The role of the antennal glands and gills in acid-base regulation and ammonia excretion of a marine osmoconforming brachyuran.

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister - an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH4+ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na+/K+-ATPase detection was observed. The V-type H+-ATPase - a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

The Combined Effects of Nicotine and Cannabis on Cortical Thickness Estimates in Adolescents and Emerging Adults.

Early life substance use, including cannabis and nicotine, may result in deleterious effects on the maturation of brain tissue and gray matter cortical development. The current study employed linear regression models to investigate the main and interactive effects of past-year nicotine and cannabis use on gray matter cortical thickness estimates in 11 bilateral independent frontal cortical regions in 223 16-22-year-olds. As the frontal cortex develops throughout late adolescence and young adulthood, this period becomes crucial for studying the impact of substance use on brain structure. The distinct effects of nicotine and cannabis use status on cortical thickness were found bilaterally, as cannabis and nicotine users both had thinner cortices than non-users. Interactions between nicotine and cannabis were also observed, in which cannabis use was associated with thicker cortices for those with a history of nicotine and tobacco product (NTP) use in three left frontal regions. This study sheds light on the intricate relationship between substance use and brain structure, suggesting a potential modulation of cannabis' impact on cortical thickness by nicotine exposure, and emphasizing the need for further longitudinal research to characterize these interactions and their implications for brain health and development.

Unveiling the Multifaceted Roles of ISG15: From Immunomodulation to Therapeutic Frontiers.

The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.

hnRNP A1 dysfunction alters RNA splicing and drives neurodegeneration in multiple sclerosis (MS).

Neurodegeneration is the primary driver of disease progression in multiple sclerosis (MS) resulting in permanent disability, creating an urgent need to discover its underlying mechanisms. Herein, we establish that dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) results in differential of binding to RNA targets causing alternative RNA splicing, which contributes to neurodegeneration in MS and its models. Using RNAseq of MS brains, we discovered differential expression and aberrant splicing of hnRNP A1 target RNAs involved in neuronal function and RNA homeostasis. We confirmed this in vivo in experimental autoimmune encephalomyelitis employing CLIPseq specific for hnRNP A1, where hnRNP A1 differentially binds and regulates RNA, including aberrantly spliced targets identified in human samples. Additionally, dysfunctional hnRNP A1 expression in neurons caused neurite loss and identical changes in splicing, corroborating hnRNP A1 dysfunction as a cause of neurodegeneration. Collectively, these data indicate hnRNP A1 dysfunction causes altered neuronal RNA splicing, resulting in neurodegeneration in MS.

Targeted Molecular Profiling of Circulating Cell-Free DNA in Patients With Advanced Hepatocellular Carcinoma.

PURPOSE: Next-generation sequencing (NGS) of tumor-derived, circulating cell-free DNA (cfDNA) may aid in diagnosis, prognostication, and treatment of patients with hepatocellular carcinoma (HCC). The operating characteristics of cfDNA mutational profiling must be determined before routine clinical implementation. METHODS: This was a single-center, retrospective study with the primary objective of defining genomic alterations in circulating cfDNA along with plasma-tissue genotype agreement between NGS of matched tumor samples in patients with advanced HCC. cfDNA was analyzed using a clinically validated 129-gene NGS assay; matched tissue-based NGS was analyzed with a US Food and Drug Administration-authorized NGS tumor assay. RESULTS: Fifty-three plasma samples from 51 patients with histologically confirmed HCC underwent NGS-based cfDNA analysis. Genomic alterations were detected in 92.2% of patients, with the most commonly mutated genes including TERT promoter (57%), TP53 (47%), CTNNB1 (37%), ARID1A (18%), and TSC2 (14%). In total, 37 (73%) patients underwent paired tumor NGS, and concordance was high for mutations observed in patient-matched plasma samples: TERT (83%), TP53 (94%), CTNNB1 (92%), ARID1A (100%), and TSC2 (71%). In 10 (27%) of 37 tumor-plasma samples, alterations were detected by cfDNA analysis that were not detected in the patient-matched tumors. Potentially actionable mutations were identified in 37% of all cases including oncogenic/likely oncogenic alterations in TSC1/2 (18%), BRCA1/2 (8%), and PIK3CA (8%). Higher average variant allele fraction was associated with elevated alpha-fetoprotein, increased tumor volume, and no previous systemic therapy, but did not correlate with overall survival in treatment-naïve patients. CONCLUSION: Tumor mutation profiling of cfDNA in HCC represents an alternative to tissue-based genomic profiling, given the high degree of tumor-plasma NGS concordance; however, genotyping of both blood and tumor may be required to detect all clinically actionable genomic alterations.

