bluuebunny/crossref_metadata_2025 huggingface dataset:
Healthcare Utilization Following Hemipelvectomy or Hip Disarticulation in the Military Health System
Authors: Wido Daniel , Harrington Colin J , Schulz Rebecca N , Jannace Kalyn C , Smith Douglas G , Pasquina Paul F
Published: July 2023

Introduction

Amputations at the hip and pelvic level are often performed secondary to high-energy trauma or pelvic neoplasms and are frequently associated with a prolonged postoperative rehabilitation course that involves a multitude of health care providers. The purpose of this study was to examine the health care utilization of patients with hip- and pelvic-level amputations that received care in the U.S. Military Health System.

Materials and Methods

We performed a retrospective review of all patients who underwent a hip- or pelvic-level amputation in the Military Health System between 2001 and 2017. We compiled and reviewed all inpatient and outpatient encounters during three time points: (1) 3 months pre-amputation to 1 day pre-amputation, (2) the day of amputation through 12 months post-amputation, and (3) 13-24 months post-amputation. Health care utilization was defined as the average number of encounter days/admissions for each patient. Concomitant diagnoses following amputation including post-traumatic stress disorder, traumatic brain injury, anxiety, depression, and chronic pain were also recorded.

Results

A total of 106 individuals with hip- and pelvic-level amputations were analyzed (69 unilateral hip disarticulation, 6 bilateral hip disarticulations, 27 unilateral hemipelvectomy, 2 bilateral hemipelvectomies, and 2 patients with a hemipelvectomy and contralateral hip disarticulation). Combat trauma contributed to 61.3% (n = 65) of all amputations. During the time period of 3 months pre-amputation, patients had an average of 3.8 encounter days. Following amputation, health care utilization increased in both the year following amputation and the time period of 13-24 months post-amputation, averaging 170.8 and 77.4 encounter days, respectively. Patients with trauma-related amputations averaged more total encounter days compared to patients with disease-related amputations in the time period of 12 months following amputation (203.8 vs.106.7, P < .001) and the time period of 13-24 months post-amputation (92.0 vs. 49.0, P = .005). PTSD (P = .02) and traumatic brain injuries (P < .001) were more common following combat-related amputations.

Conclusions

This study highlights the increased health care resource demand following hip- and pelvic-level amputations in a military population, particularly for those patients who sustained combat-related trauma. Additionally, patients with combat-related amputations had significantly higher rates of concomitant PTSD and traumatic brain injury. Understanding the extensive needs of this unique patient population helps inform providers and policymakers on the requirements for providing high-quality care to combat casualties.
Wind-reprocessed transients from stellar-mass black hole Tidal Disruption Events
Authors: Kremer Kyle , Mockler Brenna , Piro Anthony L , Lombardi James C
Published: July 2023

Tidal disruptions of stars by stellar-mass black holes are expected to occur frequently in dense star clusters. Building upon previous studies that performed hydrodynamic simulations of these encounters, we explore the formation and long-term evolution of the thick, super-Eddington accretion disks formed. We build a disk model that includes fallback of material from the tidal disruption, accretion onto the black hole, and disk mass losses through winds launched in association with the super-Eddington flow. We demonstrate that bright transients are expected when radiation from the central engine powered by accretion onto the black hole is reprocessed at large radii by the optically-thick disk wind. By combining hydrodynamic simulations of these disruption events with our disk + wind model, we compute light curves of these wind-reprocessed transients for a wide range of stellar masses and encounter penetration depths. We find typical peak bolometric luminosities of roughly $10^{41}\!-\!10^{44}\,$erg s−1 (depending mostly on accretion physics parameters) and temperatures of roughly $10^5\!-\!10^6\,$K, suggesting peak emission in the ultraviolet/blue bands. We predict all-sky surveys such as the Vera Rubin Observatory and ULTRASAT will detect up to thousands of these events per year in dense star clusters out to distances of several Gpc.
Nitrogen enrichment and clustered star formation at the dawn of the Galaxy
Authors: Belokurov Vasily , Kravtsov Andrey
Published: July 2023

