High-Throughput SFC-MS/MS Method to Measure EPSA and Predict Human Permeability.

Permeability is a key factor driving the absorption of orally administered drugs. In early discovery, the efficient evaluation of permeability, particularly for compounds violating Lipinski's Rule of 5, remains challenging. Addressing this, we established a high-throughput method to measure the experimental polar surface area (HT-EPSA) as an in vitro surrogate to measure permeability. Compared to earlier methods, HT-EPSA significantly reduces data acquisition time with enhanced sensitivity, selectivity, and data quality. In the effort of translating EPSA to human in vitro and in vivo passive permeability, we demonstrated the application of EPSA for predicting Caco-2 cell and human intestinal permeability, showing improvements over topological polar surface area and the parallel artificial membrane permeability assay for rank-ordering permeability in a proteolysis targeting chimera case study. The HT-EPSA method is expected to be highly beneficial in guiding early stage compound rank-ordering, faster decision-making, and in predicting in vitro and/or in vivo human intestinal permeability.

DNA Damage Stress Control Is a Truncated Large T Antigen and Euchromatic Histone Lysine Methyltransferase 2-Dependent Central Feature of Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. Merkel cell polyomavirus causes 80% of MCCs, encoding the viral oncogenes small T and truncated large T (tLT) antigens. These proteins impair the RB1-dependent G1/S checkpoint blockade and subvert the host cell epigenome to promote cancer. Whole-proteome analysis and proximal interactomics identified a tLT-dependent deregulation of DNA damage response (DDR). Our investigation revealed, to our knowledge, a previously unreported interaction between tLT and the histone methyltransferase EHMT2. T antigen knockdown reduced DDR protein levels and increased the levels of the DNA damage marker γH2Ax. EHMT2 normally promotes H3K9 methylation and DDR signaling. Given that inhibition of EHMT2 did not significantly change the MCC cell proteome, tLT-EHMT2 interaction could affect the DDR. With tLT, we report that EHMT2 gained DNA damage repair proximal interactors. EHMT2 inhibition rescued proliferation in MCC cells depleted for their T antigens, suggesting impaired DDR and/or lack of checkpoint efficiency. Combined tLT and EHMT2 inhibition led to altered DDR, evidenced by multiple signaling alterations. In this study, we show that tLT hijacks multiple components of the DNA damage machinery to enhance tolerance to DNA damage in MCC cells, which could explain the genetic stability of these cancers.

Corrigendum: Late-stage MC38 tumours recapitulate features of human colorectal cancer - implications for appropriate timepoint selection in preclinical studies.

[This corrects the article DOI: 10.3389/fimmu.2023.1152035.].

A Scoping Review of the Intracranial Complications of Pediatric Sinusitis.

OBJECTIVE: Intracranial complications of pediatric sinusitis are uncommon but are often associated with significant morbidity, especially when appropriate care is delayed. The present study aimed to identify commonalities for the development and progression of these complications in the pediatric population. DATA SOURCES: CENTRAL, CINAHL, Citation searching, ClinicalTrials.gov, Embase, Google Scholar, MEDLINE, PsycINFO, PubMed, Scopus, Web of Science, and World Health Organization. REVIEW METHODS: A comprehensive literature search was performed using Preferred Reporting Items for Systematic Reviews and Meta-analyses scoping review guidelines. Studies describing intracranial infections secondary to sinusitis in the pediatric population (age <18 years) were included. Studies in which adult and pediatric data were not separated and studies in which the pediatric cohort was fewer than 10 cases were excluded. Ultimately, 33 studies describing 1149 unique patient cases were included for data collection and analysis. RESULTS: Our analysis revealed intracranial complications were more common in adolescent males. Most children presented with over 1 week of vague symptoms, such as headache and fever. The majority of complications were diagnosed radiographically with computed tomography. Subdural empyema and epidural abscess were the most common intracranial complications reported. On average, patients were admitted for over 2 weeks. Most children were treated with a combination of antibiotics and surgical intervention. Complications were rare, but when present, were often associated with significant morbidity. CONCLUSION: This scoping review of the available literature has provided insight into commonalities among pediatric patients who develop intracranial complications of sinusitis, providing a foundation for further study to inform medical and surgical decision-making in this population.

