Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism.

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.

Genome sequencing identifies biallelic variants in SCLT1 in a patient with syndromic nephronophthisis: Reflections on the SCLT1-related ciliopathy spectrum.

Ciliopathies represent a major category of rare multisystem disease. Arriving at a specific diagnosis for a given patient is challenged by the significant genetic and clinical heterogeneity of these conditions. We report the outcome of the diagnostic odyssey of a child with obesity, renal, and retinal disease. Genome sequencing identified biallelic splice site variants in sodium channel and clathrin linker 1 (SCLT1), an emerging ciliopathy gene. We review the literature on all patients reported with biallelic SCLT1 variants highlighting a frequent clinical presentation that overlaps Bardet-Biedl and Senior-Loken syndromes. We also discuss current concepts in syndrome designation in light of these data.

Biomechanical comparison of titanium alloy additively manufactured and conventionally manufactured plate-screw constructs.

AIM: To biomechanically compare the bending stiffness, strength, and cyclic fatigue of titanium additively manufactured (AM) and conventionally manufactured (CM) limited contact plates (LCP) of equivalent dimensions using plate-screw constructs. METHODS: Twenty-four 1.5/2.0-mm plate constructs (CM: n = 12; AM: n = 12) were placed under 4-point bending conditions. Data were collected during quasi-static single cycle to failure and cyclic fatigue testing until implants plastically deformed or failed. Bending stiffness, bending structural stiffness, and bending strength were determined from load-displacement curves. Fatigue life was determined as number of cycles to failure. Median test variables for each method were compared using the Wilcoxon rank sum test within each group. Fatigue data was also analysed by the Kaplan-Meier estimator of survival function. RESULTS: There was no evidence for a difference in bending stiffness and bending structural stiffness between AM and CM constructs. However, AM constructs exhibited greater bending strength (median 3.07 (min 3.0, max 3.4) Nm) under quasi-static 4-point bending than the CM constructs (median 2.57 (min 2.5, max 2.6) Nm, p = 0.006). Number of cycles to failure under dynamic 4-point bending was higher for the CM constructs (median 164,272 (min 73,557, max 250,000) cycles) than the AM constructs (median 18,704 (min 14,427, max 33,228) cycles; p = 0.02). Survival analysis showed that 50% of AM plates failed by 18,842 cycles, while 50% CM plates failed by 78,543 cycles. CONCLUSION AND CLINICAL RELEVANCE: Additively manufactured titanium implants, printed to replicate a conventional titanium orthopaedic plate, were more prone to failure in a shorter fatigue period despite being stronger in single cycle to failure. Patient-specific implants made using this process may be brittle and therefore not comparable to CM orthopaedic implants. Careful selection of their use on a case/patient-specific basis is recommended.

2023 American Society of Anesthesiologists Practice Guidelines for Preoperative Fasting: Carbohydrate-containing Clear Liquids with or without Protein, Chewing Gum, and Pediatric Fasting Duration-A Modular Update of the 2017 American Society of Anesthesiologists Practice Guidelines for Preoperative Fasting.

These practice guidelines are a modular update of the "Practice guidelines for preoperative fasting and the use of pharmacologic agents to reduce the risk of pulmonary aspiration: Application to healthy patients undergoing elective procedures." The guidance focuses on topics not addressed in the previous guideline: ingestion of carbohydrate-containing clear liquids with or without protein, chewing gum, and pediatric fasting duration.

2023 American Society of Anesthesiologists Practice Guidelines for Monitoring and Antagonism of Neuromuscular Blockade: A Report by the American Society of Anesthesiologists Task Force on Neuromuscular Blockade.

These practice guidelines provide evidence-based recommendations on the management of neuromuscular monitoring and antagonism of neuromuscular blocking agents during and after general anesthesia. The guidance focuses primarily on the type and site of monitoring and the process of antagonizing neuromuscular blockade to reduce residual neuromuscular blockade.

Interactions between fitness components across the life cycle constrain competitor coexistence.