Anomalously high nitrogen-to-oxygen abundance ratios [N/O] are observed in globular clusters (GCs), among the field stars of the Milky Way (MW), and even in the gas in a z ≈ 11 galaxy. Using data from the APOGEE Data Release 17 and the Gaia Data Release 3, we present several independent lines of evidence that most of the MW’s high-[N/O] stars were born in situ in massive bound clusters during the early, pre-disc evolution of the Galaxy. Specifically, we show that distributions of metallicity [Fe/H], energy, the angular momentum Lz, and distance of the low-metallicity high-[N/O] stars match the corresponding distributions of stars of the Aurora population and of the in situ GCs. We also show that the fraction of in situ field high-[N/O] stars, fN/O, increases rapidly with decreasing metallicity. During epochs when metallicity evolves from $\rm [Fe/H]=-1.5$ to $\rm [Fe/H]=-0.9$, the Galaxy spins up and transitions from a turbulent Aurora state to a coherently rotating disc. This transformation is accompanied by many qualitative changes. In particular, we show that high N/O abundances similar to those observed in GN-z11 were common before the spin-up ($\rm [Fe/H]\lesssim -1.5$) when up to $\approx 50~{{\ \rm per\ cent}}-70~{{\ \rm per\ cent}}$ of the in situ stars formed in massive bound clusters. The dramatic drop of fN/O at $\rm [Fe/H]\gtrsim -0.9$ indicates that after the disc emerges the fraction of stars forming in massive bound clusters decreases by two orders of magnitude.
A model for GN-z11: top-heavy stellar initial mass functions in forming galactic nuclei and ultra-compact dwarfs
Authors: Bekki Kenji , Tsujimoto Takuji
Published: July 2023

Recent JWST observations of the z = 10.6 galaxy GN-z11 have revealed a very high gas-phase nitrogen abundance (higher than four times the solar value), a very small half-light radius (≈60 pc), and a large stellar mass (Ms ≈ 109 M⊙) for its size. We consider that this object is a forming galactic nucleus or ultra-compact dwarf galaxy rather than a proto globular cluster, and thereby investigate the chemical abundance pattern using one-zone chemical evolution models. The principal results of the models are as follows. The observed log (N/O) > −0.24, log (C/O) > −0.78, and 12 + log (O/H) ≈ 7.8 can be self-consistently reproduced by the models both with very short star formation time-scales (<107 yr) and with top-heavy stellar initial mass functions. The adopted assumption of no chemical enrichment by massive (m > 25 M⊙) core collapse supernovae (CCSNe) is also important for the reproduction of high gas-phase log (N/O), because such CCSNe can decrease high log (N/O) of gas polluted by OB and Wolf–Rayet stars. GN-z11 can have a significant fraction (>0.5) of nitrogen-rich ([N/Fe] > 0.5) stars, which implies a possible link between nitrogen-rich stellar populations of the inner Galaxy and giant elliptical galaxies and high-z objects with high gas-phase log (N/O) like GN-z11.
Determinants of the Intensity of Bank-Firm Relationships: Evidence from Japan
Authors: Aoki Yasuharu
Published: July 2023

Focusing on the Japanese setting, this study investigates the standard firm characteristics that affect the intensity of bank-firm relationships (IBR) and how these characteristics relate to the decline in IBR. The regression results show that firm size, credit quality, investment opportunities, and ownership concentration are negatively associated with IBR, while firm age, pledgeability, and managerial ownership are positively associated. This study also finds that (1) an increase in credit quality, (2) a decrease in the positive impact of managerial ownership, and (3) an increase in the negative impact of ownership concentration cause the decline in IBR. (JEL G21, G32, L14)