Salmonella exploits membrane reservoirs for invasion of host cells.

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

Why do second-cycle medical students choose oncology? A literature overview.

INTRODUCTION: Given the current global cancer epidemic across the world, the need for healthcare professionals in this field is crucial. Little is known about the factors that drive medical students toward oncology. METHOD: We conducted a systematic review of the literature (from 1980 to the present), using several search equations and selecting original articles written in English based on qualitative or quantitative surveys, to understand what motivates medical students to choose oncology. RESULTS: We identified only seven articles that reported quantitative surveys; no qualitative surveys were found. These seven surveys are composed of closed-ended questions and are pragmatic questionnaires based on field knowledge, but without an underlying theory. These studies most often interrogate students already oriented towards oncology. The following five concepts associated with the choice of oncology had the highest recurrence among these seven surveys, which had been conducted in different countries and at various times: interest in cancer management, initiation of the specialty during the 2nd cycle, job opportunities, low working hours, and quality of life. DISCUSSION: The literature on this topic is particularly scarce. No qualitative studies have been published in the English language. The limited data in the literature do not allow us to fully comprehend the problem.

Chromosome-level assembly of the gray fox (Urocyon cinereoargenteus) confirms the basal loss of PRDM9 in Canidae.

Reference genome assemblies have been created from multiple lineages within the Canidae family; however, despite its phylogenetic relevance as a basal genus within the clade, there is currently no reference genome for the gray fox (Urocyon cinereoargenteus). Here, we present a chromosome-level assembly for the gray fox (U. cinereoargenteus), which represents the most contiguous, non-domestic canid reference genome available to date, with 90% of the genome contained in just 34 scaffolds and a contig N50 and scaffold N50 of 59.4 and 72.9 Megabases, respectively. Repeat analyses identified an increased number of simple repeats relative to other canids. Based on mitochondrial DNA, our Vermont sample clusters with other gray fox samples from the northeastern United States and contains slightly lower levels of heterozygosity than gray foxes on the west coast of California. This new assembly lays the groundwork for future studies to describe past and present population dynamics, including the delineation of evolutionarily significant units of management relevance. Importantly, the phylogenetic position of Urocyon allows us to verify the loss of PRDM9 functionality in the basal canid lineage, confirming that pseudogenization occurred at least 10 million years ago.

Culex erraticus (Diptera: Culicidae) utilizes gopher tortoise burrows for overwintering in North Central Florida.

Mosquito-borne diseases represent a significant threat to human and animal health in the United States. Several viruses, including West Nile, Saint Louis encephalitis, and Eastern equine encephalitis are endemic. In humans, the disease is typically detected during the summer months, but not during the winter months. The ability of these viruses to reemerge year after year is still not fully understood, but typically involves persistence in a reservoir host or vector during periods of low transmission. Mosquito species are known to overwinter at different life stages (adults, larvae, or eggs) in manufactured or natural sites. Gopher tortoise burrows are known to serve as refuge for many vertebrate and invertebrate species in pine savannas. In this study, we surveyed the interior of gopher tortoise burrows for overwintering mosquitoes. We identified 4 species (Anopheles crucians s.l., Culex erraticus, Mansonia dyari, and Uranotaenia sapphirina). Cx. erraticus was the most abundant, and its presence and abundance increased in winter months, implying that this species utilized gopher tortoise burrows for overwintering. Bloodfed Cx. erraticus and An. crucians s.l. females were detected. While An. crucians s.l. fed exclusively on the white-tailed deer, Cx. erraticus had a more diverse host range but fed primarily on the gopher tortoise. Tortoises and other long-lived reptiles like the American alligator have been shown to sustain high viremia following West Nile virus (WNV) and Eastern equine encephalitis virus (EEEV) infection and therefore could play a role in the maintenance of these viruses. In addition, Cx. erraticus is naturally infected with WNV and is a known bridge vector for EEEV. As such, these overwintering sites may play a role in perpetuating over-winter arboviral activity in Florida.

