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This study aims to develop a thin-film nanofibrous composite (TFNC) membrane for removing fermentation inhibitors from a hydrolysate solution. Such membrane was fabricated via interfacial polymerization of a polyamide thin-film layer on top of a solution blow-spun polyacrylonitrile nanofiber scaffold. The effects of pH (3.29, 4.29, 8.26) and pressure (4, 6.67, 9.33, 12 bar) on the membrane’s performance were investigated. The TFNC membrane had a high pure water permeability of 21.06 L/m2-h-bar and was able to deliver satisfactory rejection coefficients at pH 3.29 and pressure of 4 bar (glucose − 91.84%, xylose − 86.06%, hydroxymethylfurfural − 20.52%, furfural − 21.32%). Different pH settings resulted in similar permeate fluxes, but increasing the pH from acidic to basic reduced the rejection coefficients due to electrostatic repulsion. Increasing the pressure increased the permeate flux but decreased the rejection coefficients. The developed membrane showed remarkable permeability and effectively removed inhibitors, useful in biorefineries.

Abstract Background Fatal opioid-involved overdose rates increased precipitously from 5.0 per 100,000 population to 33.5 in Massachusetts between 1999 and 2022. Methods We used spatial rate smoothing techniques to identify persistent opioid overdose-involved fatality clusters at the ZIP Code Tabulation Area (ZCTA) level. Rate smoothing techniques were employed to identify locations of high fatal opioid overdose rates where population counts were low. In Massachusetts, this included areas with both sparse data and low population density. We used Local Indicators of Spatial Association (LISA) cluster analyses with the raw incidence rates, and the Empirical Bayes smoothed rates to identify clusters from 2011 to 2021. We also estimated Empirical Bayes LISA cluster estimates to identify clusters during the same period. We constructed measures of the socio-built environment and potentially inappropriate prescribing using principal components analysis. The resulting measures were used as covariates in Conditional Autoregressive Bayesian models that acknowledge spatial autocorrelation to predict both, if a ZCTA was part of an opioid-involved cluster for fatal overdose rates, as well as the number of times that it was part of a cluster of high incidence rates. Results LISA clusters for smoothed data were able to identify whether a ZCTA was part of a opioid involved fatality incidence cluster earlier in the study period, when compared to LISA clusters based on raw rates. PCA helped in identifying unique socio-environmental factors, such as minoritized populations and poverty, potentially inappropriate prescribing, access to amenities, and rurality by combining socioeconomic, built environment and prescription variables that were highly correlated with each other. In all models except for those that used raw rates to estimate whether a ZCTA was part of a high fatality cluster, opioid overdose fatality clusters in Massachusetts had high percentages of Black and Hispanic residents, and households experiencing poverty. The models that were fitted on Empirical Bayes LISA identified this phenomenon earlier in the study period than the raw rate LISA. However, all the models identified minoritized populations and poverty as significant factors in predicting the persistence of a ZCTA being part of a high opioid overdose cluster during this time period. Conclusion Conducting spatially robust analyses may help inform policies to identify community-level risks for opioid-involved overdose deaths sooner than depending on raw incidence rates alone. The results can help inform policy makers and planners about locations of persistent risk.

Diabetes mellitus is a chronic metabolic disease that is mainly characterized by hyperglycemia. Chalcones and their derivatives have demonstrated promising pharmacological potential for the treatment of diabetes. The aim of the study was to evaluate antidiabetic activities and analyze 4-methoxychalcone (MPP) using GC-MS. The compound was characterized using mass spectroscopy, nuclear magnetic resonance and headspace with gas chromatography coupled to mass spectrometry (HS-GC-MS). MPP was evaluated via the inhibition of the alpha-glucosidase enzyme, cell viability and antiglycation and hemolytic activities in vitro. The study of the interaction between the bovine serum albumin protein and MPP was investigated via molecular docking. Oral sucrose tolerance and oral glucose tolerance tests were performed in streptozotocin (STZ)-induced diabetic mice. The HS-GC-MS method was able to accurately detect and characterize the compound, and the interaction between MPP and BSA revealed the remarkable affinity for the two main binding sites of BSA. This was confirmed by the in vitro antiglycation test, since MPP showed activity through both oxidative and non-oxidative stress. MPP significantly attenuated the increase in glycemia after glucose loading in STZ-induced diabetic mice. These results confirm that MPP has antihyperglycemic activity and may be an alternative for the treatment of diabetes mellitus.