Received March 15, 2023; editorial decision July 11, 2023 by Editor Isil Erel. Authors have furnished an Internet Appendix, which is available on the Oxford University Press Web site next to the link to the final published paper online.
Symbolic and Transformative: Alignments Toward Feminicídio Legal Reform inside the Brazilian Police
Authors: Pamplona Roberta S
Published: July 2023

Feminism travels unevenly through state structures, and the state’s incorporation of feminist ideas remains controversial within feminist movements. This study uses the Brazilian Feminicídio Law, which increases punishment for gender-based homicides, as a case study to ask how law enforcement actors adopt a feminist legal reform. Data come from one year of in-depth fieldwork across police stations in a major Brazilian city and from newspaper articles. I show that state actors accept the feminist legal frame based on previous understandings of police practices. However, institutional divisions led them to develop different alignments toward the new legal reform. While police officers from the homicide division symbolically align toward feminicídio and do not aim to change police practices, officers from the women’s division perceive feminicídio as an opportunity to claim significant changes in police practices, revealing a transformative alignment. Struggles over legitimation and diverging professional beliefs and interests explain the two distinct responses.
Revisiting evidential presumptions of undue influence in Hong Kong
Authors: Cheng Yutong
Published: July 2023

The usefulness of evidential presumptions in establishing undue influence in equity has long been debated. This article revisits this debate in the Hong Kong context, arguing evidential presumptions are, in essence, of little use in establishing undue influence because there is no necessary causation between evidential presumptions and the establishment of undue influence. This article examines the fiduciary and evidentiary nature of the evidential presumptions and compares them as tools with tests of establishing undue influence throughout judicial history. Social implications behind evidential presumptions have been considered, aiming to adopt a flexible approach based on the maxim of equity.
XVIAmerican Literature: The Twentieth Century
Authors: Gifford James , Rudder Adam , Velásquez Antonio
Published: July 2023

This chapter has eight sections: 1. Poetry; 2. Fiction 1900–1945; 3. Fiction since 1945; 4. Genre Fiction; 5. Drama; 6. Comics; 7. African American Writing; 8. Native Writing; 9. Latino/a, Asian American, and General Ethnic Writing. Section 1 is by James Gifford; section 2 will resume next year; section 3 will resume next year; section 4 is by James Gifford; section 5 will resume next year; section 6 will resume next year; section 7 is by Adam Rudder; section 8 will resume next year; section 9 is by Antonio Velásquez.
Playing with FIRE: How an RXLR Oomycete Effector Fuels Disease by Hijacking 14-3-3 Proteins
Authors: Tiwari Manish , Lovelace Amelia H.
Published: July 2023

The plant pathogen Phytophthora palmivora causes rot disease in several monocots and dicots. The plant 14-3-3 proteins are targets of different types of effector molecules secreted by the pathogens. An RXLR-type effector FIRE (14-3-3 interacting RXLR effector) and its target 14-3-3 proteins that localize to haustoria have been identified, pointing to a potential site of interaction. The pathogen hijacks the host 14-3-3 proteins through FIRE-mediated interaction and lowers the immunity for disease progression. The effector FIRE and 14-3-3 interaction deciphered in this study could pave the way for genetic modification of plants with altered 14-3-3 protein for broad host resistance.

[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
sTRBC1 and cyTRBC1 Expression Distinguishes Indolent T-Lymphoblastic Proliferations From T-Lymphoblastic Leukemia/Lymphoma
Authors: Liu Juan , Li Mingyong , Fu Jun , Dong Min , Fan Xiaoming , Zhong Ling , Xu Gang , Li Yanxin , Xi Qian
Published: July 2023

