaks around 700 and 2000 . If at least 4 Gray can be delivered each cell cycle, the excess risk of malignancy should be less than the expected lifetime risk, even for pessimistic estimates of see Fig. .Slow-growing schwannomas can probably be treated safely at a minimum dose rate of 1 Gray per day. Faster growing tumours could justify more aggressive treatment: the 3 mm/yr cases from Paldor et al. 2016 might require 4 Gray per day to ensure that we are on the right side of the peak risk from Fig. This does assume that solid growth can be reliably distinguished from inflammation and “pseudo-progression”, which may not always be possible.For completeness, we also studied the “worst case scenario” in which fractionated therapy is delivered in independent doses several days apart. This would maximise the chance that tumour cells “recover” from the deterministic effects of cell death, and go on to divide again. This should not be the case in practice .F ≈10 fractions, and may grow as high as ≈10%  could be improved upon. This may affect our conclusions regarding the “worst-case scenario”, but probably not best practice.These results must be qualified by the uncertainties in the identity of the hypothetical tumour suppressor (or suppressors) patients . In addiIn addition to improved estimates of the underlying mutation rates and dynamics of LOH in Schwann cells, our mechanistic approach to modelling schwannomas has enabled new theoretical estimates of the excess risk of malignant transformation in radiosurgery. This shows that the modelling approach of branching processes on graphs generalises to other neoplasias, enabling new connections and inferences to be made . FurtherLZTR1 as one of the first hits, as well as explicitly studying multiple candidate third-hit oncogenes. This could be coupled with a comprehensive study of copy number alterations in VS biopsies, as well as the status of SMARCB1, NF2 and LZTR1. This might be achieved by coupling a copy number analysis with an SNP array, or a higher resolution approach leveraging next-generation sequencing [fgene to those predicted by the model.Firstly, a better picture of genetic diversity in sporadic VS may be achievable with a more detailed model. This more detailed mechanistic model could include quencing –85. A mou does not distinguish between transitions and transversions, and the underlying model assumes all substitutions are equally likely [u reported here.One important limitation of our approach is that the mutation rate y likely , 29. Dify likely . In the y likely . This woTSX. Our estimates of nTSX in section “Parameter estimation” suggest that a promising experimental approach could be to look for LOH in at least ten samples of malignant schwannomas. This might uncover new risk factors, new roles for risk factors known from other tumours such as TP53, as well as markers of malignant transformation. The rarity of malignancy in vestibular schwannoma could also provide an opportunity to observe malignant transformation “in slow motion”, with a large and definite reference population of benign tumours.When applied to malignant transformation, our approach showed that the observed lifetime risk can be explained by a simple two-hit model in the growing tumour see Fig. . This thThe extension of this modelling approach to radiotherapy suggested that at tumour sizes and radiation doses typical of therapy, the excess risk of malignancy for sporadic schwannoma is negligible. Cells that receive high doses, and thus have the highest likelihood of induced mutations, will be the least likely to survive. Stochastic effects, which include malignancy, should peak at intermediate doses of around 1 Gray. Above about 4 Gray, deterministic effects dominate, and cell survival falls off very rapidly. This dose is well below recommended prescriptions , 89.The excess risk should also be very small for fractionated therapies, as long as dose rates are higher than 4 Gray over the course of one tumour cell cycle, which we estimate at around 4 days (see section “Dose fractionation”). This is broadly in line with current recommendations. Current clinical practice is therefore expected to have a negligible excess risk of radiation-induced malignancy, consistent with previous empirical studies , 9, 86.The highly pessimistic worst-case scenario in which tumour cells fully recover in between doses should be completely avoidable if the most rapidly growing tumours are treated more aggressively (3mm/year expansion = 4 Gray/day) than more common cases. This pessimistic scenario, detailed in section “Dose fractionation”, is unlikely. Nonetheless, it may be interesting to investigate whether there is an association between malignancy and failure to complete treatment. It should also be cautioned that we do not consider cases of familial NF2.These findings need to be qualified by the uncertainties both in the identity of the relevant genes. These are questions for which experimental answers are urgently needed. As well as revealing new connections between genomics, epidemiology, and microscopic mechanisms, this work underscores the critical need to identify the genes responsible for both tumour initiation and malignant transformation.counter.pybootstrap.pyMathematical and methodological appendicesReproducibility checklistLegends to the supplemental material


Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of  Historically, natural products extracted from plants, herbs, animals, and microorganisms have been a reliable source of bioactive molecules for pharmaceutical discovery. Despite this complexity, recent technological advances and the advent of new methods for high-throughput screening have contributed to the resurgence of pharmaceutical interest in natural products or their direct derivatives for developing new drugs . Plant eAmblyomma cajennense, currently Amblyomma sculptum  generation  and first (TFPI-2 KD1) KDs of TFPI-1, and TFPI-2, respectively, to evaluate its relatedness to TFPI-like inhibitors . As expeee loops  (Mesquitee loops . Althougee loops .via a substrate-like mechanism (NCBI Conserved Domain Database CDD:238057).The charge distributions at the proteins’ molecular surfaces were inspected because electrostatic interactions are particularly important for protein-protein recognition/interaction . This anIn this context, it is worth mentioning that even minor alterations in the L1 loop can modify the molecule’s inhibitory ability/specificity, not only in this region. For instance, these differences can, at least in part, help explain why TFPI-2 presents as a distinct inhibitory target from TFPI-1 .Ornithodoros moubata is a low molecular weight Kunitz-related FXa inhibitor  . The secThe KD of Amblyomin-X is more similar to Ixolaris K2 , 3B, butIt is important to consider Amblyomin-X as a whole molecule, presenting an additional C-terminal portion near the size of its KD. Amblyomin-X’s C-terminal lacks homology to proteins in public databases, and it is assumed that it is potentially unstructured and possibly very flexible. Regarding Amblyomin-X’s inhibitory features, the C-terminal can potentially favor and/or modulate interactions in the whole molecule, which can’t be assessed by analyzing just the structured portion of the molecule. Additional experimental approaches will be used in order to clarify the contribution of the C-terminus to Amblyomin-X activities.Hence, it is reasonable to assume that Amblyomin-X is a peculiar molecule that carries a particular KD at the N-terminal and a unique C-terminal. At the moment, the elements regarding protease inhibition and antitumoral mechanisms of action are still under investigation.Hematophagous animals contain several molecules with inhibitory activity towards serine proteinases or coagulation system complexes, in addition to vasodilators and platelet aggregation inhibitors . These mA. sculptum (former A. cajennense) is classified as a hard tick and is one of the most studied because of its ability to transmit the bacterium Rickettsia rickettsii, which causes the Brazilian spotted fever (BSF), the most lethal rickettsiosis in Brazil  as a 13.5 kDa inhibitor composed of a single Kunitz-type homologous domain in the N-terminal and a C-terminal with no similarity to any protease inhibitor or other sequences (The recombinant Amblyomin-X was expressed in equences . Like otequences .in vitro). In ex vivo experiments, PT and APTT in Amblyomin-X-treated mice were not altered at doses of 1 mg/Kg, although a higher concentration of the inhibitor (2 mg/Kg) altered APTT , prothrombin time (PT), and procoagulant activity (PCA), in assays using human plasma  and primary fibroblasts, Amblyomin-X induced time- and concentration-dependent death, cell cycle arrest, and apoptosis only in tumor cells. Amblyomin-X did not affect non-tumor cells, suggesting its selectivity for cancer cells. Furthermore, in an in vivo assay, Amblyomin-X treatment (1 mg/kg daily for 14 days) induced tumoral mass regression and a considerable reduction in the percentage of internal metastasis in murine melanomas generated with the B16F10 cell line. Metastatic nodules in the lungs, kidneys, and lymph nodes decreased by 60% in treated animals compared to the metastatic lesions distributed in the internal organs of the control group  and small GTPase production and MMP-9 secretion, leading to disruption of the actin cytoskeleton and reduced cell migration . Anotherin vivo assay in a mouse renal orthotopic model demonstrated that Amblyomin-X treatment significantly inhibited metastasis formation, and histological analyses showed that Amblyomin-X cytotoxicity was restricted to the tumor area, reinforcing the selective anti-tumor effect of the in vivo treatment (in vivo results were consistent with those of previous studies. The results demonstrated that after 21 days of daily treatment with Amblyomin-X (1 mg/kg), the EPN xenograft model displayed significant tumor mass regression compared to the control  expression in endothelial cells. PECAM-1 expression levels are important markers in endothelial cell-cell junctions, indicating tube organization during new vessel formation . The effin vivo and in vitro assays to explore the molecular Amblyomin-X action mechanisms on endothelial cell functions during angiogenesis. Using a dorsal chamber model, this study showed that Amblyomin-X reduced angiogenesis without any biological or chemical stimulation, such as VEGF-A. Mice dorsal skin was topically treated with Amblyomin-X . The treatments were administered once a day, every two days, for a total of three applications. The local angiogenic effects of