Hypervascular floor of mouth tumor: Rare presentation of oral cavity squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignancy affecting the oral cavity and commonly presents as an exophytic lesion with red or white granular ulcerations. Most diagnoses are confirmed by biopsy and clinical features; however, early SCC has been shown to hide within benign appearing lesions, such as vascular tumors, resulting in missed diagnoses and delay in treatment. The following case report will discuss a patient who presented with a mass in the floor of the mouth which appeared as a vascular tumor on exam and imaging. This was originally thought to be benign based on FNA findings however was found to harbor invasive squamous cell carcinoma on final pathology. The goal of this case report is to provide a background on the variable presentations of OSCC, vascular tumors, and uncommon presentations for which specialists should be aware of in their practice.

Efficacy and safety of long-acting cabotegravir compared with daily oral tenofovir disoproxil fumarate plus emtricitabine to prevent HIV infection in cisgender men and transgender women who have sex with men 1 year after study unblinding: a secondary analysis of the phase 2b and 3 HPTN 083 randomised controlled trial.

BACKGROUND: Injectable cabotegravir was superior to daily oral tenofovir disoproxil fumarate plus emtricitabine for HIV prevention in two clinical trials. Both trials had the primary aim of establishing the HIV prevention efficacy of long-acting injectable cabotegravir pre-exposure prophylaxis (PrEP) compared with tenofovir disoproxil fumarate plus emtricitabine daily oral PrEP. Long-acting PrEP was associated with diagnostic delays and integrase strand-transfer inhibitor (INSTI) resistance. This report presents findings from the first unblinded year of the HIV Prevention Trials Network (HPTN) 083 study. METHODS: The HPTN 083 randomised controlled trial enrolled HIV-uninfected cisgender men and transgender women at elevated HIV risk who have sex with men, from 43 clinical research sites in Africa, Asia, Latin America, and the USA. Inclusion criteria included: a negative HIV serological test at the screening and study entry, undetectable HIV RNA levels within 14 days of study entry, age 18 years or older, overall good health as determined by clinical and laboratory evaluations, and a creatinine clearance of 60 mL/min or higher. Participants were randomly allocated to receive long-acting injectable cabotegravir or daily oral tenofovir disoproxil fumarate plus emtricitabine PrEP. After study unblinding, participants remained on their original regimen awaiting an extension study. HIV infections were characterised retrospectively at a central laboratory. Here we report the secondary analysis of efficacy and safety for the first unblinded year. The primary outcome was incident HIV infection. Efficacy analyses were done on the modified intention-to-treat population using a Cox regression model. Adverse events were compared across treatment groups and time periods (blinded vs unblinded). This trial is registered with ClinicalTrials.gov, NCT02720094. FINDINGS: Of the 4488 participants who contributed person-time to the blinded analysis, 3290 contributed person-time to the first unblinded year analysis between May 15, 2020, and May 14, 2021. Updated HIV incidence in the blinded phase was 0·41 per 100 person-years for long-acting injectable cabotegravir PrEP and 1·29 per 100 person-years for daily oral tenofovir disoproxil fumarate plus emtricitabine PrEP (hazard ratio [HR] 0·31 [95% CI 0·17-0·58], p=0·0003). HIV incidence in the first unblinded year was 0·82 per 100 person-years for long-acting PrEP and 2·27 per 100 person-years for daily oral PrEP (HR 0·35 [0·18-0·69], p=0·002). Adherence to both study products decreased after study unblinding. Additional infections in the long-acting PrEP group included two with on-time injections; three with one or more delayed injections; two detected with long-acting PrEP reinitiation; and 11 more than 6 months after their last injection. Infection within 6 months of cabotegravir exposure was associated with diagnostic delays and INSTI resistance. Adverse events were generally consistent with previous reports; incident hypertension in the long-acting PrEP group requires further investigation. INTERPRETATION: Long-acting injectable cabotegravir PrEP retained high efficacy for HIV prevention in men and transgender women who have sex with men during the first year of open-label follow-up, with a near-identical HR for HIV risk reduction between long-acting injectable cabotegravir and daily oral tenofovir disoproxil fumarate plus emtricitabine PrEP during the first year after unblinding compared with the blinded period. Extended follow-up further defined the risk period for diagnostic delays and emergence of INSTI resistance. FUNDING: Division of AIDS at the National Institute of Allergy and Infectious Diseases, ViiV Healthcare, and Gilead Sciences.