Indolent T-lymphoblastic proliferation (iT-LBP) consists of a proliferation of non-neoplastic TdT+ T cells in extrathymic tissues, requiring no treatment. However, due to overlapping clinical and histologic features, distinguishing iT-LBP from T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL) can be challenging. Recently, flow cytometry–based evaluation of TRBC1 has been used to detect of T-cell clonality in TCRαβ+ mature T-cell lymphomas and aid in the differential diagnosis between T-ALL and normal thymocytes. We present a case of iT-LBP with high-grade serous ovarian carcinoma (HGSOC). To investigate the potential utility of TRBC1 expression in distinguishing iT-LBP from T-ALL/LBL, we assessed both surface (s) and cytoplasmic (cy) TRBC1 expression patterns on blast cells from the patient with iT-LBP and HGSOC as well as 11 patients diagnosed with T-ALL/LBL. The results revealed that sTRBC1 and cyTRBC1 exhibited polytypic expression patterns in patient with iT-LBP and HGSOC, while cyTRBC1 showed monotypic expression in those with T-ALL/LBL. This suggests that evaluation of sTRBC1 and cyTRBC1 expression can serve as a simple, rapid, and effective approach to differentiate between iT-LBP and T-ALL/LBL.
CytoPb: Segmentation-free and threshold-free analysis of highly multiplexed images of biological tissue
Authors: Barber Paul R , Dolcetti Luigi , Yeh Chen-An , Ng Tony
Published: July 2023

We present a novel method and code, CytoPb, for the analysis of multiplexed images that is segmentation-free and threshold-free and produces results that correlate well with existing methods but is faster to run, needs minimal training and removes subjectivity associated with the validation of single-cell segmentation and thresholding. In three data sets cell abundance measures correlate with previous methods (Pearson’s coef. >0.75). Neighbourhood analysis and cell-cell proximity are possible.
GAPiM: Discovering Genetic Variations on a Real Processing-in-Memory System
Authors: Abecassis Naomie , Gómez-Luna Juan , Mutlu Onur , Ginosar Ran , Moisson-Franckhauser Aphélie , Yavits Leonid
Published: July 2023

Variant calling is a fundamental stage in genome analysis that identifies mutations (variations) in a sequenced genome relative to a known reference genome. Pair-HMM is a key part of the variant calling algorithm and its most compute-intensive part. In recent years, Processing-in-Memory (PiM) solutions, which consist of placing compute capabilities near/inside memory, have been proposed to speed up the genome analysis pipeline. We implement the Pair-HMM algorithm on a commercial PiM platform developed by UPMEM. We modify the Pair-HMM algorithm to make it more suitable for PiM execution with acceptable loss of accuracy. We evaluate our implementation on single chromosomes and whole genome sequencing datasets, demonstrating up to 2x speedup compared to existing CPU accelerations and up to 3x speedup compared to FPGA accelerations.
Directly selecting differentially expressed genes for single-cell clustering analyses
Authors: Chen Zihao , Wang Changhu , Huang Siyuan , Shi Yang , Xi Ruibin
Published: July 2023

In single-cell RNA sequencing (scRNA-seq) studies, cell-types and their associated marker genes are often identified by clustering and differential expression gene (DEG) analysis. scRNA-seq data contain many genes not relevant to cell-types and gene selection procedures are needed for more accurate clustering. An ideal gene selection procedure should select all DEGs between cell-types for best cell-type identification. However, because cell-types are unknown, gene selection and DEG analysis are performed separately using different methods. Genes are selected using surrogate criteria not directly related with clustering, which often miss important genes or select unimportant genes. Clustering accuracy could be seriously influenced because of the inferior gene selection. DEGs are often detected by comparing different clusters, leading to many false DEGs due to the selection bias problem. In this paper, we present Festem, a unified method for gene selection and DEG analysis in scRNA-seq studies. Festem investigates gene’s clustering information based on the observation that marginal distributions of DEGs are mixtures of their different cell-type-conditional distributions, and can directly select the clustering-informative DEGs and avoid the selection bias problem. Extensive simulation and real data analyses show that Festem achieves high precision and recall for DEG detection, and enables more accurate clustering and cell-type identification. Applications to several scRNA-seq datasets demonstrate that Festem can identify cell-types that are often missed by other methods. In a large intrahepatic cholangiocarcinoma dataset, we identify CD8+ T cell-types and find that their marker genes are novel prognostic biomarkers.
Generating serum-based antibodies from tumor-exposed mice: a new potential strategy for cutaneous squamous cell carcinoma treatment
Authors: Liu Zheng
Published: July 2023