Amblyomin-X on dorsal subcutaneous tissue were measured by intravital microscopy and PECAM-1 labeling. The data obtained showed that topically applying Amblyomin-X significantly reduced the number of vessels in the subcutaneous tissue of mice compared to phosphate-buffered saline (PBS) treatment, corroborating the results of a previous study  and prostaglandin E2 (PGE2). These results indicated that Amblyomin did not affect vascular permeability or endothelial contractile mechanisms.The authors also investigated the effects of proteins on vascular permeability. Intradermally injecting Amblyomin-X  did not affect the microvascular permeability measured by Evans blue extravasation into the tissue. Furthermore, treating t-End endothelial cells with Amblyomin-X  in vitro. Analysis of tube formation showed that Amblyomin-X treatment (100 ng/ml) reduced the organization of new vessels in Matrigel® in t-End cells. Treatment with Amblyomin-X (100 ng/ml) reduced the expression of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and β3 integrin, with no alterations in PECAM-1 or β1 integrin expression levels.Moreover, the authors showed that Amblyomin-X (10 ng/ml and 1000 ng/ml) treatments reduced cell migration and adhesion in the Matrigel® matrix (10 and 1000 ng/ml) Together, these findings highlight that Amblyomin-X has antiangiogenic properties that directly reduce neo-vessel formation and inhibit VEGF-A-induced angiogenesis. Amblyomin-X reduces endothelial cell migration and tube formation and modulates angiogenic adhesion molecules, such as VCAM, integrins, and PECAM (induced by VEGF). The molecular mechanisms underlying these effects and their roles in antitumor activity need to be explored and clarified.Amblyomin-X is a promising drug candidate for cancer treatment because of its antitumor potential. Before human clinical trials, new drug candidates must undergo preclinical animal studies to predict drug behavior in patients and ensure their safety profile . BoufeurAmblyomin-X was observed in the plasma 15 min after injection and was detected for up to 60 min. However, it was not detected in plasma samples collected 24 h after repeated daily administration. The surface plasmon resonance assay showed that Amblyomin-X could not bind to albumin, corroborating its rapid clearance from plasma because albumin is the main protein responsible for drug transportation in the blood . In addi−1. AUC values are expected to be higher in humans because small animals possess higher metabolic rates , followed by near-infrared photoimmunotherapy (NIR-PIT) of tumor lead to the death of cancer cells  through its first Kunitz-domain  after 24 h, the lethal dose (LD50) was not determined. While there were no deaths in the acute dose group treated with a dose range of 0.25 mg/kg to 256 mg/kg, doses higher than 64 mg/kg promoted alterations in motor and sensorial signals that lasted until day six after treatment. Animals in the subacute dose group treated with 1 mg/kg of Amblyomin-X also showed motor alterations that disappeared after treatment . CompareNone of the groups showed any irreversible body mass loss. Despite a slight decrease in platelets and leukocytes in the acute dose group after 24 h of treatment, which was reversed after day 14, there were no alterations in blood cell morphology, coagulation, or PT in either group. Alterations in platelet and leukocyte counts observed at the acute dose were also observed upon bortezomib treatment . While AFinally, histological analysis of the internal organs  showed no alterations upon Amblyomin-X treatment, except with the 256 mg/kg dose after 24 h of treatment, which was reversed after 14 days. These results enabled the determination of the maximum tolerable dose and the dose with no observed adverse effects, 16 mg/kg and 0.57 mg/kg, respectively, showing that Amblyomin-X is a promising drug with low toxicity and reversible side effects .in vitro and likely in vivo.In 2010, Chudzinski-Tavassi et al. conducted one of the first studies to explore the molecular mechanism by which Amblyomin-X drives tumor cell death. In this study, microarray analysis showed that 24 genes were modulated in human melanoma (SK-MEL-28) and human pancreatic adenocarcinoma (Mia-PaCa-2) cells after treatment with Amblyomin-X. Among these genes, the most upregulated was dynein cytoplasmic 1 light intermediate chain 2 (DYNC1LIC2), followed by proteasome beta-type subunit 2 (PSMB2) . AmblyomProteasomes are multimeric proteolytic complexes responsible for degrading ubiquitinated proteins . Correctvia interaction with the LIC2 of dynein  activation assisted by dynein transportation , mitochondrial dysfunction, poly adenosine diphosphate-ribose polymerase (PARP) cleavage, and caspase-3 activation in tumor cells. Interestingly, few or no changes were observed in the normal human and mouse fibroblasts. Amblyomin-X did not induce PI, ER stress, or ROS production in fibroblasts, highlighting the selectivity of this molecule for tumor cells  stress-related cell death , the effpoptosis . The samor cells . Non-tumor cells . The mecor cells . In geneor cells  and alsoor cells , immune or cells  and blooor cells . In contor cells . PrelimiAmblyomin-X is a protein that selectively leads to cell death by inducing ER stress, caspase activation, mitochondrial