Abstract Tumor-associated microglia and blood-derived macrophages (TAMs) play a central role in modulating the immune suppressive microenvironment in glioma. Here, we show that GPNMB is predominantly expressed by TAMs in human glioblastoma multiforme and the murine RCAS-PDGFb high grade glioma model. Loss of GPNMB in the in vivo tumor microenvironment results in significantly smaller tumor volumes and generates a pro-inflammatory innate and adaptive immune cell microenvironment. The impact of host-derived GPNMB on tumor growth was confirmed in two distinct murine glioma cell lines in organotypic brain slices from GPNMB-KO and control mice. Using published data bases of human glioma, the elevated levels in TAMs could be confirmed and the GPNMB expression correlated with a poorer survival.

Background: Anaemia is a common but treatable condition that predicts adverse clinical outcomes. However, standards of anaemia management vary considerably. Aim: To estimate the prevalence of anaemia and extent of screening for common underlying causes in the healthcare system in the Republic of Ireland. Design & setting: We conducted a retrospective cohort study of 112 181 adult patients, aged ≥18 years, who had a full blood count performed in 2013, using data from the National Kidney Disease Surveillance System. Method: The prevalence of anaemia was determined across demographic and clinical subgroups, according to World Health Organization (WHO) definitions. The proportion screened for iron, vitamin B12, and folate deficiency was determined within a 3-month follow-up period and the corresponding prevalence for each deficiency determined. Results: The overall prevalence of anaemia was 12.0% (95% confidence interval [CI] = 11.8% to 12.2%) and was higher in women than men (13.2% versus 10.5%, P75 years) and worsening kidney function (8.2%, 10.9%, 33.2%, and 63.8% for each estimated glomerular filtration rate [eGFR] categories >90, 60–89, 30–59 and

Abstract Recent advances in solar‐driven interfacial evaporation (SDIE) have led to high evaporation rates that open promising avenues for practical utilization in freshwater production and industrial application for pollutant and nutrient concentration, and resource recovery. Breakthroughs in overcoming the theoretical limitation of 2D interfacial evaporation have allowed for developing systems with high evaporation rates. This study presents a comprehensive review of various evaporator designs that have achieved pure evaporation rates beyond 4 kg m−2 h−1, including structural and material designs allowing for rapid evaporation, passive 3D designs, and systems coupled with alternative energy sources of wind and joule heating. The operational mechanisms for each design are outlined together with discussion on the current benefits and areas for improvement. The overarching challenges encountered by SDIE concerning the feasibility of direct integration into contemporary practical settings are assessed, and issues relating to sustaining elevated evaporation rates under diverse environmental conditions are addressed.

Background: Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. Methods: We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Results: Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Conclusions: Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.