Current cancer treatment strategies continue to face significant challenges, primarily due to tumor relapse, drug resistance, and low treatment efficiency. These issues arise because certain tumor cells adapt to the host immune microenvironment and evade the immune system. This study presents a new cancer immunotherapy strategy using serum-based antibodies from mice exposed to mouse cutaneous squamous cell carcinoma (mCSCC). The experiment was conducted in three stages. In the first stage, mCSCC cells were isolated and expanded cultured from DMBA/TPA-induced mCSCC. In the second stage, these expanded tumor cells were injected into healthy mice to stimulate the production of anti-tumor antibodies. In the final stage, therapeutic serum was extracted from these healthy mice and reintroduced into the tumor-bearing mice. An ELISA assay was utilized to analyze the levels of p53, Bcl-xL, NF-κB, and Bax. The results showed that the serum treatment not only reduced tumor volume but also reversed changes in p53, Bcl-xL, NF-κB, and Bax. In conclusion, this study developed a new immunotherapeutic strategy for treating mCSCC. However, further research is needed to fully comprehend the mechanism of this serum treatment.
Assessing the basal gene expression of cancer cell lines for in vitro transcriptomic toxicology screening
Authors: Black Michael B. , Efremenko Alina Y. , McMullen Patrick D. , Barutcu A. Rasim , Nong Andy
Published: July 2023

In vitro toxicology has used immortalized cancer cell lines as model human systems for decades. However, these cell lines pose problems in designing toxicity testing programs as they inherently do not represent normal human biology. There is also a huge number of such cell lines to choose from, derived from human cancer cells from nearly every tissue. We explored the idea of using available basal gene expression data (NCI-60 cell line panel and Human GTEx tissue data) to determine if there was sufficient variability in cell line gene expression to group cell lines by relevance to specific human tissues. The transcriptomic analysis suggests that the variability in gene expression in cancer cell lines and in normal human tissue is minimal. The overall basal gene expression of cancer cells lines even overlapped normal human tissue gene expression. While some human tissues (e.g., lung) have basal expression profiles that do not appear to be like any cancer cell line, including cancers that may be derived from the same tissue, most human tissues show basal expression profiles comparable to several cancer cell lines. These results are important to address the genomic baseline and variability of cancer cell lines used for new approach methods of toxicity testing.
Sensitive remote homology search by local alignment of small positional embeddings from protein language models
Authors: Johnson Sean R. , Peshwa Meghana , Sun Zhiyi
Published: July 2023

Accurately detecting distant evolutionary relationships between proteins remains an ongoing challenge in bioinformatics. Search methods based on primary sequence struggle to accurately detect homology between sequences with less than 20% amino acid identity. Profile- and structure-based strategies extend sensitive search capabilities into this twilight zone of sequence similarity but require slow pre-processing steps. Recently, whole-protein and positional embeddings from deep neural networks have shown promise for providing sensitive sequence comparison and annotation at long evolutionary distances. Embeddings are generally faster to compute than profiles and predicted structures but still suffer several drawbacks related to the ability of whole-protein embeddings to discriminate domain-level homology, and the database size and search speed of methods using positional embeddings. In this work, we show that low-dimensionality positional embeddings can be used directly in speed-optimized local search algorithms. As a proof of concept, we use the ESM2 3B model to convert primary sequences directly into the 3Di alphabet or amino acid profiles and use these embeddings as input to the highly optimized Foldseek, HMMER3, and HH-suite search algorithms. Our results suggest that positional embeddings as small as a single byte can provide sufficient information for dramatically improved sensitivity over amino acid sequence searches without sacrificing search speed.
Mcadet: a feature selection method for fine-resolution single-cell RNA-seq data based on multiple correspondence analysis and community detection
Authors: Cui Saishi , Nassiri Sina , Zakeri Issa
Published: July 2023