dysfunction, and PI in tumor cells .STX17 (syntaxin-17) causes the tumor phenotype, which constitutes a cis-acting regulatory mutation (BRAF and NRAS, as well as the newly discovered PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2 (A recent study showed that this molecule could significantly reduce melanoma progression in horses . Mutatiomutation . Mutatednd ARID2 . Althougnd ARID2 .In vivo treatment of encapsulated horse melanomas via intratumoral Amblyomin-X injections significantly reduced tumor size (mor size . Histolomor size . Since mmor size . EnrichmMetaCore analyses showed that the “immune system” was the class with the most enriched pathways, followed by the “innate immune system,” “inflammation,” “cancer,” and “adhesion/ECM/cytoskeleton” . SimilarLichtenstein et al. (According to n et al. , in silin et al. . It is pn et al. . In summn et al. .Cellular interaction partners of Amblyomin-X were identified by interactome analysis, which showed that it potentially interacts with key transcriptomic elements . The equAmblyomma sculptum, which displays anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X presents a Kunitz-type domain that shares ∼40% similarity with the Kunitz domains of endogenous TFPI and Ixolaris, a well-known FXa inhibitor from Ixodes scapularis. Amblyomin-X’s cytotoxic activity in tumor cells and its ability to reduce tumor growth and metastasis in in vivo models has led to the vast exploration of its functions, action mechanisms, structures, pharmacokinetics, and biodistribution. Amblyomin-X drives tumor cell death via PI and appears to play a role in the tumor microenvironment by activating the immune system against tumor progression. A remarkable feature of this recombinant protein is that it seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making it an attractive molecule for anticancer therapy, where the challenge is not only to suppress tumor growth but also to decrease treatment side effects. Therefore, considering the current efforts to develop effective anticancer therapies, Amblyomin-X could be a promising new antitumor drug candidate.Amblyomin-X is a recombinant protein derived from a cDNA library prepared from the salivary gland of the tick


Background#OrthoTwitter has evolved to disseminate findings and engage the public. However, the academic impact of Twitter utilization in orthopaedic surgery is unknown.Questions/purposesThe purpose of the study was to evaluate relationships between the author and manuscript Twitter activity and citations.MethodsManuscripts in 17 orthopaedic journals from 2018 were identified. Citations, online mentions, impact factors, and subspecialties were obtained. H-index and Twitter account details for authors were obtained for a subset of manuscripts. Relationships between Twitter activity and citations were evaluated.Resultsβest=0.13, p<0.05), Twitter mentions , impact factors , and having a first  or senior author  on Twitter. Articles published in arthroplasty , general interest , sports , and non-open access journals  were cited more. H-index correlated with followers for first  and senior authors .2,473/4,224 (58.5%) manuscripts were mentioned on Twitter , with Twitter manuscripts cited more frequently . Twitter mentions, impact factors, non-open-access status, and subspecialties were associated with citation counts. Articles mentioned in 10, 100, and 1,000 Tweets were observed to have a 1.1-fold, 1.7-fold, and 245-fold increase in citations. In author-level analyses, 156 (20.0%) first and 216 (27.7%) senior authors had Twitter accounts. Citation count was associated with increasing senior author H-index (ConclusionAuthor Twitter utilization is independently associated with manuscript citations. Authors should be aware of the potential association between social media utilization and traditional academic impact. Understanding the relationship between social media utilization and academic impact is necessary to effectively disseminate research. Social media is transforming the way academic medical research is shared. While articles are shared on a variety of platforms, more than four out of five online mentions of academic manuscripts occur on Twitter . With ovOrthopaedic surgery is also comprised of multiple diverse subspecialties - each with its own subset of journals and communities - which may influence the relationship between Twitter utilization and citations. While citation counts and online mentions have previously been reported for hand surgery  and sporFurthermore, while alternative metrics  have been proposed to modernize the estimated impact of manuscripts , there iUnderstanding the association between social media utilization and academic impact and how this association may vary across subspecialties is increasingly necessary to effectively disseminate research and assess research impact. The purpose of the study was to evaluate the relationship between citation count and both manuscript-related Twitter activity and author Twitter utilization among orthopaedic surgery subspecialties. We hypothesized that author Twitter utilization and manuscript-related Twitter activity would be associated with greater citation counts and that there would be variability among orthopaedic subspecialties.Data collectionBecause this study utilized publicly available