CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations.

Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight the risks associated with genome editing techniques involving dsDNA breaks in E. coli and likely other bacteria and emphasize the importance of sequencing the genomes of edited cells to ensure the absence of unintended mutations.

Neuropsychological task outcomes among survivors of childhood acute lymphoblastic leukemia in Malaysia.

This study intended to explore the neuropsychological ramifications in childhood acute lymphoblastic leukemia (ALL) survivors in Malaysia and to examine treatment-related sequelae. A case-control study was conducted over a 2-year period. Seventy-one survivors of childhood ALL who had completed treatment for a minimum of 1 year and were in remission, and 71 healthy volunteers were enlisted. To assess alertness (processing speed) and essential executive functioning skills such as working memory capacity, inhibition, cognitive flexibility, and sustained attention, seven measures from the Amsterdam Neuropsychological Tasks (ANT) program were chosen. Main outcome measures were speed, stability and accuracy of responses. Mean age at diagnosis was 4.50 years (SD ± 2.40) while mean age at study entry was 12.18 years (SD ± 3.14). Survivors of childhood ALL underperformed on 6 out of 7 ANT tasks, indicating poorer sustained attention, working memory capacity, executive visuomotor control, and cognitive flexibility. Duration of treatment, age at diagnosis, gender, and cumulative doses of chemotherapy were not found to correlate with any of the neuropsychological outcome measures. Childhood ALL survivors in our center demonstrated significantly poorer neuropsychological status compared to healthy controls.

Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Differential structure and function of phosphorus-mineralizing microbial communities in organic and upper mineral soil horizons across a temperate rainforest chronosequence.

Microbial community structure and function were assessed in the organic and upper mineral soil across a ~4000-year dune-based chronosequence at Big Bay, New Zealand, where total P declined and the proportional contribution of organic soil in the profile increased with time. We hypothesized that the organic and mineral soils would show divergent community evolution over time with a greater dependency on the functionality of phosphatase genes in the organic soil layer as it developed. The structure of bacterial, fungal, and phosphatase-harbouring communities was examined in both horizons across 3 dunes using amplicon sequencing, network analysis, and qPCR. The soils showed a decline in pH and total phosphorus (P) over time with an increase in phosphatase activity. The organic horizon had a wider diversity of Class A (phoN/phoC) and phoD-harbouring communities and a more complex microbiome, with hub taxa that correlated with P. Bacterial diversity declined in both horizons over time, with enrichment of Planctomycetes and Acidobacteria. More complex fungal communities were evident in the youngest dune, transitioning to a dominance of Ascomycota in both soil horizons. Higher phosphatase activity in older dunes was driven by less diverse P-mineralizing communities, especially in the organic horizon.

Which surgical technique may yield the best results in large, infected, segmental non-unions of the tibial shaft? A scoping review.

PURPOSE: Infected nonunion of the tibia with a large segmental bone defect is a complex and challenging condition for the patient and surgeon. This scoping review was conducted to identify existing evidence and knowledge gaps regarding this clinical scenario. Secondly, the objective of this study was to search for a valid recommendation on the optimal treatment. METHODS: A comprehensive search was conducted in the bibliographic databases: PubMed, Embase.com, and Web of Science Core Collection. Studies reporting on bone transport techniques, the Masquelet technique, and vascularized fibular grafts in bone defects greater than 5 cm were included. Bone healing results and functional results were compared according to duration of nonunion, infection recurrence, bone consolidation, complication rate, external fixation time, and time until full weight-bearing. RESULTS: Of the 2753 articles retrieved, 37 studies could be included on bone transport techniques (n = 23), the Masquelet technique (n = 7), and vascularized fibular grafts (n = 7). Respective bone union percentages were 94.3%, 89.5%, and 96.5%. The percentages of infection recurrence respectively were 1.6%, 14.4% and 7.0%, followed by respectively 1.58, 0.78, and 0.73 complications per patient. CONCLUSION: Bone transport was found to be the most widely studied technique in the literature. Depending on the surgeon's expertise, vascularized fibular grafts may be held as a favourable alternative. This review indicates that further high-quality research on large bone defects ( ≥ 5 cm) in patients with infected tibial nonunions is necessary to gain more insight into the potentially beneficial results of vascularized fibular grafts and the Masquelet technique.