ABSTRACTSalmonella serovars Typhi and Paratyphi cause a prolonged illness known as enteric fever, whereas other serovars cause acute gastroenteritis. Mechanisms responsible for the divergent clinical manifestations of nontyphoidal and enteric fever Salmonella infections have remained elusive. Here, we show that S. Typhi and S. Paratyphi A can persist within human macrophages, whereas S. Typhimurium rapidly induces apoptotic macrophage cell death that is dependent on Salmonella pathogenicity island 2 (SPI2). S. Typhi and S. Paratyphi A lack 12 specific SPI2 effectors with pro-apoptotic functions, including nine that target nuclear factor κB (NF-κB). Pharmacologic inhibition of NF-κB or heterologous expression of the SPI2 effectors GogA or GtgA restores apoptosis of S. Typhi-infected macrophages. In addition, the absence of the SPI2 effector SarA results in deficient signal transducer and activator of transcription 1 (STAT1) activation and interleukin 12 production, leading to impaired TH1 responses in macrophages and humanized mice. The absence of specific nontyphoidal SPI2 effectors may allow S. Typhi and S. Paratyphi A to cause chronic infections.IMPORTANCESalmonella enterica is a common cause of gastrointestinal infections worldwide. The serovars Salmonella Typhi and Salmonella Paratyphi A cause a distinctive systemic illness called enteric fever, whose pathogenesis is incompletely understood. Here, we show that enteric fever Salmonella serovars lack 12 specific virulence factors possessed by nontyphoidal Salmonella serovars, which allow the enteric fever serovars to persist within human macrophages. We propose that this fundamental difference in the interaction of Salmonella with human macrophages is responsible for the chronicity of typhoid and paratyphoid fever, suggesting that targeting the nuclear factor κB (NF-κB) complex responsible for macrophage survival could facilitate the clearance of persistent bacterial infections.

Background: Lutetium-177-prostate-specific membrane antigen- 617 (Lu-PSMA) is an effective therapy for metastatic castration-resistant prostate cancer (mCRPC). However, treatment responses are heterogeneous despite stringent positron emission tomography (PET)-based imaging selection criteria. Molecularly based biomarkers have potential to refine patient selection and optimise outcomes. Objective: To identify circulating tumour DNA (ctDNA) features associated with treatment outcomes for men treated with Lu-PSMA. Design, setting, and participants: ctDNA from men treated with Lu-PSMA in combination with idronoxil for progressive mCRPC were analysed using an 85-gene customised sequencing assay. ctDNA fractions, molecular profiles, and the presence of alterations in aggressive-variant prostate cancer (AVPC) genes were analysed at baseline, cycle 3 and at disease progression. Intervention: Men received Lu-PSMA with idronoxil every 6 wk for up to six cycles. Outcome measurements and statistical analysis: Baseline and exit PSMA and fluorodeoxyglucose PET/computed tomography (CT) imaging was conducted at baseline and study exit. Single-photon emission CT (SPECT) scans were performed 24 h after Lu-PSMA. Blood samples were collected at baseline,cycle 3 and at disease progression. Cox proportional-hazards models were used to assess associations and derive hazard ratios (HRs) and confidence intervals (CIs) for associations between molecular factors, imaging features, and clinical outcomes. Results and limitations: Sixty samples from 32 men were sequenced (32 at baseline, 24 at cycle 3, four from patients with disease progression); two samples (baseline, on-treatment) from one individual were excluded from analysis owing to poor quality of the baseline sequencing data. Alterations in AVPC genes were associated with shorter prostate-specific antigen (PSA) progression-free survival (PFS) and overall survival (OS) in univariate (HR 3.4, 95% CI 1.5–7.7; p = 0.0036; and HR 3.3, 95% CI 1.4–7.7; p = 0.0063, respectively) and multivariate analyses (HR 4.8, 95% CI 1.8–13; p = 0.0014; and HR 4.1, 95% CI 1.6–11; p = 0.004). Conclusions: ctDNA alterations in AVPC genes were associated with shorter PSA PFS and OS among men treated with Lu-PSMA and intermittent idronoxil. These candidate molecular biomarkers warrant further study to determine whether they have predictive value and potential to guide synergistic combination strategies to enhance outcomes for men treated with Lu-PSMA for mCRPC. Patient summary: Certain DNA/gene changes detected in the blood of men with advanced prostate cancer were associated with shorter benefit from lutetium PSMA, a targeted radioactive therapy. This information may be useful in determining which men may benefit most from this treatment, but additional research is needed.