data, no Institutional Review Board approval was sought. No funding was received for this research.Article Selectionhttps://pubmed.ncbi.nlm.nih.gov/) [Seventeen high-impact orthopaedic surgery journals, representing a range of subspecialties, were selected. Open access status, subspecialty, and impact factor were recorded Table . Articleih.gov/) . The autih.gov/) ,12. Artiih.gov/) .Article Online Activitywww.altmetric.com) is an online platform and service that queries various online sources, including Twitter, Facebook, and mainstream media, for article mentions according to the National Information Standards Organization Data Quality Code of Conduct. Altmetrics automatically collects Tweets, Retweets, and quoted Tweets that contain a direct link to manuscripts. Twitter likes are not included. Altmetrics.com was selected as a source because it has been shown to have superior reliability in identifying Tweets compared to alternate sources [rAltmetric R package was used to query the Altmetrics database for the AAS and online article mentions [Altmetrics  was employed to obtain author-level account details from the author account handles, including the number of account followers, the number of accounts followed, the number of tweets, and the account date of creation.Statistical analysesData were analyzed using R version 4.1.2  . NormallThe relationship between citations and manuscript-level data was assessed for the full set of manuscripts. The relationship between citations and manuscript- and author-level data were assessed for the specified randomized subset of 20% of manuscripts. Negative binomial logistic regression models were generated via generalized linear modelling to assess the relationship between citation count and author- and manuscript-level data. Negative binomial logistic regression models were selected as citation counts were over-dispersed. P-values were generated using Wald tests. In this approach, the presented beta coefficients represent the expected change in the natural log of citation count for a given one-unit increase in the predictor. A forced entry approach to variable selection was employed because associations between citation count and h-index, open access status, journal impact factor, Twitter mentions, and subspecialty have previously been suggested or hypothesized ,18,19. FFinally, the relationship between citations and author Twitter account details was assessed for both first and senior authors. Twitter account details were compared between first and senior authors, including the association between H-index and Twitter followers. The ratio of Twitter followers to H-index was compared among subspecialties .After exclusion criteria were applied, 4,224 manuscripts remained, which had been cited a total of 53,635 times. Of the articles, 62.1% were mentioned online, and 94.2% of these  were mentioned on Twitter , journal impact factor , and non-open access status  were associated with greater citation count. For purposes of interpretation, the log of the number of citations was observed to increase by 0.0055 with each additional tweet mentioning the article. For context, 10 tweets would be associated with a 1.1-fold increase in citations, 100 would be associated with a 1.7-fold increase, and 1,000 tweets would be associated with a 245-fold increase. Compared to articles published in pediatrics-related journals, articles published in all other subspecialty-related journals except trauma received more citations  was selected for author-level analyses after exclusion. Three hundred and twenty (41%) of these manuscripts featured either a first or senior author with a Twitter account, with 156 first-author accounts and 216 senior-author accounts Table . Articleβest = 0.13, p < 0.05), number of Twitter mentions , journal impact factor , and having a first  or senior author  with a Twitter account. Compared to pediatrics-related journals, articles published in arthroplasty , general interest , sports-related journals , as well as non-open access articles , were associated with more citations , the number of following accounts , and the number of tweets . Among senior authors, there was a slight positive correlation between citations and the number of account followers , but there was no significant correlation between citations and tweets  or following accounts . There was also no relationship between account age and citations for either the first  or senior  authors.Senior authors had higher H-indices than the first authors . Senior author Twitter accounts had more followers , posted more tweets , and had older Twitter accounts . First and senior author accounts were following similar numbers of accounts . The correlation between the H-index and Twitter followers was fair  for the first authors and moderate  for senior authors . This corroborates a recent study that found arthroplasty, oncology, and sports medicine to be the three highest-cited orthopaedic subspecialties . AdditioWhile Twitter previously accounted for 82% of the online article mentions in orthopaedics , the preThe study corroborated the findings of prior studies by demonstrating positive correlations between Twitter mentions (rho = 0.25) and AAS rho = 0.31) and citations. In 2017, Evanvew et al. analyzed the relationship between AAS and citations