Quantitative MRI of Gd-DOTA Accumulation in the Mouse Brain After Intraperitoneal Administration: Validation by Mass Spectrometry.

BACKGROUND: In mice, intraperitoneal (ip) contrast agent (CA) administration is convenient for mapping microvascular parameters over a long-time window. However, continuous quantitative MRI of CA accumulation in brain over hours is still missing. PURPOSE: To validate a quantitative time-resolved MRI technique for mapping the CA kinetics in brain upon ip administration. STUDY TYPE: Prospective, animal model. SPECIMEN: 25 C57Bl/6JRj mice underwent MRI. FIELD STRENGTH/SEQUENCE: 7-T, gradient echo sequence. ASSESSMENT: Gd-DOTA concentration was monitored by MRI (25 s/repetition) over 135 minutes with (N = 15) and without (N = 10) ip mannitol challenge (5 g/kg). After the final repetition, the brains were sampled to quantify gadolinium by mass spectrometry (MS). Upon manual brain segmentation, the average gadolinium concentration was compared with the MS quantification in transcardially perfused (N = 20) and unperfused (N = 5) mice. Precontrast T1 -maps were acquired in 8 of 25 mice. STATISTICAL TESTS: One-tailed Spearman and Pearson correlation between gadolinium quantification by MRI and by MS, D'Agostino-Pearson test for normal distribution, Bland-Altman analysis to evaluate the agreement between MRI and MS. Significance was set at P-value <0.05. RESULTS: MRI showed that ip administered CA reached the blood compartment (>5 mM) within 10 minutes and accumulated continuously for 2 hours in cerebrospinal fluid (>1 mM) and in brain tissue. The MRI-derived concentration maps showed interindividual differences in CA accumulation (from 0.47 to 0.81 mM at 2 hours) with a consistent distribution resembling the pathways of the glymphatic system. The average in-vivo brain concentration 2 hours post-CA administration correlated significantly (r = 0.8206) with the brain gadolinium quantification by MS for N = 21 paired observations available. DATA CONCLUSION: The presented experimental and imaging protocol may be convenient for monitoring the spatiotemporal pattern of CA uptake and clearance in the mouse brain over 2 hours. The quantification of the CA from the MRI signal in brain is corroborated by MS. EVIDENCE LEVEL: N/A TECHNICAL EFFICACY: Stage 1.

Risk Factors for Hyperosmolar Hyperglycemic State in Pediatric Type 2 Diabetes.

Background: There is a paucity of data on the risk factors for the hyperosmolar hyperglycemic state (HHS) compared with diabetic ketoacidosis (DKA) in pediatric type 2 diabetes (T2D). Methods: We used the national Kids' Inpatient Database to identify pediatric admissions for DKA and HHS among those with T2D in the years 2006, 2009, 2012, and 2019. Admissions were identified using ICD codes. Those aged <9yo were excluded. We used descriptive statistics to summarize baseline characteristics and Chi-squared test and logistic regression to evaluate factors associated with admission for HHS compared with DKA in unadjusted and adjusted models. Results: We found 8,961 admissions for hyperglycemic emergencies in youth with T2D, of which 6% were due to HHS and 94% were for DKA. These admissions occurred mostly in youth 17-20 years old (64%) who were non-White (Black 31%, Hispanic 20%), with public insurance (49%) and from the lowest income quartile (42%). In adjusted models, there were increased odds for HHS compared to DKA in males (OR 1.77, 95% CI 1.42-2.21) and those of Black race compared to those of White race (OR 1.81, 95% CI 1.34-2.44). Admissions for HHS had 11.3-fold higher odds for major or extreme severity of illness and 5.0-fold higher odds for mortality. Conclusion: While DKA represents the most admissions for hyperglycemic emergencies among pediatric T2D, those admitted for HHS had higher severity of illness and mortality. Male gender and Black race were associated with HHS admission compared to DKA. Additional studies are needed to understand the drivers of these risk factors.