A rare soccer-related injury: Traumatic posterior hip fracture-dislocation - Case series and overview of the literature.

BACKGROUND: Soccer is one of the most popular sports with millions of active professional and non-professional players worldwide. Traumatic hip dislocations are rare in soccer but can lead to major sequelae both physically and psychologically. The aim of this review was to obtain insight into the outcomes after surgerically repaired hip fracture-dislocation in soccer players as well as rehabilitation and prevention. METHODS: Two cases of a posterior hip fracture-dislocation that occurred during an amateur soccer match are presented and mechanism of injury, complications and rehabilitation were analysed. Follow-up of both patients was at least one year after surgery. Questionnaires and physical examinations were obtained to quantify and qualify outcome. RESULTS: In both cases the hip-dislocations were reduced within 3 h after injury. Semi-elective open reduction and internal fixation was performed within seven days. In one case, there was a concomitant Pipkin fracture and sciatic nerve neuropathy. There were no postoperative complications. Follow-up showed full of range of motion and normal hip functionality in both cases. However, both patients indicated a reduced quality of life and anxiety related to the accident. CONCLUSION: Traumatic hip fracture-dislocations during soccer practice are extremely rare. Despite uncomplicated fracture healing after surgery and return of hip function, both patients still suffer from psychological problems resulting in a decreased quality of life. Further research is required to enhance psychological outcomes, as well as to facilitate return to pre-injury levels of participation and engagement in sports following traumatic hip fracture-dislocations related to soccer.

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function.

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.

RNAi-mediated knockdown of exportin 1 negatively affected ovary development, survival and maize mosaic virus accumulation in its insect vector Peregrinus maidis.

Exportin 1 (XPO1) is the major karyopherin-ß nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

Cost effectiveness analysis for commonly used human cell and tissue products in the management of diabetic foot ulcers.

Background and Aims: This study considers the cost-effectiveness of commonly used cellular, acellular, and matrix­like products (CAMPs) of human origin also known as human cell and tissue products (HCT/Ps) in the management of diabetic foot ulcers. Methods: We developed a 1-year economic model assessing six CAMPs [cryopreserved placental membrane with viable cells (vCPM), bioengineered bilayered living cellular construct (BLCC), human fibroblast dermal substitute (hFDS), dehydrated human amnion chorion membrane (dHACM), hypothermically stored amniotic membrane (HSAM) and human amnion membrane allograft (HAMA) which had randomized controlled trial evidence compared with standard of care (SoC). CAMPs were compared indirectly and ranked in order of cost-effectiveness using SoC as the baseline, from a CMS/Medicare's perspective. Results: The mean cost, healed wounds (hw) and QALYs per patient for vCPM is $10,907 (0.914 hw, 0.783 QALYs), for HAMA $11,470 (0.903 hw, 0.780 QALYs), for dHACM $15,862 (0.828 hw, 0.764 QALYs), for BLCC $18,430 (0.816 hw, 0.763 QALYs), for hFDS $19,498 (0.775 hw, 0.757 QALYs), for SoC $19,862 (0.601 hw, 0.732 QALYs) and $24, 214 (0.829, 0.763 QALYs) for HSAM respectively. Over 1 year, vCPM results in cheaper costs overall and better clinical outcomes compared to other CAMPs. Following probabilistic sensitivity analysis, vCPM has a 60%, HAMA 40% probability of being cost-effective then dHACM, hFDS, BLCC, and lastly HSAM using a $100,000/healed wound or QALY threshold. Conclusions: All CAMPs were shown to be cost-effective when compared to SoC in managing DFUs. However, vCPM appears to be the most cost-effective CAMP over the modelled 52 weeks followed by HAMA, dHACM, hFDS, BLCC, and HSAM. We urge caution in interpreting the results because we currently lack head-to-head evidence comparing all these CAMPs and therefore suggest that this analysis be updated when more direct evidence of CAMPs becomes available.

Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1ß/CXCR2 Axis in Generation of Metastasis-specific Neutrophils.

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand-receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1ß/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1ß/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states. SIGNIFICANCE: We identify two recurring neutrophil populations and demonstrate their staged evolution from health to malignancy through the IL1ß/CXCL8/CXCR2 axis, allowing for immunotherapeutic neutrophil-targeting approaches to counteract immunosuppressive subtypes that emerge in metastasis.

An All-Suture-Based Technique for Meniscal Repair Is Cost-Effective in Comparison to Partial Meniscectomy for Horizontal Cleavage Tears.

Purpose: To determine the cost-effectiveness of meniscal repair (MR) using an all-suture-based technique when compared to partial meniscectomy (PM) for horizontal cleavage tears (HCTs) from a payor's perspective in the United States. Methods: A state-transition model and cost-utility analysis were developed from a US payor's perspective to project treatment costs and quality-adjusted life-years (QALYs) in a cohort of 35-year-old patients without osteoarthritis at baseline and presenting with either a lateral or medial HCT. Two outpatient costing perspectives were used, namely ambulatory surgical centers (ASCs) and hospitals. The state-transition model had 7 health states with transition probabilities, costs, and utilities obtained from the existing literature. Cost-effectiveness was assessed using a willingness-to-pay threshold of $100,000/QALY, and sensitivity analysis considered the effects of parameter uncertainty on model results. MR failure rates were focused on an all-suture-based technique; however, in a separate scenario, this study considered effectiveness data from various MR techniques and devices. Results: MR dominated PM over a lifetime horizon, increasing QALYs by 0.43 per patient and decreasing the cost by $12,227 per patient within a hospital setting (and by $12,570 within an ASC). MR with an all-suture-based technique continued to be the dominant treatment when age at primary treatment was varied between 30 and 60 years. Sensitivity analysis showed that MR was not cost-effective in year 1, was cost-effective from year 2, and was cost-saving from year 6 onward from both ASC and hospital perspectives. Probabilistic sensitivity analysis found that MR was cost-effective over a lifetime horizon in 99% of 10,000 iterations on base-case analysis. Conclusions: Using a lifetime horizon, this study found that from a payor's perspective, MR is a cost-saving intervention when compared with PM in patients with an HCT. Level of Evidence: Level III, economic analysis.

Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.

CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis.

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, -34b-3p, -212-3p, -449a-5p, -21a-3p, -376b-3p, -376b-5p and - 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-ß1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-ß, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

Donor pregnancies and transfusion recipient mortality: A role for red blood cell storage?

BACKGROUND AND OBJECTIVES: Donor characteristics have been implicated in transfusion-related adverse events. Uncertainty remains about whether sex, and specifically pregnancy history of the blood donor, could affect patient outcomes. Whether storage duration of the blood product could be important for patient outcomes has also been investigated, and a small detrimental effect of fresh products remains a possibility. Here, we hypothesize that fresh red blood cell products donated by ever-pregnant donors are associated with mortality in male patients. MATERIALS AND METHODS: We used data from a cohort study of adult patients receiving a first transfusion between 2005 and 2015 in the Netherlands. The risk of death after receiving a transfusion from one of five exposure categories (female never-pregnant stored ≤10 days, female never-pregnant stored >10 days, female ever-pregnant stored ≤10 days, female ever-pregnant stored >10 days and male stored for ≤10 days), compared to receiving a unit donated by a male donor, which was stored for >10 days (reference), was calculated using a Cox proportional hazards model. RESULTS: The study included 42,456 patients who contributed 88,538 person-years in total, of whom 13,948 died during the follow-up of the study (33%). Fresh units (stored for ≤10 days) from ever-pregnant donors were associated with mortality in male patients, but the association was not statistically significant (hazard ratio 1.39, 95% confidence interval 0.97-1.99). Sensitivity analyses did not corroborate this finding. CONCLUSION: These findings do not consistently support the notion that the observed association between ever-pregnant donor units and mortality is mediated by blood product storage.

Inhibition of ATR opposes glioblastoma invasion through disruption of cytoskeletal networks and integrin internalization via macropinocytosis.