for 1,675 randomized controlled trials and estimated the rho to be 0.11  and cita. Also inInterestingly, articles published in open-access journals received fewer citations, even when controlling for the impact factor. Prior literature on the impact of open access status has been conflicting. For example, Silva et al. identified a weakly positive association among sports medicine articles , but an The strengths of this article include the incorporation of author-level social media utilization, a large sample size of manuscripts, and the inclusion of a variety of journals representing different subspecialties. There are several limitations to our analysis of the author Twitter profiles, however. For example, some authors may run pseudonymous Twitter accounts that cannot be identified. The content of the author's tweets may also be unrelated to medicine. The exact mechanism of the Twitter feed-generating algorithm is not known and may bias the presentation of certain articles. Finally, an author's propensity for Twitter utilization may not be independent of other personal or professional factors related to academic productivity. Additionally, aside from applying exclusion criteria to certain types of articles, this study did not adjust for specific study design factors such as sample size or risk of bias. Finally, journal-specific Twitter activity and other factors may also be associated with citations ,24. UltiAuthor Twitter utilization is associated with academic citation count independently of other author and manuscript factors. Future studies should examine the content of tweets, the scientific quality of manuscripts, and the Twitter activity of specific journals. As research dissemination rapidly evolves online, authors should be aware of the impact of social media utilization on academic impact in order to effectively share findings and assess research impact.Twitter utilization and manuscript citations are independently associated with authors who publish in orthopaedic journals. Those who appreciate this potential link between social media implementation and traditional academic impact can benefit from more effective dissemination of their research. Additionally, understanding this association is essential for appraising the impact of evolving online platforms for research circulation. Future studies should examine Twitter activity within specific institutions and journals, the content of tweets, and the scientific quality of the manuscripts shared on the platform.


Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-β-D-glucanase activity was confirmed in the supernatant of the insect’s digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications.Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in  Hoplasoma unicolor.Presence of cellulolytic endoglucanase activity was detected in digestive fluid of the beetle Quantitative estimation showed a relatively high cellulase activity with specific activity measured as 0.69 (±0.01) units per mg of protein.Zymogram analysis revealed the presence of two active cellulolytic proteins in the gut extract.Cellulases are hydrolytic enzymes that catalyse the cleavage of 1,4-β-glycosidic bonds between glucose residues present in cellulose, the most abundant biopolymer produced on earth and the major constituent of agricultural and industrial wastes  as standard (The enzyme extract was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method as described by We carried out zymogram analysis to detect the specific proteins having cellulolytic activity in the gut extract as described in a previous work  assay . Enzyme H. unicolor. To this end, we collected digestive fluid from 20 adult insects and obtained its protein fraction by centrifugation. This partially purified gut extract was examined for the presence of endoglucanase activity in an agar plate assay using CMC as the substrate. A distinct zone of clear halo was formed in the agar media when spotted with the gut extract that clearly indicated depolymerisation of CMC in the media confirming presence of cellulolytic proteins in the sample [H. unicolor, to that list. Although most of the previous studies didn’t confirm whether the respective enzymes are encoded within the insect genome or of microbial origin . The gut extract was used at two different concentrations, 0.8 μg or 4 μg of protein, in the analysis. Five distinct protein bands were detected at both concentrations with the MW ranging from ~25 to 65 kDa , suggestTribolium castaneum, Syrbula admirabilis and Tenebrio molitor  U/mg of protein which seems quite high as compared to those reported in the insects of the same order  and in oH. unicolor. The endo-1,4-β-D-glucanases of the gut fluid showed prominent efficiency in digestion of cellulose with specific activity higher than those of most other insects. Further research for purification and complete characterisation of the endoglucanases are, therefore, needed to understand their origin and catalytic efficacy which can provide foundation for development of an efficient and inexpensive system for biofuel production and waste management based on the hydrolytic enzymes of insect digestive fluid.Findings of this paper represent the first description and preliminary characterisation of the cellulolytic activity in the digestive fluid of