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Introduction: The amphibian skin microbiome is an important line of defense against pathogens including the deadly chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd is known to preferentially infect ventral skin surfaces and feet of host amphibians, often leaving dorsal surfaces like the back uninfected. Within-individual variation in infection distribution across the skin, therefore, may relate to differences in microbiomes among skin regions. However, microbiome heterogeneity within amphibian individuals remains poorly characterized. Methods: We utilized 16S rRNA gene amplicon sequencing to compare microbiomes of 10 body regions from nine captive Rana sierrae individuals and their tank environments. These individuals were naive to Bd, allowing us to assess whether microbiomes differed among body regions prior to any impacts that may be caused by infection. Results: We found that frog skin and tank environments harbored distinct microbial communities. On frog skin, the bacterial families Burkholderiaceae (phylum Proteobacteria) and Rubritaleaceae (phylum Verrucomicrobia) were dominant, driven in large part by relative abundances of undescribed members of these families that were significantly higher on frogs than in their environment. Within individuals, we detected differences between microbiomes of body regions where Bd infection would be expected compared to regions that infrequently experience infection. Notably, putative Bd-inhibitory relative abundance was significantly higher on body regions where Bd infection is often localized. Discussion: These findings suggest that microbiomes in certain skin regions may be predisposed for interactions with Bd. Further, our results highlight the importance of considering intraindividual heterogeneities, which could provide insights relevant to predicting localized interactions with pathogens.

The global impact of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), persists in part due to the emergence of new variants. Understanding variant-specific infection dynamics and pathogenesis in murine models is crucial for identifying phenotypic changes and guiding the development of countermeasures. To address the limitations of earlier studies that investigated only a few variants or used small sample sizes, we evaluated clinical disease, infection kinetics, viral titers, cellular localization, and histopathologic changes in the lungs and brains of transgenic B6.Cg-Tg(K18-ACE2)2Prlmn/J ("K18") and corresponding genetic control (C57BL/6J) mice expressing human angiotensin-converting enzyme 2 (hACE2). Six SARS-CoV-2 variants were assessed: B.1 (WA1-like), alpha, beta, delta, omicron, and omicron XBB.1.5, using cohorts of ≥18 mice. Following intranasal inoculation with B.1, alpha, beta, or delta variants, K18 mice experienced rapid weight loss and reached euthanasia criteria by 5-6 days post-inoculation (dpi). In contrast, K18 mice inoculated with both omicron variants recovered to their starting weight within 4-6 dpi. Infectious SARS-CoV-2 was detected in the oropharynx at 1 and2 dpi, in the lungs at 2, 4, and 6 dpi, and in the brain at 4 and 6 dpi for all variants except omicron. SARS-CoV-2 nucleoprotein was detected, and interstitial pneumonia of varying severity was observed in K18 mice infected with all variants. Brain lesions were identified in mice infected with the B.1, beta, and delta variants 6 dpi. As K18 mice express hACE2 in the brain-a feature not present in humans-we also compared infection dynamics of three variants to those of a mouse-adapted WA1 strain in C57BL/6J mice lacking the human ACE2 gene. C57BL/6J mice did not experience lethal disease, exhibited milder pneumonia, and had no evidence of neuroinvasion despite similar infection kinetics to K18 mice. These findings demonstrate contrasting phenotypes across the two models and reduced tropism and pathology of omicron compared to earlier variants in both models. This comprehensive analysis of SARS-CoV-2 variants in two mouse models provides valuable insights for model and variant selection for future studies.

Grades are a staple of education and a gateway to future career opportunities. Yet, grading practices can (re)produce inequities and cause students to feel inadequate and unmotivated. Alternative grading practices may address these problems, but these strategies are often time intensive and impractical in larger classroom settings. In this study, we explore an easy-to-implement grading practice, in-class quiz retakes, to conceptualize how grades motivate learning and impact well-being for undergraduate students in science, technology, engineering, and mathematics (STEM). Through the lens of self-determination theory, we conducted semistructured interviews with undergraduates who experienced quiz retakes in two STEM courses. Our results revealed that retakes largely improved students' perceptions of their competence in the subject matter, autonomy in grade outcomes, feelings of relatedness to the instructors, and overall motivation to learn. The majority of students also expressed how traditional grading practices negatively impacted their motivation and well-being. In addition, a quantitative analysis revealed that quiz retakes particularly benefitted students who scored lower on their initial quizzes. We aspire for this study to prompt educators to reconsider traditional grading practices by opting for more equitable and just alternative grading approaches that motivate student learning and mitigate systemic barriers in education.