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl-) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca2+)-activated Cl- currents in endothelial cells from control (TMEM16Afl/fl) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A (TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca2+ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Sex Differences in Outcome of Trauma Patients Presented with Severe Traumatic Brain Injury: A Multicenter Cohort Study.

The objective of this study was to determine whether there is an association between sex and outcome in trauma patients presented with severe traumatic brain injury (TBI). A retrospective multicenter study was performed in trauma patients aged ≥ 16 years who presented with severe TBI (Head Abbreviated Injury Scale (AIS) ≥ 4) over a 4-year-period. Subgroup analyses were performed for ages 16-44 and ≥45 years. Also, patients with isolated severe TBI (other AIS ≤ 2) were assessed, likewise, with subgroup analysis for age. Sex differences in mortality, Glasgow Outcome Score (GOS), ICU admission/length of stay (LOS), hospital LOS, and mechanical ventilation (MV) were examined. A total of 1566 severe TBI patients were included (831 patients with isolated TBI). Crude analysis shows an association between female sex and lower ICU admission rates, shorter ICU/hospital LOS, and less frequent and shorter MV in severe TBI patients ≥ 45 years. After adjusting, female sex appears to be associated with shorter ICU/hospital LOS. Sex differences in mortality and GOS were not found. In conclusion, this study found sex differences in patient outcomes following severe TBI, potentially favoring (older) females, which appear to indicate shorter ICU/hospital LOS (adjusted analysis). Large prospective studies are warranted to help unravel sex differences in outcomes after severe TBI.

Programming of neural progenitors of the adult subependymal zone towards a glutamatergic neuron lineage by neurogenin 2.

Although adult subependymal zone (SEZ) neural stem cells mostly generate GABAergic interneurons, a small progenitor population expresses the proneural gene Neurog2 and produces glutamatergic neurons. Here, we determined whether Neurog2 could respecify SEZ neural stem cells and their progeny toward a glutamatergic fate. Retrovirus-mediated expression of Neurog2 induced the glutamatergic lineage markers TBR2 and TBR1 in cultured SEZ progenitors, which differentiated into functional glutamatergic neurons. Likewise, Neurog2-transduced SEZ progenitors acquired glutamatergic neuron hallmarks in vivo. Intriguingly, they failed to migrate toward the olfactory bulb and instead differentiated within the SEZ or the adjacent striatum, where they received connections from local neurons, as indicated by rabies virus-mediated monosynaptic tracing. In contrast, lentivirus-mediated expression of Neurog2 failed to reprogram early SEZ neurons, which maintained GABAergic identity and migrated to the olfactory bulb. Our data show that NEUROG2 can program SEZ progenitors toward a glutamatergic identity but fails to reprogram their neuronal progeny.

Validated extended multiplexed LC-MS/MS assay for the quantification of adagrasib and sotorasib in human plasma, together with four additional SMIs.

Recently, two small molecular inhibitors (SMIs) -adagrasib and sotorasib- have been introduced for targeting Kirsten rat sarcoma (KRAS) p.G12C mutations in patients with non-small cell lung cancer (NSCLC). In order to support pharmacokinetic research as well as clinical decision making, we developed and validated a simple and accurate liquid chromatography-tandem mass spectrometry method for the multiplexed quantification of adagrasib and sotorasib. This assay was co-validated with the quantification for brigatinib, lorlatinib, pralsetinib and selpercatinib. Methanol was used for single-step protein precipitation. Chromatographic separation was performed using an Acquity® HSS C18 UPLC column, with an elution gradient of ammonium formate 0.1 % v/v in water and acetonitrile. In K2-EDTA plasma, adagrasib was found to be stable for at least seven days at room temperature and 4 °C, and at least 3 months at -80 °C. Sotorasib was found to be stable for at least three days at room temperature, seven days at 4 °C and at least 3 months at -80 °C. The method was validated over a linear range of 80-4000 ng/mL for adagrasib and 25-2500 ng/mL for sotorasib. The assay is therefore well-equipped for determining plasma concentrations in clinical practice.