Abstract Background Abnormalities of serum sodium are associated with increased mortality risk in hospitalised patients, but it is unclear whether, and to what extent other factors influence this relationship. We investigated the impact of dysnatraemia on total and cause-specific mortality in the Irish health system while exploring the concurrent impact of age, kidney function and designated clinical work-based settings. Methods A retrospective cohort study of 32,666 participants was conducted using data from the National Kidney Disease Surveillance System. Hyponatraemia was defined as 145 mmol/L with normal range 135–145 mmol/L. Multivariable Cox proportional hazards regression was used to estimate hazard ratios (HR’s) and 95% Confidence Intervals (CIs) while penalised spline models further examined patterns of risk. Results There were 5,114 deaths (15.7%) over a median follow up of 5.5 years. Dysnatraemia was present in 8.5% of patients overall. In multivariable analysis, both baseline and time-dependent serum sodium concentrations exhibited a U-shaped association with mortality. Hyponatremia was significantly associated with increased risk for cardiovascular [HR 1.38 (1.18–1.61)], malignant [HR: 2.49 (2.23–2.78)] and non-cardiovascular/non-malignant causes of death [1.36 (1.17–1.58)], while hypernatremia was significantly associated with cardiovascular [HR: 2.16 (1.58–2.96)] and non-cardiovascular/ non-malignant deaths respectively [HR: 3.60 (2.87–4.52)]. The sodium-mortality relationship was significantly influenced by age, level of kidney function and the clinical setting at baseline (P

Background: Recent studies indicate that COVID-19 has had a significant impact on access and continuity to opioid and benzodiazepine medications; little is known about its effect on access to and utilization of stimulant medications. Objective: To investigate trends of dispensed stimulant medications in relation to the COVID-19 pandemic response. Methods: Stimulant prescriptions dispensed during 2011–2021 were analyzed using the Massachusetts Prescription Drug Monitoring Program (PDMP), the state's data repository for all controlled substance medications dispensed to residents from retail pharmacies and out of state mail-order pharmacies. Statewide trends were estimated by age group, sex, and stimulant-naïve patients (individuals with no stimulant prescription in the prior one-year period). Results: Overall, stimulant prescriptions increased 70% from 2011 to 2021. Wide differences by sex and age groups were found pre and post COVID response periods. Between 2019 and 2021, stimulant prescriptions for males 12–18 years old decreased 14.6% compared to 0.9% for females. Female stimulant-naïve patients ages 25–34 increased more than males between 2019 and 2021 (11.6% compared to

Background and Objectives: Neurosurgery has been underrepresented in the medical school curriculum. Advances in augmented reality and 3D printing have opened the way for early practical training through simulations. We assessed the usability of the UpSurgeOn simulation-based training model and report first experiences from a hands-on neurosurgery course for medical students. Materials and Methods: We organized a two-day microneurosurgery simulation course tailored to medical students. On day one, three neurosurgeons demonstrated anatomical explorations with the help of life-like physical simulators (BrainBox, UpSurgeOn). The surgical field was projected onto large high-definition screens by a robotic-assisted exoscope (RoboticScope, BHS Technologies). On day two, the students were equipped with microsurgical instruments to explore the surgical anatomy of the pterional, temporal and endoscopic retrosigmoid approaches. With the help of the RoboticScope, they simulated five clipping procedures using the Aneurysm BrainBox. All medical students filled out a digital Likert-scale-based questionnaire to evaluate their experiences. Results: Sixteen medical students participated in the course. No medical students had previous experience with UpSurgeOn. All participants agreed that the app helped develop anatomical orientation. They unanimously agreed that this model should be part of residency training. Fourteen out of sixteen students felt that the course solidified their decision to pursue neurosurgery. The same fourteen students rated their learning experience as totally positive, and the remaining two rated it as rather positive. Conclusions: The UpSurgeOn educational app and cadaver-free models were perceived as usable and effective tools for the hands-on neuroanatomy and neurosurgery teaching of medical students. Comparative studies may help measure the long-term benefits of UpSurgeOn-assisted teaching over conventional resources.