Numerous mechanisms can promote competitor coexistence. Yet, these mechanisms are often considered in isolation from one another. Consequently, whether multiple mechanisms shaping coexistence combine to promote or constrain species coexistence remains an open question. Here, we aim to understand how multiple mechanisms interact within and between life stages to determine frequency-dependent population growth, which has a key role stabilizing local competitor coexistence. We conducted field experiments in three lakes manipulating relative frequencies of two Enallagma damselfly species to evaluate demographic contributions of three mechanisms affecting different fitness components across the life cycle: the effect of resource competition on individual growth rate, predation shaping mortality rates, and mating harassment determining fecundity. We then used a demographic model that incorporates carry-over effects between life stages to decompose the relative effect of each fitness component generating frequency-dependent population growth. This decomposition showed that fitness components combined to increase population growth rates for one species when rare, but they combined to decrease population growth rates for the other species when rare, leading to predicted exclusion in most lakes. Because interactions between fitness components within and between life stages vary among populations, these results show that local coexistence is population specific. Moreover, we show that multiple mechanisms do not necessarily increase competitor coexistence, as they can also combine to yield exclusion. Identifying coexistence mechanisms in other systems will require greater focus on determining contributions of different fitness components across the life cycle shaping competitor coexistence in a way that captures the potential for population-level variation.

Quantitative Neuromuscular Monitoring in Clinical Practice: A Professional Practice Change Initiative.

BACKGROUND: Residual neuromuscular blockade can be avoided with quantitative neuromuscular monitoring. The authors embarked on a professional practice initiative to attain documented train-of-four ratios greater than or equal to 0.90 in all patients for improved patient outcomes through reducing residual paralysis. METHODS: The authors utilized equipment trials, educational videos, quantitative monitors in all anesthetizing locations, and electronic clinical decision support with real-time alerts, and initiated an ongoing professional practice metric. This was a retrospective assessment (2016 to 2020) of train-of-four ratios greater than or equal to 0.9 that were documented before extubation. Anesthesia records were manually reviewed for neuromuscular blockade management details. Medical charts of surgical patients who received a neuromuscular blocking drug were electronically searched for patient characteristics and outcomes. RESULTS: From pre- to postimplementation, more patients were assigned American Society of Anesthesiologists Physical Status III to V, fewer were inpatients, the rocuronium average dose was higher, and more patients had a prereversal train-of-four count less than 4. Manually reviewed anesthesia records (n = 2,807) had 2 of 172 (1%) cases with documentation of train-of-four ratios greater than or equal to 0.90 in November 2016, which was fewer than the cases in December 2020 (250 of 269 [93%]). Postimplementation (February 1, 2020, to December 31, 2020), sugammadex (650 of 935 [70%]), neostigmine (195 of 935 [21%]), and no reversal (90 of 935 [10%]) were used to attain train-of-four ratios greater than or equal to 0.90 in 856 of 935 (92%) of patients. In the electronically searched medical charts (n = 20,181), postimplementation inpatients had shorter postanesthesia care unit lengths of stay (7% difference; median [in min] [25th, 75th interquartile range], 73 [55, 102] to 68 [49, 95]; P < 0.001), pulmonary complications were less (43% difference; 94 of 4,138 [2.3%] to 23 of 1,817 [1.3%]; P = 0.010; -1.0% difference [95% CI, -1.7 to -0.3%]), and hospital length of stay was shorter (median [in days] [25th, 75th], 3 [2, 5] to 2 [1, 4]; P < 0.001). CONCLUSIONS: In this professional practice initiative, documentation of train-of-four ratios greater than or equal to 0.90 occurred for 93% of patients in a busy clinical practice. Return-of-strength documentation is an intermediate outcome, and only one of many factors contributing to patient outcomes.

The Aryl Hydrocarbon Receptor Undergoes Chaperone-Mediated Autophagy in Triple-Negative Breast Cancer Cells.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.

Gut microbiome profiling of a rural and urban South African cohort reveals biomarkers of a population in lifestyle transition.