Single-cell RNA sequencing (scRNA-seq) data analysis faces numerous challenges, including high sparsity, a high-dimensional feature space, technical biases, and biological noise. These challenges hinder downstream analysis, necessitating the use of feature selection methods to address technical biases, identify informative genes, and reduce data dimensionality. However, existing methods for selecting highly variable genes (HVGs) exhibit limited overlap and inconsistent clustering performance across benchmark datasets. Moreover, these methods often struggle to accurately select HVGs from fine-resolution scRNA-seq datasets and rare cell types, raising concerns about the reliability of their results. To overcome these limitations, we propose a novel feature selection framework for unique molecular identifiers (UMIs) scRNA-seq data called Mcadet. Mcadet integrates Multiple Correspondence Analysis (MCA), graph-based community detection, and a novel statistical testing approach. To assess the effectiveness of Mcadet, we conducted extensive evaluations using both simulated and real-world data, employing unbiased metrics for comparison. Our results demonstrate the superior performance of Mcadet in the selection of HVGs in scenarios involving fine-resolution scRNA-seq datasets and datasets containing rare cell populations. By addressing the challenges of feature selection in scRNA-seq analysis, Mcadet provides a valuable tool for improving the reliability and accuracy of downstream analyses in single-cell transcriptomics.
Early life lipid overload in Native American myopathy is phenocopied bystac3knock out in zebrafish
Authors: Donaka Rajashekar , Zheng Houfeng , Karasik David
Published: July 2023

Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving skeletal muscle performance by the functional integrity of cytoskeleton. SH3 and cysteine-rich domain 3 (Stac3) is a protein involved in nutrient sensing, and is an essential component of the excitation-contraction (EC) coupling machinery for Ca2+releasing. A mutation inSTAC3causes debilitating Native American myopathy (NAM) in humans, and loss of this gene in mice and zebrafish resulted in death in early life. Previously, NAM patients demonstrated increased lipids in skeletal muscle biopsy. However, elevated neutral lipids could alter muscle function in NAM disease via EC coupling apparatus is yet undiscovered in early development.

Here, using a CRISPR/Cas9 inducedstac3knockout (KO) zebrafish model, we determined that loss ofstac3led to muscle weakness, as evidenced by delayed larval hatching. We observed decreased whole-body Ca2+level at 5 days post-fertilization (dpf) and defects in the skeletal muscle cytoskeleton, i.e., F-actin and slow muscle fibers at 5 and 7 dpf. Homozygous larvae exhibited elevated neutral lipid levels at 5 dpf, which persisted beyond 7 dpf. Myogenesis regulators such asmyoDandmyf5, were significantly altered instac3-/-larvae at 5 dpf, thus a progressive death of the KO larva by 11 dpf.

In summary, the presented findings suggest thatstac3-/-can serve as a non-mammalian model to identify lipid-lowering molecules for refining muscle function in NAM patients.
New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity
Authors: Vargas Adrian M. Molina , Osborn Raven , Sinha Souvik , Arantes Pablo R. , Patel Amun , Dewhurst Stephen , Palermo Giulia , O’Connell Mitchell R.
Published: July 2023

The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics.

KEY POINTS

Certain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches.

Understanding Cas13a’s allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants.

These Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains.

GRAPHICAL ABSTRACT

New strategies to improve Cas13a RNA-detection specificity developed via mismatch tolerance profiling, uncovering features that modulate specificity, and structure-guided engineering of LbuCas13a.
Interpreting roles of mutations associated with the emergence ofS. aureusUSA300 strains using transcriptional regulatory network reconstruction
Authors: Poudel Saugat , Hyun Jason , Hefner Ying , Monk Jon , Nizet Victor , Palsson Bernhard O.
Published: July 2023