Abstract Targeted drug delivery systems hold the remarkable potential to improve the therapeutic index of anticancer medications markedly. Here, we report a targeted delivery platform for cancer treatment using clathrin light chain (CLC)‐conjugated drugs. We conjugated CLC to paclitaxel (PTX) through a glutaric anhydride at high efficiency. Labeled CLCs localized to 4T1 tumors implanted in mice, and conjugation of PTX to CLC enhanced its delivery to these tumors. Treatment of three different mouse models of cancer—melanoma, breast cancer, and lung cancer—with CLC‐PTX resulted in significant growth inhibition of both the primary tumor and metastatic lesions, as compared to treatment with free PTX. CLC‐PTX treatment caused a marked increase in apoptosis of tumor cells and reduction of tumor angiogenesis. Our data suggested HSP70 as a binding partner for CLC. Our study demonstrates that CLC‐based drug‐conjugates constitute a novel drug delivery platform that can augment the effects of chemotherapeutics in treating a variety of cancers. Moreover, conjugation of therapeutics with CLC may be used as means by which drugs are delivered specifically to primary tumors and metastatic lesions, thereby prolonging the survival of cancer patients.

Chronic consumption of sugar- and artificially-sweetened beverages (SSB and ASB) are associated with an increased risk of stroke but it is unclear how acute consumption influences cerebral vascular function.Purpose: We hypothesized that: (1) acute consumption of SSB and ASB would augment dynamic cerebral autoregulation (dCA) and attenuate cerebral vascular reactivity to hypercapnia (CVRCO2) compared to water; and (2) dCA and CVRCO2 would be attenuated with SSB compared to ASB and water.Methods: Twelve healthy adults (age: 23 ± 2 years, four females) completed three randomized trials where they drank 500 ml of water, SSB (Mountain Dew®), or ASB (Diet Mountain Dew®). We measured mean arterial pressure (MAP), middle and posterior cerebral artery blood velocities (MCAv and PCAv), and end-tidal CO2 tension (PETCO2). Cerebral vascular conductance was calculated as cerebral artery blood velocity/MAP (MCAc and PCAc). Twenty min after consumption, participants completed a 5 min baseline, and in a counterbalanced order, a CVRCO2 test (3%, 5%, and 7% CO2 in 3 min stages) and a dCA test (squat-stand tests at 0.10 Hz and 0.05 Hz for 5 min each) separated by 10 min. CVRCO2 was calculated as the slope of the linear regression lines of MCAv and PCAv vs. PETCO2. dCA was assessed in the MCA using transfer function analysis. Coherence, gain, and phase were determined in the low frequency (LF; 0.07–0.2 Hz) and very low frequency (VLF; 0.02–0.07 Hz).Results: MCAv and MCAc were lower after SSB (54.11 ± 12.28 cm/s, 0.58 ± 0.15 cm/s/mmHg) and ASB (51.07 ± 9.35 cm/s, 0.52 ± 1.0 cm/s/mmHg) vs. water (62.73 ± 12.96 cm/s, 0.67 ± 0.11 cm/s/mmHg; all P < 0.035), respectively. PCAc was also lower with the ASB compared to water (P = 0.007). MCA CVRCO2 was lower following ASB (1.55 ± 0.38 cm/s/mmHg) vs. water (2.00 ± 0.57 cm/s/mmHg; P = 0.011) but not after SSB (1.90 ± 0.67 cm/s/mmHg; P = 0.593). PCA CVRCO2 did not differ between beverages (P > 0.853). There were no differences between beverages for coherence (P ≥ 0.295), gain (P ≥ 0.058), or phase (P ≥ 0.084) for either frequency.Discussion: Acute consumption of caffeinated SSB and ASB resulted in lower intracranial artery blood velocity and conductance but had a minimal effect on cerebral vascular function as only MCA CVRCO2 was altered with the ASB compared to water.