BACKGROUND: Comparisons of traditional hunter-gatherers and pre-agricultural communities in Africa with urban and suburban Western North American and European cohorts have clearly shown that diet, lifestyle and environment are associated with gut microbiome composition. Yet, little is known about the gut microbiome composition of most communities in the very diverse African continent. South Africa comprises a richly diverse ethnolinguistic population that is experiencing an ongoing epidemiological transition and concurrent spike in the prevalence of obesity, largely attributed to a shift towards more Westernized diets and increasingly inactive lifestyle practices. To characterize the microbiome of African adults living in more mainstream lifestyle settings and investigate associations between the microbiome and obesity, we conducted a pilot study, designed collaboratively with community leaders, in two South African cohorts representative of urban and transitioning rural populations. As the rate of overweight and obesity is particularly high in women, we collected single time-point stool samples from 170 HIV-negative women (51 at Soweto; 119 at Bushbuckridge), performed 16S rRNA gene sequencing on these samples and compared the data to concurrently collected anthropometric data. RESULTS: We found the overall gut microbiome of our cohorts to be reflective of their ongoing epidemiological transition. Specifically, we find that geographical location was more important for sample clustering than lean/obese status and observed a relatively higher abundance of the Melainabacteria, Vampirovibrio, a predatory bacterium, in Bushbuckridge. Also, Prevotella, despite its generally high prevalence in the cohorts, showed an association with obesity. In comparisons with benchmarked datasets representative of non-Western populations, relatively higher abundance values were observed in our dataset for Barnesiella (log2fold change (FC) = 4.5), Alistipes (log2FC = 3.9), Bacteroides (log2FC = 4.2), Parabacteroides (log2FC = 3.1) and Treponema (log2FC = 1.6), with the exception of Prevotella (log2FC = - 4.7). CONCLUSIONS: Altogether, this work identifies putative microbial features associated with host health in a historically understudied community undergoing an epidemiological transition. Furthermore, we note the crucial role of community engagement to the success of a study in an African setting, the importance of more population-specific studies to inform targeted interventions as well as present a basic foundation for future research.

Potency Analysis of Mesenchymal Stromal Cells Using a Phospho-STAT Matrix Loop Analytical Approach.

Potency assays for mesenchymal stromal cells (MSCs) need to be defined in advanced clinical trials. Here, we have developed an assay matrix approach that captures the signal transducer and activator of transcription (STAT) phosphorylation of MSCs upon stimulation with their combined secretome that arose with the interaction of activated peripheral blood mononuclear cells (PBMCs). Secretome of heat-inactivated (HI) MSCs cocultured with and without activated PBMCs was used as an internal reference. We have compared the short-term phosphorylation status of STAT1, STAT3, STAT4, STAT5, and STAT6 on MSCs derived from human bone marrow, adipose tissue, and umbilical cord using phosflow technology. Secretome of live MSCs cocultured with activated PBMCs downregulate STAT1 and STAT3 phosphorylation on MSCs, whereas the secretome of HI-MSCs or PBMCs do not. Thus, investigation of the combined secretome of MSC and PBMC interaction on MSCs determine the potency of MSCs as the generator and sensor of the secretome. Bone marrow, adipose, and umbilical cord MSCs are comparable in modulating STAT1 and STAT3 responses. Measurements of STAT1 and STAT3 phosphorylation on MSCs as responder cells correlate and predict allogeneic T-cell suppression. Our comparative phosphomatrix approach between live and reference HI-MSCs defines the potency of MSCs as both stimulators and responders as part of a robust platform for predictive potency analysis. Stem Cells 2019;37:1119-1125.

Functional trait similarity predicts survival in rare plant reintroductions.

Rare species reintroductions are an increasingly common conservation strategy, but often result in poor survival of reintroduced individuals. Reintroduction sites are chosen primarily based on historical occupancy and/or abiotic properties of the site, with much less consideration given to properties of the larger biotic community. However, ecological niche theory suggests that the ability to coexist with other species is determined in part by the degree of functional similarity between species. The degree to which functional similarity affects the survival of reintroduced plants is poorly understood, but has important implications for the allocation of limited conservation resources. We collected a suite of abiotic, biotic, and functional trait variables centered on outplanted individuals from four reintroduced rare plant species and used logistic regression and model selection to assess their influence on individual survival. We show that higher functional similarity between reintroduced individuals and the local community, measured by differences between their multivariate functional traits and the community-weighted mean traits of their immediate neighbors, increases survival and is a stronger predictor of survival than local variation in abiotic factors, suggesting that the functional composition of the biotic community should be incorporated into site selection to improve reintroduction success.

Pharmacological Inhibition of CDK8 in Triple-Negative Breast Cancer Cell Line MDA-MB-468 Increases E2F1 Protein, Induces Phosphorylation of STAT3 and Apoptosis.