The stage-specific toxicity of per- and polyfluoroalkyl substances (PFAS) in nematode Caenorhabditis elegans.

Per- and Polyfluoroalkyl Substances (PFAS) are a diverse class of industrial chemicals that have been used for decades in industrial and commercial applications. Due to their widespread usages, persistence in the environment, and bioaccumulation in animals and humans, great public health concerns have been raised on adverse health risks of PFAS. In this study, ten PFAS were selected according to their occurrence in different water bodies. The wild-type worms were exposed to individual PFAS at 0, 0.1, 1,10, 100, and 200 µM, and the toxic effects of PFAS on growth, development, fecundity, and behavior at different life stages were investigated using a high-throughput screening (HTS) platform. Our results showed that perfluorooctanesulfonic acid (PFOS), 1H,1H, 2H, 2H-perfluorooctanesulfonamidoacetic acid (NEtFOSAA), perfluorobutanesulfonic (PFBS), and perfluorohexanesulfonic acid (PFHxS) exhibited significant inhibitive effects on the growth in the L4 larva and later stages of worms with concentrations ranging from 0.1 to 200 µmol/L. PFOS and PFBS significantly decreased the brood size of worms across all tested concentrations (p < 0.05), and the most potent PFAS is PFOS with BMC of 0.02013 µM (BMCL, 1.6e-06 µM). During adulthood, all PFAS induced a significant reduction in motility (p < 0.01), while only PFOS can significantly induce behavior alteration at the early larvae stage. Furthermore, the adverse effects occurred in larval stages were found to be the most susceptible to the PFAS exposure. These findings provide valuable insights into the potential adverse effects associated with PFAS exposure and show the importance of considering developmental stages in toxicity assessments.

Genomic and ultrastructural analysis of monkeypox virus in skin lesions and in human/animal infected cells reveals further morphofunctional insights into viral pathogenicity.

Monkeypox (MPOX) is a zoonotic disease that affects humans and other primates, resulting in a smallpox-like illness. It is caused by monkeypox virus (MPXV), which belongs to the Poxviridae family. Clinically manifested by a range of cutaneous and systemic findings, as well as variable disease severity phenotypes based on the genetic makeup of the virus, the cutaneous niche and respiratory mucosa are the epicenters of MPXV pathogenicity. Herein, we describe the ultrastructural features of MPXV infection in both human cultured cells and cutaneous clinical specimens collected during the 2022-2023 MPOX outbreak in New York City that were revealed through electron microscopy. We observed typical enveloped virions with brick-shaped morphologies that contained surface protrusions, consistent with the classic ultrastructural features of MPXV. In addition, we describe morpho-functional evidence that point to roles of distinct cellular organelles in viral assembly during clinical MPXV infection. Interestingly, in skin lesions, we found abundant melanosomes near viral assembly sites, particularly in the vicinity of mature virions, which provides further insight into virus-host interactions at the subcellular level that contribute to MPXV pathogenesis. These findings not only highlight the importance of electron microscopic studies for further investigation of this emerging pathogen but also in characterizing MPXV pathogenesis during human infection.

An MVA-based vector expressing cell-free ISG15 increases IFN-I production and improves HIV-1-specific CD8 T cell immune responses.

The human immunodeficiency virus (HIV), responsible of the Acquired Immune Deficiency Syndrome (AIDS), continues to be a major global public health issue with any cure or vaccine available. The Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is induced by interferons and plays a critical role in the immune response. ISG15 is a modifier protein that covalently binds to its targets via a reversible bond, a process known as ISGylation, which is the best-characterized activity of this protein to date. However, ISG15 can also interact with intracellular proteins via non-covalent binding or act as a cytokine in the extracellular space after secretion. In previous studies we proved the adjuvant effect of ISG15 when delivered by a DNA-vector in heterologous prime-boost combination with a Modified Vaccinia virus Ankara (MVA)-based recombinant virus expressing HIV-1 antigens Env/Gag-Pol-Nef (MVA-B). Here we extended these results evaluating the adjuvant effect of ISG15 when expressed by an MVA vector. For this, we generated and characterized two novel MVA recombinants expressing different forms of ISG15, the wild-type ISG15GG (able to perform ISGylation) or the mutated ISG15AA (unable to perform ISGylation). In mice immunized with the heterologous DNA prime/MVA boost regimen, the expression of the mutant ISG15AA from MVA-Δ3-ISG15AA vector in combination with MVA-B induced an increase in the magnitude and quality of HIV-1-specific CD8 T cells as well as in the levels of IFN-I released, providing a better immunostimulatory activity than the wild-type ISG15GG. Our results confirm the importance of ISG15 as an immune adjuvant in the vaccine field and highlights its role as a potential relevant component in HIV-1 immunization protocols.