Abstract Background Buprenorphine is a widely used treatment option for patients with opioid use disorder (OUD). Premature discontinuation from this treatment has many negative health and societal consequences. Objective To develop and evaluate a machine learning based two-stage clinical decision-making framework for predicting which patients will discontinue OUD treatment within less than a year. The proposed framework performs such prediction in two stages: (i) at the time of initiating the treatment, and (ii) after two/three months following treatment initiation. Methods For this retrospective observational analysis, we utilized Massachusetts All Payer Claims Data (MA APCD) from the year 2013 to 2015. Study sample included 5190 patients who were commercially insured, initiated buprenorphine treatment between January and December 2014, and did not have any buprenorphine prescription at least one year prior to the date of treatment initiation in 2014. Treatment discontinuation was defined as at least two consecutive months without a prescription for buprenorphine. Six machine learning models (i.e., logistic regression, decision tree, random forest, extreme-gradient boosting, support vector machine, and artificial neural network) were tested using a five-fold cross validation on the input data. The first-stage models used patients’ demographic information. The second-stage models included information on medication adherence during the early phase of treatment based on the proportion of days covered (PDC) measure. Results A substantial percentage of patients (48.7%) who started on buprenorphine discontinued the treatment within one year. The area under receiving operating characteristic curve (C-statistic) for the first stage models varied within a range of 0.55 to 0.59. The inclusion of knowledge regarding patients’ adherence at the early treatment phase in terms of two-months and three-months PDC resulted in a statistically significant increase in the models’ discriminative power (p-value

Thermally localized solar-driven water evaporation (SWE) in recent years has increasingly been developed due to the potential of cost-efficient freshwater production from small-scale portable devices. In particular, the multistage SWE has attracted much attention as the systems possess mostly a simple foundational structure and high solar-to-thermal conversion output rates, enough to produce freshwater from 1.5 L m−2h−1 (LMH) to 6 LMH. In this study, the currently designed multistage SWE devices were reviewed and examined based on their unique characteristics as well as their performances in freshwater production. The main distinguishing factors in these systems were the condenser staging design and the spectrally selective absorbers either in a form of high solar absorbing material, photovoltaic (PV) cells for water and electricity co-production, and coupling of absorber and solar concentrator. Other elements of the devices involved differences such as the direction of water flow, the number of layers constructed, and the materials used for each layer of the system. The key factors to consider for these systems include the heat and mass transport in the device, solar-to-vapor conversion efficiency, gain output ratio (representing how many times the latent heat has been reused), water production rate/number of stages, and kWh/number of stages. It was evident that most of the studied devices involved slightly different mechanisms and material compositions to draw out higher efficiency rates from the current limitations. The reviewed designs showed the ability to be adopted into small-scale solar desalination allowing for accessibility of sufficient freshwater in needing regions.

The role of neutrophils in the regulation of tumour growth and metastasis remains controversial. Here, the authors demonstrate that neutrophils, by exerting inhibitory effects on cytotoxic NK cells, show a net pro-metastatic effect in immune-competent mice, while they are tumoricidal and anti-metastatic in NK cell-deficient hosts.

Abstract Microglia are the primary immune-competent cells of the central nervous system (CNS) and sense both pathogen- and host-derived factors through several receptor systems including the Toll-like receptor (TLR) family. Although TLR5 has previously been implicated in different CNS disorders including neurodegenerative diseases, its mode of action in the brain remained largely unexplored. We sought to determine the expression and functional consequences of TLR5 activation in the CNS. Quantitative real-time PCR and immunocytochemical analysis revealed that microglia is the major CNS cell type that constitutively expresses TLR5. Using Tlr5 −/− mice and inhibitory TLR5 antibody we found that activation of TLR5 in microglial cells by its agonist flagellin, a principal protein component of bacterial flagella, triggers their release of distinct inflammatory molecules, regulates chemotaxis, and increases their phagocytic activity. Furthermore, while TLR5 activation does not affect tumor growth in an ex vivo GL261 glioma mouse model, it triggers microglial accumulation and neuronal apoptosis in the cerebral cortex in vivo. TLR5-mediated microglial function involves the PI3K/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, as specific inhibitors of this signaling pathway abolish microglial activation. Taken together, our findings establish TLR5 as a modulator of microglial function and indicate its contribution to inflammatory and injurious processes in the CNS.

Gallbladder cancer incidence shows characteristic geographic patterns. Here the authors perform a genomic analysis of gallbladder cancers in patients from countries with high incidence (South Korea, India and Chile) and identify ELF3 and other significantly mutated genes not previously associated with gallbladder cancer.