TheStaphylococcus aureusclonal complex 8 (CC8) is made up of several subtypes with varying levels of clinical burden; from community-associated methicillin resistantS. aureus(CA-MRSA) USA300 strains to hospital-associated (HA-MRSA) USA500 strains and ancestral methicillin susceptible (MSSA) strains. This phenotypic distribution within a single clonal complex makes CC8 an ideal clade to study the emergence of mutations important for antibiotic resistance and community spread. Gene level analysis comparing USA300 against MSSA and HA-MRSA strains have revealed key horizontally acquired genes important for its rapid spread in the community. However, efforts to define the contributions of point mutations and indels have been confounded by strong linkage disequilibrium resulting from clonal propagation. To break down this confounding effect, we combined genetic association testing with a model of the transcriptional regulatory network (TRN) to find candidate mutations that may have led to changes in gene regulation. First, we used a De Bruijn graph genome-wide association study (DBGWAS) to enrich mutations unique to the USA300 lineages within CC8. Next, we reconstructed the TRN by using Independent Component Analysis on 670 RNA sequencing samples from USA300 and non-USA300 CC8 strains which predicted several genes with strain-specific altered expression patterns. Examination of the regulatory region of one of the genes enriched by both approaches,isdH,revealed a 38 base pair deletion containing a Fur binding site and a conserved Single Nucleotide Polymorphism (SNP) which likely led to the altered expression levels in USA300 strains. Taken together, our results demonstrate the utility of reconstructed TRNs to address the limits of genetic approaches when studying emerging pathogenic strains.
Restriction site associated DNA sequencing for tumour mutation burden estimation and mutation signature analysis
Authors: McGuinness Conor F , Black Michael A , Dunbier Anita K
Published: July 2023

Genome-wide measures of genetic disruption such as tumour mutation burden (TMB) and mutation signatures are emerging as useful biomarkers to stratify patients for treatment. Clinicians commonly use cancer gene panels for tumour mutation burden estimation, and whole genome sequencing is the gold standard for mutation signature analysis. However, the accuracy and cost associated with these assays limits their utility at scale. Usingin silicolibrary simulations we demonstrate that restriction enzyme associated DNA sequencing (RADseq) may be a cost-effective solution to improve accuracy of TMB estimation and derivation of mutation profiles when compared to a derived FDA approved cancer gene panel TMB score. Using simulated immune checkpoint blockade (ICB) trials, we show that inaccurate tumour mutation burden estimation leads to a reduction in power for deriving an optimal TMB cutoff to stratify patients for immune checkpoint blockade treatment. Additionally, prioritisation of APOBEC hypermutated tumours in these trials optimises TMB cutoff determination for breast cancer. Finally, the utility of RADseq in an experimental setting is also demonstrated, based on characterisation of an APOBEC mutation signature in anAPOBEC3Atransfected mouse cell line. In conclusion, our work demonstrates that RADseq has the potential to be used as a cost-effective, accurate solution for TMB estimation and mutation signature analysis by both clinicians and basic researchers.
Multiscale modelling of chromatin 4D organization in SARS-CoV-2 infected cells
Authors: Chiariello Andrea M. , Abraham Alex , Bianco Simona , Esposito Andrea , Vercellone Francesca , Conte Mattia , Fontana Andrea , Nicodemi Mario
Published: July 2023

SARS-CoV-2 is able to re-structure chromatin organization and alters the epigenomic landscape of the host genome, though the mechanisms that produce such changes are still poorly understood. Here, we investigate with polymer physics chromatin re-organization of the host genome, in space and time upon SARS-CoV-2 viral infection. We show that re-structuring of A/B compartments is well explained by a re-modulation of intra-compartment homotypic affinities, which leads to the weakening of A-A interactions and enhances A-B mixing. At TAD level, re-arrangements are physically described by a general reduction of the loop extrusion activity coupled with an alteration of chromatin phase-separation properties, resulting in more intermingling between different TADs and spread in space of TADs themselves. In addition, the architecture of loci relevant to the antiviral interferon (IFN) response, such as DDX58 or IFIT, results more variable within the 3D single-molecule population of the infected model, suggesting that viral infection leads to a loss of chromatin structural specificity. Analysis of time trajectories of pairwise gene-enhancer and higher-order contacts reveals that such variability derives from a more fluctuating dynamics in infected case, suggesting that SARS-CoV-2 alters gene regulation by impacting the stability of the contact network in time.