The evolution of oxygenic photosynthesis in the Cyanobacteria was one of the most transformative events in Earth history, eventually leading to the oxygenation of Earth's atmosphere. However, it is difficult to understand how the earliest Cyanobacteria functioned or evolved on early Earth in part because we do not understand their ecology, including the environments in which they lived. Here, we use a cutting-edge bioinformatics tool to survey nearly 500,000 metagenomes for relatives of the taxa that likely bookended the evolution of oxygenic photosynthesis to identify the modern environments in which these organisms live. Ancestral state reconstruction suggests that the common ancestors of these organisms lived in terrestrial (soil and/or freshwater) environments. This restricted distribution may have increased the lag between the evolution of oxygenic photosynthesis and the oxygenation of Earth's atmosphere.IMPORTANCECyanobacteria generate oxygen as part of their metabolism and are responsible for the rise of oxygen in Earth's atmosphere over two billion years ago. However, we do not know how long this process may have taken. To help constrain how long this process would have taken, it is necessary to understand where the earliest Cyanobacteria may have lived. Here, we use a cutting-edge bioinformatics tool called branch water to examine the environments where modern Cyanobacteria and their relatives live to constrain those inhabited by the earliest Cyanobacteria. We find that these species likely lived in non-marine environments. This indicates that the rise of oxygen may have taken longer than previously believed.

Necrobiosis is a histologic term used to describe abnormal deposits of "degenerating" collagen within the skin. It can be found as an incidental finding in various granulomatous conditions, but is a hallmark of necrobiosis lipoidica (NL) and necrobiotic xanthogranuloma (NXG). There is limited prior research on necrobiosis. Here, we employed single-cell analysis of lesional and nonlesional skin to study the pathophysiology of necrobiosis. Our findings demonstrate that necrobiotic lesional skin is characterized by SPP1hi macrophages expressing MARCO; NKG7-expressing effector CD8+ T cells coexpressing CCL5, IFNG, GZMs, and PRF1; CCL5hi fibroblasts coexpressing CXCL9, diverse collagens (e.g., COL4A4, COL11A1, COL8A1), and TIMP1; and IGHM-expressing plasma cells. Integrative analysis of signaling ligands and receptor expression identified strong cell-cell communication between NKG7+ T cells, CCL5hi fibroblasts, and SPP1-expressing macrophages. In contrast, these cell populations were not dominant features of systemic sclerosis, another collagen deposition disease. Furthermore, although SPP1-expressing macrophages were detectable in sarcoidosis, IFNG-expressing T cells were a more defining feature of sarcoidosis compared with NL and NXG. From these findings, we speculate that necrobiosis results from the deposition of diverse collagens and ECM proteins through a process driven by CCL5-expressing fibroblasts and SPP1-expressing macrophages.

Repairing DNA double-strand breaks is crucial for maintaining genome integrity, which occurs primarily through homologous recombination (HR) in Saccharomyces cerevisiae. Nucleosomes, composed of DNA wrapped around a histone octamer, present a natural barrier to end resection to initiate HR, but the impact on the downstream HR steps of homology search, DNA strand invasion, and repair synthesis remain to be determined. Displacement loops (D-loops) play a pivotal role in HR, yet the influence of chromatin dynamics on D-loop metabolism remains unclear. Using the physical D-loop capture and D-loop extension (DLE) assays to track HR intermediates, we employed genetic analysis to reveal that H2B mono-ubiquitylation (H2Bubi) affects multiple steps during HR repair. We infer that H2Bubi modulates chromatin structure, not only promoting histone degradation for nascent D-loop formation but also stabilizing extended D-loops through nucleosome assembly. Furthermore, H2Bubi regulates DNA resection via Rad9 recruitment to suppress a feedback control mechanism that dampens D-loop formation and DLE at hyper-resected ends. Through physical and genetic assays to determine repair outcomes, we demonstrate that H2Bubi plays a crucial role in preventing break-induced replication and thus promoting genomic stability.