Abstract Background Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies—definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9. Results We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes. Conclusions Exon skipping is a widespread phenomenon occurring across the genome. These findings have significant implications for the application of genome editing in both basic research and clinical practice.

Abstract Background PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. Methods A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. Results We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial–mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. Conclusions Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

INTRODUCTION: Herpes zoster is the common reactivation of latent varicella zoster virus infection and is associated with substantial morbidity and costs. Age, immunosuppression, diabetes and active cancer are important risk factors for developing herpes zoster. The recombinant herpes zoster vaccine is highly effective in preventing this infection. To inform cost-effectiveness analyses of vaccination in risk groups, it is important to define the rates of complications, mortality, and hospitalisation and its costs. METHODS: In this single-centre, investigator-initiated, retrospective study we analysed herpes zoster patients treated in our tertiary dermatology referral centre between 2005 and 2019. Case costs were calculated using health insurance invoices and by estimating the drug-related costs. The associations between patients' characteristics and complications were assessed using logistic regression models. RESULTS: A total of 355 herpes zoster patients were analysed (mean age 61.5 ± 18.8 years; 56.6% women). The complication rate was 30.1% (n = 107) with post-herpetic neuralgia being the most frequent (37/355, 10.4%). The herpes zoster-related hospitalisation rate was 19.2% (n = 68), the herpes zoster-related mortality rate 0.85% (n = 3). Of all patients, 22.8% (n = 81) had herpes zoster risk factors. The odds ratio for complications (multivariate analysis) was 2.5 (95% confidence interval [CI] 1.12–5.72) in cancer patients and 1.04 (1.02–1.05) for each additional year of age (1.41 for 10 years). The cost ratio (median) of in- vs outpatient treatment was 26.9 (CHF/case 9029 vs 335), of all complicated vs non-complicated herpes zoster patients 6.6 (CHF/case 2203 vs 332) and of complicated vs non-complicated herpes zoster outpatients 4.3 (CHF/case 1331 vs 306). CONCLUSION: In this herpes zoster population at a university hospital, one in three patients was affected by herpes zoster-related complications. Age and cancer were independently associated with a high complication rate, suggesting a need to vaccinate cancer patients. Herpes zoster-related complications and the need for inpatient treatment escalated the costs per case.

ABSTRACT Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

Given the oppressive nature of higher education environments, educators must support students in making sense of their experiences—especially minoritized students. Although this kind of sensemaking often happens informally, college educators are primed to support students in understanding: (1) their experiences within and their interactions with higher education environments; (2) how those experiences and interactions reflect and are shaped by systemic issues; and (3) the implications of these experiences and interactions during college and beyond. Accordingly, we introduce critical sensemaking (CSM) as a pedagogical tool that educators can expose students to and create a more equitable college environment. CSM is a framework that encourages students to interrogate their collegiate environments and experiences, focusing on power and systems of oppression and how they interpret and negotiate their environments. Only by gaining this understanding can students press for institutional transformation in a manner that reflects various aspects of their experiences; hence, creating an institutional accountability structure. Moreover, educators supporting students’ acquisitions of CSM skills provides opportunities to build trust and meaningful coalitions toward creating more inclusive college environments.

Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.

FK506 is a potential antifungal compound that inhibits calcineurin, but it also has immunosuppressive activity. Here, Juvvadi et al. report the structure of FK506 in complex with the FK506-binding protein FKPB12 and calcineurin, and design a less immunosuppresive FK506 analog with antifungal activity in mice.

Abstract Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host’s response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14–15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual’s recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4–5, 2019.