Cyclin-dependent kinase 8 (CDK8) has been identified as a colon cancer oncogene. Since this initial observation, CDK8 has been implicated as a potential driver of other cancers including acute myelogenous leukemia (AML) and some breast cancers. Here, we observed different biological responses to CDK8 inhibition among colon cancer cell lines and the triple-negative breast cancer (TNBC) cell line MDA-MB-468. When treated with CDK8 inhibitor 4, all treated cell lines responded with decreased cell viability and increased apoptosis. In the MDA-MB-468 cell line, the decrease in cell viability was dependent on increased phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is not observed in the colon cancer cell lines. Furthermore, increased STAT3 phosphorylation in 4 treated MDA-MB-468 cells was dependent on increased transcription factor E2F1 protein. These results are consistent with previous reports of exogenous expression of E2F1-induced apoptosis in MDA-MB-468 cells.

Longitudinal proton spectroscopy study of the prefrontal cortex in youth at risk for bipolar disorder before and after their first mood episode.

OBJECTIVES: To investigate neurochemical abnormalities in the left and right ventrolateral prefrontal cortex (VLPFC) and anterior cingulate cortex (ACC) of youth at risk for bipolar disorder using proton magnetic resonance spectroscopy before and after their first mood episode. METHODS: Children and adolescents offspring of parents with bipolar I disorder (at-risk group, n = 117) and matched healthy controls (HC group, n = 61) were recruited at the University of Cincinnati. At-risk subjects had no lifetime major mood and psychotic disorders at baseline, and were followed up every 4 months to monitor for development of a major depressive, manic, hypomanic, or mixed mood episode. Levels of N-acetyl-aspartate (NAA), phosphocreatine plus creatine (PCr + Cr), choline-containing compounds, myo-inositol, and glutamate were determined using LCModel and corrected for partial volume effects. RESULTS: There were no baseline differences in metabolite levels for any of the brain regions between at-risk and HC youth. Nineteen at-risk subjects developed a first mood episode during follow-up. Survival analyses showed that baseline PCr + Cr levels in the left VLPFC significantly predicted a mood episode during follow-up in the at-risk group (HR: 0.47, 95% CI: 0.27-0.82, P = 0.008). There were no longitudinal changes in metabolites levels in the VLPFC and ACC before and after a mood episode in at-risk subjects. CONCLUSIONS: We found no evidence for abnormal proton spectroscopy metabolite levels in the VLPFC and ACC of at-risk youth, prior and after the development of their first mood episode. Preliminary findings of association between baseline PCr + Cr levels in the left VLPFC and risk to develop a mood episode warrant further investigation.

Collective decision-making appears more egalitarian in populations where group fission costs are higher.

Collective decision-making is predicted to be more egalitarian in conditions where the costs of group fission are higher. Here, we ask whether Trinidadian guppies (Poecilia reticulata) living in high or low predation environments, and thereby facing differential group fission costs, make collective decisions in line with this prediction. Using a classic decision-making scenario, we found that fish from high predation environments switched their positions within groups more frequently than fish from low predation environments. Because the relative positions individuals adopt in moving groups can influence their contribution towards group decisions, increased positional switching appears to support the prediction of more evenly distributed decision-making in populations where group fission costs are higher. In an agent-based model, we further identified that more frequent, asynchronous updating of individuals' positions could explain increased positional switching, as was observed in fish from high predation environments. Our results are consistent with theoretical predictions about the structure of collective decision-making and the adaptability of social decision-rules in the face of different environmental contexts.

The homologous recombination protein RAD51D protects the genome from large deletions.

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that protects the genome from chromosomal instability. RAD51 mediator proteins (i.e. paralogs) are critical for efficient HR in mammalian cells. However, how HR-deficient cells process DSBs is not clear. Here, we utilized a loss-of-function HR-reporter substrate to simultaneously monitor HR-mediated gene conversion and non-conservative mutation events. The assay is designed around a heteroallelic duplication of the Aprt gene at its endogenous locus in isogenic Chinese hamster ovary cell lines. We found that RAD51D-deficient cells had a reduced capacity for HR-mediated gene conversion both spontaneously and in response to I-SceI-induced DSBs. Further, RAD51D-deficiency shifted DSB repair toward highly deleterious single-strand annealing (SSA) and end-joining processes that led to the loss of large chromosomal segments surrounding site-specific DSBs at an exceptionally high frequency. Deletions in the proximity of the break were due to a non-homologous end-joining pathway, while larger deletions were processed via a SSA pathway. Overall, our data revealed that, in addition to leading to chromosomal abnormalities, RAD51D-deficiency resulted in a high frequency of deletions advancing our understanding of how a RAD51 paralog is involved in maintaining genomic stability and how its deficiency may predispose cells to tumorigenesis.