Artificial light impairs local attraction to females in male glow-worms.

The negative effects of artificial lighting at night (ALAN) on insects are increasingly recognised and have been postulated as one possible cause of declines in insect populations. Yet, the behavioural mechanisms underpinning ALAN effects on insects remain unclear. ALAN interferes with the bioluminescent signal female glow-worms use to attract males, disrupting reproduction. To determine the behavioural mechanisms that underpin this effect of ALAN, we quantified the effect of white illumination on males' ability to reach a female-mimicking LED within a Y-maze. We show that as the intensity of illumination increases, the proportion of males reaching the female-mimicking LED declines. Brighter illumination also increases the time taken by males to reach the female-mimicking LED. This is a consequence of males spending more time: (i) in the central arm of the Y-maze; and (ii) with their head retracted beneath their head shield. These effects reverse rapidly when illumination is removed, suggesting that male glow-worms are averse to white light. Our results show that ALAN not only prevents male glow-worms from reaching females, but also increases the time they take to reach females and the time they spend avoiding exposure to light. This demonstrates that the impacts of ALAN on male glow-worms extend beyond those previously observed in field experiments, and raises the possibility that ALAN has similar behavioural impacts on other insect species that remain undetected in field experiments.

Methanotrophic oxidation of organic micropollutants and nitrogen upcycling in a hybrid membrane biofilm reactor (hMBfR) for simultaneous O2 and CH4 supply.

Pharmaceuticals and other organic micropollutants (OMPs) present in wastewater effluents are of growing concern, as they threaten environmental and human health. Conventional biological treatments lead to limited removal of OMPs. Methanotrophic bacteria can degrade a variety of OMPs. By employing a novel bubble-free hybrid membrane biofilm bioreactor (hMBfR), we grew methanotrophic bacteria at three CH4 loading rates. Biomass productivity and CH4 loading showed a linear correlation, with a maximum productivity of 372 mg-VSS·L-1·d-1, with corresponding biomass concentration of 1117.6 ± 56.4 mg-VSS·L-1. Furthermore, the biodegradation of sulfamethoxazole and 1H-benzotriazole positively correlated with CH4 oxidation rates, with highest biodegradation kinetic constants of 3.58 L·g-1·d-1 and 5.42 L·g-1·d-1, respectively. Additionally, the hMBfR recovered nutrients as microbial proteins, with an average content 39% DW. The biofilm community was dominated by Methylomonas, while the bulk was dominated by aerobic heterotrophic bacteria. The hMBfR removed OMPs, allowing for safer water reuse while valorising CH4 and nutrients.

Late-stage MC38 tumours recapitulate features of human colorectal cancer - implications for appropriate timepoint selection in preclinical studies.

Anti-tumour T cell responses play a crucial role in controlling the progression of colorectal cancer (CRC), making this disease a promising candidate for immunotherapy. However, responses to immune-targeted therapies are currently limited to subpopulations of patients and specific types of cancer. Clinical studies have therefore focussed on identifying biomarkers that predict immunotherapy responses and elucidating the immunological landscapes of different cancers. Meanwhile, our understanding of how preclinical tumour models resemble human disease has fallen behind, despite their crucial role in immune-targeted drug development. A deeper understanding of these models is therefore needed to improve the development of immunotherapies and the translation of findings made in these systems. MC38 colon adenocarcinoma is a widely used preclinical model, yet how it recapitulates human colorectal cancer remains poorly defined. This study investigated the tumour-T cell immune landscape of MC38 tumours using histology, immunohistochemistry, and flow cytometry. We demonstrate that early-stage tumours exhibit a nascent TME, lacking important immune-resistance mechanisms of clinical interest, while late-stage tumours exhibit a mature TME resembling human tumours, with desmoplasia, T cell exhaustion, and T cell exclusion. Consequently, these findings clarify appropriate timepoint selection in the MC38 model when investigating both immunotherapies and mechanisms that contribute to immunotherapy resistance. Overall, this study provides a valuable resource that will enable appropriate application of the MC38 model and expedite the development and clinical translation of new immunotherapies.