Background: Graft-versus host disease (GVHD) is a complication of stem cell transplantation associated with significant morbidity and mortality. Non-specific immune-suppression, the mainstay of treatment, may result in immune-surveillance dysfunction and disease recurrence. Methods: We created humanised mice model for chronic GVHD (cGVHD) by injecting cord blood (CB)-derived human CD34+CD38−CD45RA− haematopoietic stem/progenitor cells (HSPCs) into hIL-6 transgenic NOD/SCID/Il2rgKO (NSG) newborns, and compared GVHD progression with NSG newborns receiving CB CD34− cells mimicking acute GVHD. We characterised human immune cell subsets, target organ infiltration, T-cell repertoire (TCR) and transcriptome in the humanised mice. Findings: In cGVHD humanised mice, we found activation of T cells in the spleen, lung, liver, and skin, activation of macrophages in lung and liver, and loss of appendages in skin, obstruction of bronchioles in lung and portal fibrosis in liver recapitulating cGVHD. Acute GVHD humanised mice showed activation of T cells with skewed TCR repertoire without significant macrophage activation. Interpretation: Using humanised mouse models, we demonstrated distinct immune mechanisms contributing acute and chronic GVHD. In cGVHD model, co-activation of human HSPC-derived macrophages and T cells educated in the recipient thymus contributed to delayed onset, multi-organ disease. In acute GVHD model, mature human T cells contained in the graft resulted in rapid disease progression. These humanised mouse models may facilitate future development of new molecular medicine targeting GVHD. Keywords: Acute GVHD, Chronic GVHD, IL-6, Humanised mouse

Abstract Background Infections are the second leading cause of death and hospitalisation among haemodialysis (HD) patients. Rates of access-related bloodstream infections (AR-BSI) are influenced by patient characteristics and local protocols. We explored factors associated with AR-BSI in a contemporary cohort of HD patients at a tertiary nephrology centre. Methods A retrospective cohort of 235 chronic HD patients was identified from a regional dialysis programme between Jan 2015 and Dec 2016. Data on demographics, primary renal disease, comorbid conditions and dialysis access type were obtained from the Kidney Disease Clinical Patient Management System (KDCPMS). Data on blood cultures were captured from the microbiology laboratory. Poisson regression with robust variance estimates was used to compare infection rates and relative risk of AR-BSI according to the site and type of vascular access. Results The mean age was 65 (± 15) years, 77% were men, and the median follow up was 19 months (IQR: 10–24 months), accumulating 2030 catheter-months and 1831 fistula-months. Overall rates of AR-BSI were significantly higher for central venous catheter (CVC) compared to arteriovenous fistula (AVF), (2.22, 95% (CI): 1.62–2.97) versus 0.11 (0.01–0.39) per 100 patient-months respectively), with a rate ratio of 20.29 (4.92–83.66), p

Background: Nanomedicine offers an excellent opportunity to tackle treatment-refractory malignancies by enhancing the delivery of therapeutics to the tumor site. High endothelial venules (HEVs) are found primarily in lymph nodes or formed de novo in peripheral tissues during inflammatory responses. They express peripheral node addressin (PNAd), which is recognized by the monoclonal antibody MECA79. Methods: Here, we demonstrated that HEVs form de novo in human pancreatic ductal adenocarcinoma (PDAC). We engineered MECA79 coated nanoparticles (MECA79-NPs) that recognize these ectopic HEVs in PDAC. Findings: The trafficking of MECA79-NPs following intravenous delivery to human PDAC implanted in a humanized mouse model was more robust than non-conjugated NPs. Treatment with MECA79-Taxol-NPs augmented the delivery of Paclitaxel (Taxol) to the tumor site and significantly reduced the tumor size. This effect was associated with a higher apoptosis rate of PDAC cells and reduced vascularization within the tumor. Interpretation: Targeting the HEVs of PDAC using MECA79-NPs could lay the ground for the localized delivery of a wide variety of drugs including chemotherapeutic agents. Fund: National Institutes of Health (NIH) grants: T32-EB016652 (B·B.), NIH Cancer Core Grant CA034196 (L.D.S.), National Institute of Allergy and Infectious Diseases grants R01-AI126596 and R01-HL141815 (R.A.). Keywords: Pancreatic ductal adenocarcinoma, High endothelial venules, Peripheral node addressin, MECA79 coated nanoparticles, Taxol