The University of South Dakota Physician Assistant Studies Program.

The University of South Dakota Physician Assistant Studies Program (USD PA Program) is in its 26th year of operation. The mission remains the same: to provide "a comprehensive primary care education that prepares graduates to deliver high-quality health care to meet the needs of patients in South Dakota and the surrounding region." The inaugural class graduated in 1995 making the class of 2018 our 24th. Graduates now number 462. The purpose of this article is to provide a brief historical background and to describe the evolution of the program and its contribution to the health care workforce of South Dakota and the region.

Aiming for elimination: Outcomes of a consultation pathway supporting regional general practitioners to prescribe direct-acting antiviral therapy for hepatitis C.

To increase access to treatment, the Australian government enabled general practitioners (GPs) to prescribe direct-acting antivirals (DAAs) to treat hepatitis C virus (HCV)-in consultation with a specialist if inexperienced in HCV management. This study describes the establishment and outcomes of a remote consultation pathway supporting GPs to treat HCV. Key stakeholders from primary and tertiary healthcare services in the Barwon South Western region developed and implemented an HCV remote consultation pathway. Pharmaceutical Benefits Schedule prescription data were used to evaluate GP DAA prescription 12 months pre-and post- pathway implementation. A retrospective review of patients referred for remote consultation for 12 months post- pathway inception was undertaken to determine the care cascade. HCV treatment initiation by GPs increased after implementation of the remote consultation pathway. In the 12-month study period, 74 GPs referred 169 people for remote consultation; 114 (67%) were approved for GP DAA treatment; 48 (28%) were referred for specialist assessment. In total, 119 (71%) patients commenced DAA; 69 were eligible for SVR12 assessment. Post-treatment HCV RNA data were available for 52 (75%) people; 37 achieved SVR12; 15 achieved SVR ranging from week 5 to 11 post-treatment. No treatment failure was detected. Collaborative development and implementation of a remote consultation pathway has engaged regional GPs in managing HCV. Follow-up post-treatment could be improved; however, no treatment failure has been documented. To eliminate HCV as a public health threat, it is vital that specialists support GPs to prescribe DAA.

Preliminary in vitro and in vivo investigation of a potent platelet derived growth factor receptor (PDGFR) family kinase inhibitor.

Aberrant expression of wild-type and mutant forms of the platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases has been implicated in various oncologic indications such as leukemias, gliomas, and soft tissue sarcomas. Clinically used kinase inhibitors imatinib and sunitinib are potent inhibitors of wild-type PDGFR family members, but show reduced binding to mutant forms. Here we describe compound 5 which binds to both wild-type and oncogenic mutant forms of PDGFR family members, and demonstrates both cellular and in vivo activity.

Comparison of Recent Oil and Gas, Wind Energy, and Other Anthropogenic Landscape Alteration Factors in Texas Through 2014.

Recent research assessed how hydrocarbon and wind energy expansion has altered the North American landscape. Less understood, however, is how this energy development compares to other anthropogenic land use changes. Texas leads U.S. hydrocarbon production and wind power generation and has a rapidly expanding population. Thus, for ~47% of Texas (~324,000 km2), we mapped the 2014 footprint of energy activities (~665,000 oil and gas wells, ~5700 wind turbines, ~237,000 km oil and gas pipelines, and ~2000 km electrical transmission lines). We compared the footprint of energy development to non-energy-related activities (agriculture, roads, urbanization) and found direct landscape alteration from all factors affects ~23% of the study area (~76,000 km2), led by agriculture (~16%; ~52,882 km2). Oil and gas activities altered <1% of the study area (2081 km2), with 838 km2 from pipelines and 1242 km2 from well pad construction-and that the median Eagle Ford well pad is 7.7 times larger than that in the Permian Basin (16,200 vs. 2100 m2). Wind energy occupied <0.01% (~24 km2), with ~14 km2 from turbine pads and ~10 km2 from power transmission lines. We found that edge effects of widely-distributed energy infrastructure caused more indirect landscape alteration than larger, more concentrated urbanization and agriculture. This study presents a novel technique to quantify and compare anthropogenic activities causing both direct and indirect landscape alteration. We illustrate this landscape-mapping framework in Texas for the Spot-tailed Earless Lizard (Holbrookia lacerata); however, the approach can be applied to a range of species in developing regions globally.