Identification of different physiological functions within the gills and epipodites of the American lobster: Differences in metabolism, transbranchial transport, and mRNA expression.

Transbranchial transport processes are responsible for the homeostatic regulation of most essential physiological functions in aquatic crustaceans. Due to their widespread use as laboratory models, brachyuran crabs are commonly used to predict how other decapod crustaceans respond to environmental stressors including ocean acidification and warming waters. Non-brachyuran species such as the economically-valuable American lobster, Homarus americanus, possess trichobranchiate gills and epipodites that are known to be anatomically distinct from the phyllobranchiate gills of brachyurans; however, studies have yet to define their potential physiological differences. Our results indicate that the pleuro-, arthro-, and podobranch gills of the lobster are functionally homogenous and similar to the respiratory gills of brachyurans as indicated by equivalent rates of H+Eq., CO2, HCO3-, and ammonia transport and mRNA expression of related transporters and enzymes. The epipodites were found to be functionally distinct, being capable of greater individual rates of H+Eq., CO2, and ammonia transport despite mRNA transcript levels of related transporters and enzymes being only a fraction found in the gills. Collectively, mathematical estimates infer that the gills are responsible for 91% of the lobster's branchial HCO3- accumulation whereas the epipodites are responsible for 66% of branchial ammonia excretion suggesting different mechanisms exist in these tissues. Furthermore, the greater metabolic rate and amino acid catabolism in the epipodites suggest that the tissue much of the CO2 and ammonia excreted by this tissue originates intracellularly rather than systemically. These results provide evidence that non-brachyuran species must be carefully compared to brachyuran models.

Chemistry and lung toxicity of particulate matter emitted from firearms.

Smoke emissions produced by firearms contain hazardous chemicals, but little is known if their properties change depending on firearm and ammunition type and whether such changes affect toxicity outcomes. Pulmonary toxicity was assessed in mice exposed by oropharyngeal aspiration to six different types of smoke-related particulate matter (PM) samples; (1) handgun PM, (2) rifle PM, (3) copper (Cu) particles (a surrogate for Cu in the rifle PM) with and without the Cu chelator penicillamine, (4) water-soluble components of the rifle PM, (5) soluble components with removal of metal ions, and (6) insoluble components of the rifle PM. Gun firing smoke PM was in the respirable size range but the chemical composition varied with high levels of Pb in the handgun and Cu in the rifle smoke. The handgun PM did not induce appreciable lung toxicity at 4 and 24 h post-exposure while the rifle PM significantly increased lung inflammation and reduced lung function. The same levels of pure Cu particles alone and the soluble components from the rifle fire PM increased neutrophil numbers but did not cause appreciable cellular damage or lung function changes when compared to the negative (saline) control. Penicillamine treated rifle PM or Cu, slightly reduced lung inflammation and injury but did not improve the lung function decrements. Chelation of the soluble metal ions from the rifle fire PM neutralized the lung toxicity while the insoluble components induced the lung toxicity to the same degree as the rifle PM. The results show that different firearm types can generate contrasting chemical spectra in their emissions and that the rifle PM effects were mostly driven by water-insoluble components containing high levels of Cu. These findings provide better knowledge of hazardous substances in gun firing smoke and their potential toxicological profile.

Inpatient Diabetes Management.

Management of diabetes in hospitalized patients requires interdisciplinary, coordinated care that includes communication between physicians in the hospital and primary care providers. As the clinical condition of hospitalized patients can change quickly, insulin dosing must be altered in a timely manner to avoid adverse events.

Gender disparities in the National Institutes of Health funding for hematologic malignancies and cellular therapies.

We investigated gender inequality in the National Institutes of Health (NIH) funding for hematologic malignancies and cellular therapies (HMCT). The data were retrieved from the NIH Research Portfolio Online Reporting Tools (RePORT). In 2018-2019, 1834 grants totaling $799 million were awarded (men 71% vs. women 29%) to 975 principal investigators (PIs), including 680 (70%) male PIs and 295 (30%) female PIs. There was no significant gender difference in the mean grant amount per PI. Male PIs as compared to female PIs had a higher h-index (44 vs 31, p < 0.001), a higher number of publications (159.5 vs 94, p < 0.001), and higher years of active research (26 vs 21, p < 0.001). In multivariate analyses, a higher h-index independently predicted a higher mean grant amount per PI (p = 0.010), and female PIs were independently less likely to have a higher h-index (p < 0.001). Our study shows significant gender disparity in the NIH funding for HMCT research.