Reviewing the External Jugular to Internal Jugular Vein Bypass for Circumventing Venous Congestion: Technique Description And Case Correlates.

This study reviews the operative technique of external jugular vein to the internal jugular vein (IJV) bypass and discusses its advantages of decreasing postoperative complications in bilateral neck dissection patients. A retrospective chart review was performed on 2 patients at a single institution with prior bilateral neck dissection and jugular vein bypass. The tumor resection, reconstruction, bypass, and postoperative management were led by the listed senior author (S.P.K). An 80-year-old (case 1) and a 69-year-old (case 2) underwent bilateral neck dissection with the creation of a micro-venous anastomosis. This bypass allowed for improved venous drainage without adding significant time or difficulty to the procedure. Both patients recovered well in the initial postoperative period with maintained venous drainage. This study describes an additional technique that the trained microsurgeon can consider during the index procedure and reconstruction that can benefit the patient without adding significant time or technical challenges to the remaining portion of the procedure.

Integrity and efficiency: AbbVie's journey of building an integrated nonregulated bioanalytical laboratory.

While bioanalytical outsourcing is widely adopted in the pharmaceutical industry, AbbVie is one of the few large biopharmaceutical companies having an internal bioanalytical unit to support nearly all its drug metabolism and pharmacokinetic studies. This article highlights our experience and perspective in building an integrated and centralized laboratory to provide early discovery and preclinical-stage bioanalytical support with high operational efficiency, cost-effectiveness and data integrity. The advantages of in-house nonregulated bioanalytical support include better control of data quality, faster turnaround times, real-time knowledge sharing and troubleshooting, and lower near- and long-term costs. The success of an in-house model depends upon a comprehensively optimized and streamlined workflow, fueled by continuous improvements and implementation of innovative technologies.

Orthoplastic Treatment of Open Lower-Limb Fractures Improves Outcomes: A 12-Year Review.

BACKGROUND: The British Orthopaedic Association Standards for Orthopaedics and Trauma 4 (BOAST 4) inform the management of open lower-limb fractures. The authors conducted repeated reviews of performance against these standards over a 12-year period. This latest iteration has shown further improvements in outcomes concomitant with changes in service delivery. METHODS: Data on Gustilo-Anderson grade IIIB or IIIC open lower-limb fractures were collected from a prospectively constructed departmental database and analyzed using Excel. Outcomes assessed included time to stabilization, time to definitive soft-tissue coverage, and deep infection rates. RESULTS: A total of 69% of patients in our cohort received care that aligned with BOAST 4 guidelines. Median time to stabilization was 14.2 hours and to soft-tissue coverage was 47 hours, with 71% of cases compliant with BOAST 4 guidelines. The overall deep infection rate was 6.5% in our cohort. There was a significantly lower deep infection rate in BOAST 4-compliant cases (2%) versus noncompliant cases (16%), respectively (P = 0.05). A total of 41 of 61 patients had fixation and soft-tissue coverage in a single operation (fix and flap), eight had staged operations, and 12 required local flap closure. There was no significant difference in deep infection rates among these approaches. CONCLUSIONS: Compliance with the BOAST 4 guidelines and time to definitive soft-tissue coverage have improved at our center since the last review. Deep infection rates were significantly lower in BOAST 4-compliant cases, further validating this approach. The fix and flap technique was introduced during the study period and reduces operative burden for patients. These results support a joint orthoplastic approach as the optimal management for these complex injuries. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.