Homologous recombination (HR) is a high-fidelity repair mechanism for double-strand breaks. Rad51 is the key enzyme that forms filaments on single-stranded DNA (ssDNA) to catalyze homology search and DNA strand exchange in recombinational DNA repair. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) to ascertain the density map of the wild-type budding yeast Rad51-ssDNA filament bound to ADP-AlF3, achieving a resolution of 2.35 Å without imposing helical symmetry. The model assigned 6 Rad51 protomers, 24 nt of DNA, and 6 bound ADP-AlF3. It shows 6-fold symmetry implying monomeric building blocks, unlike the structure of the Rad51-I345T mutant filament with three-fold symmetry implying dimeric building blocks, for which the structural comparisons provide a satisfying mechanistic explanation. This image analysis enables comprehensive comparisons of individual Rad51 protomers within the filament and reveals local conformational movements of amino acid side chains. Notably, R293 in Loop 1 adopts multiple conformations to facilitate L296 and V331 in separating and twisting the DNA triplets. We also analyzed the crystal structure of Rad51-I345T and the predicted structure of yeast Rad51-K342E using the Rad51-ssDNA structure from this study as a reference.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGOs) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotically-arrested NGOs. In male germ cells, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes, allowing germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

Background: We previously reported that machine learning could be used to predict conversion to psychosis in individuals at clinical high risk (CHR) for psychosis with up to 90% accuracy using the North American Prodrome Longitudinal Study-3 (NAPLS-3) dataset. A definitive test of our predictive model that was trained on the NAPLS-3 data, however, requires further support through implementation in an independent dataset. In this report we tested for model generalization using the previous iteration of NAPLS-3, the NAPLS-2, using the identical machine learning algorithms employed in our previous study. Method: Standard machine learning algorithms were trained to predict conversion to psychosis in clinical high risk individuals on the NAPLS-3 dataset and tested on the NAPLS-2 dataset. Results: NAPLS-2 and -3 individuals significantly differed on most features used in machine learning models. All models performed above chance, with Naive Bayes and random forest methods showing the best overall performance. Importantly, however, overall performance did not match those previously observed when using only NAPLS-3 data. Conclusion: The results of this study suggest that a machine learning model trained to predict conversion to psychosis on one dataset can be used to train an independent dataset. Performance on the test set was not in the range necessary for clinical application, however. Possible reasons that limited performance are discussed.

Treatment for enteric parasites is a common practice in beef cattle, yet little data is known about the prevalence of nematode and trematode parasite infections in beef cattle in the western United States. Likewise, the data on the efficacy of deworming practices and the presence of anthelmintic resistance (AR) of these parasites in this region is sparse. The current study collected evidence for the presence of nematode and trematode parasites in 18 herds of young beef cattle grazing either dryland or irrigated pasture in northern California as well as on efficacy and evidence of AR in a subgroup of herds. We found variable levels of fecal egg counts (FEC) ranging from 6 to 322 for the arithmetic mean eggs per gram (EPG) in the tested cattle groups. There was no difference in the number of EPG between herds grazing dryland or irrigated pasture (P = 0.54). We did not find any evidence for liver flukes or lungworms in the tested cattle. There was evidence of AR to macrocyclic lactones in all eight herds where fecal egg count reduction tests (FECRT) were performed, however due to types and execution of treatment applications and sample sizes, these results need to be interpreted with caution. The most common genus of third stage larvae in coproculture testing before treatment was Cooperia (between 55% and 98% of larvae) as well as post treatment for those herds undergoing FECRT (between 50% and 96%). Ostertagia was the second most frequent genus of larvae found in coproculture testing making up between 0% and 27% of larvae before treatment and between 5% and 50% of larvae after treatment. Anthelmintic practices in beef herds in northern California and likely in a larger geographic area in the western United States need to be updated in order to continue effective use of the currently available drugs.