Analytical validation of a novel multi-target blood-based test to detect hepatocellular carcinoma.

INTRODUCTION: Surveillance is essential to diagnose and more effectively treat hepatocellular carcinoma (HCC) in at-risk patients. However, the performance of currently recommended surveillance strategies is suboptimal, particularly for early-stage detection, and patient adherence remains low. Here, we establish the analytical performance of a novel liquid biopsy test to evaluate the presence of HCC. METHODS: The multi-target HCC blood test (mt-HBT) integrates results from three DNA methylation markers (HOXA1, TSPYL5, and B3GALT6), the protein biomarker α-fetoprotein (AFP), and patient sex. The methylation markers are quantified from cell-free DNA extracted from plasma, and AFP is measured from serum. We conducted analytical validation studies on the mt-HBT, including analytical sensitivity, linearity, cross-contamination, interference, analytical accuracy, and precision. RESULTS: The mt-HBT performance met all pre-specified analytical performance criteria. The test demonstrated high reproducibility, with ≥97% concordance relative to the expected results for six categories of surrogate samples across the test's dynamic range. Of 17 candidate interfering substances, none caused significant interference to biomarker quantitation, and no occurrences of sample-to-sample cross-contamination were observed. CONCLUSION: These data demonstrate that the mt-HBT can produce consistent, reliable results for patients in the intended-use population, for whom surveillance is recommended.

hnRNP A/B Proteins: An Encyclopedic Assessment of Their Roles in Homeostasis and Disease.

The hnRNP A/B family of proteins is canonically central to cellular RNA metabolism, but due to their highly conserved nature, the functional differences between hnRNP A1, A2/B1, A0, and A3 are often overlooked. In this review, we explore and identify the shared and disparate homeostatic and disease-related functions of the hnRNP A/B family proteins, highlighting areas where the proteins have not been clearly differentiated. Herein, we provide a comprehensive assembly of the literature on these proteins. We find that there are critical gaps in our grasp of A/B proteins' alternative splice isoforms, structures, regulation, and tissue and cell-type-specific functions, and propose that future mechanistic research integrating multiple A/B proteins will significantly improve our understanding of how this essential protein family contributes to cell homeostasis and disease.

Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation.

Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.

Intravascular lithotripsy-assisted balloon angioplasty to facilitate transfemoral transcatheter aortic valve implantation in a patient with coral reef aorta.

This case describes the management of a woman in her 70s with severe symptomatic aortic stenosis and concomitant severe stenosis of the suprarenal abdominal aorta due to 'coral reef' calcification of the aortic wall and lumen (CRA). Due to her religious beliefs as a Jehovah's Witness regarding the use of blood products, she rejected the option of surgical aortic valve replacement. Transfemoral (TF) delivery of a transcatheter aortic valve was challenged by the presence of CRA. A successful TF transcatheter aortic valve implantation (TAVI) was achieved by the treatment of the CRA with intravascular lithotripsy-assisted angioplasty, followed by delivery and deployment of a self-expanding TAVI valve.

Effects of emersion on acid-base regulation, osmoregulation, and nitrogen physiology in the semi-terrestrial mangrove crab, Helice formosensis.

Emersion limits water availability and impairs the gill function of water-breathing animals resulting in a reduced capacity to regulate respiratory gas exchange, acid-base balance, and nitrogenous waste excretion. Semi-terrestrial crustaceans such as Helice formosensis mitigate these physiological consequences by modifying and recycling urine and branchial water shifting some branchial workload to the antennal glands. To investigate how this process occurs, Helice formosensis were emersed for up to 160 h and their hemolymph and urinary acid-base, nitrogenous waste, free amino acids, and osmoregulatory parameters were investigated. Upon emersion, crabs experienced a respiratory acidosis that is restored by bicarbonate accumulation and ammonia reduction within the hemolymph and urine after 24 h. Prolonged emersion caused an overcompensatory metabolic alkalosis potentially limiting the crab's ability to remain emersed. During the alkalosis, hemolymph ammonia was maintained at control levels while urinary ammonia remained reduced by 60% of control values. During emersion, ammonia may be temporarily converted to alanine as part of the Cahill cycle until re-immersion where crabs can revert alanine to ammonia for excretion coinciding with the crabs' observed delayed ammonia excretion response. The presence of high hemolymph alanine concentrations even when immersed may indicate this cycle's use outside of emersion or in preparation for emersion. Furthermore, H. formosensis appears to be uniquely capable of actively suppressing its rate of desiccation in absence of behavioral changes, in part by creating hyperosmotic urine that mitigates evaporative water loss.