Overall, our study provides the first polymer-physics based 4D reconstruction of SARS-CoV-2 infected genome with mechanistic insights on the consequent gene mis-regulation.
Hypoxia-sensing by the Histone Demethylase UTX (KDM6A) Controls Colitogenic CD4+T cell Fate and Mucosal Inflammation
Authors: Cheng Mandy I. , Hong Lee , Chen Bryan , Chin Scott , Luthers Christopher R. , Bustillos Christian , Sheikh Shehzad Z. , Su Maureen A.
Published: July 2023

Hypoxia is a feature of inflammatory conditions [e.g., inflammatory bowel disease (IBD)] and can exacerbate tissue damage in these diseases. To counteract hypoxia’s deleterious effects, adaptive responses have evolved which protect against hypoxia-associated tissue injury. To date, much attention has focused on hypoxia-activated HIF (hypoxia-inducible factor) transcription factors in these responses. However, recent work has identified epigenetic regulators that are also oxygen-sensitive, but their role in adaptation to hypoxic inflammation is currently unclear. Here, we show that the oxygen-sensing epigenetic regulator UTX is a critical modulator of colitis severity. Unlike HIF transcription factors that act on gut epithelial cells, UTX functions in colitis through its effects on immune cells. Hypoxia results in decreased CD4+T cell IFN-γ production and increased CD4+regulatory T cells, and these findings are recapitulated by T cell-specific UTX deficiency. Hypoxia impairs the histone demethylase activity of UTX, and loss of UTX function leads to accumulation of repressive H3K27me3 epigenetic marks at IL12/STAT4 pathway genes (Il12rb2, Tbx21,andIfng). In a colitis mouse model, T cell-specific UTX deletion ameliorates colonic inflammation, protects against weight loss, and increases survival. Together these findings implicate UTX’s oxygen-sensitive histone demethylase activity in mediating protective, hypoxia-induced pathways in colitis.
Splam: a deep-learning-based splice site predictor that improves spliced alignments
Authors: Chao Kuan-Hao , Mao Alan , Salzberg Steven L , Pertea Mihaela
Published: July 2023

The process of splicing messenger RNA to remove introns plays a central role in creating genes and gene variants. Here we describe Splam, a novel method for predicting splice junctions in DNA based on deep residual convolutional neural networks. Unlike some previous models, Splam looks at a relatively limited window of 400 base pairs flanking each splice site, motivated by the observation that the biological process of splicing relies primarily on signals within this window. Additionally, Splam introduces the idea of training the network on donor and acceptor pairs together, based on the principle that the splicing machinery recognizes both ends of each intron at once. We compare Splam’s accuracy to recent state-of-the-art splice site prediction methods, particularly SpliceAI, another method that uses deep neural networks. Our results show that Splam is consistently more accurate than SpliceAI, with an overall accuracy of 96% at predicting human splice junctions. Splam generalizes even to non-human species, including distant ones like the flowering plantArabidopsis thaliana. Finally, we demonstrate the use of Splam on a novel application: processing the spliced alignments of RNA-seq data to identify and eliminate errors. We show that when used in this manner, Splam yields substantial improvements in the accuracy of downstream transcriptome analysis of both poly(A) and ribo-depleted RNA-seq libraries. Overall, Splam offers a faster and more accurate approach to detecting splice junctions, while also providing a reliable and efficient solution for cleaning up erroneous spliced alignments.
Use of linear features by red-legged partridges in an intensive agricultural landscape: implications for landscape management in farmland
Authors: Perrot Charlotte , Séranne Léo , Berceaux Antoine , Noel Mathias , Arroyo Beatriz , Bacon Léo
Published: July 2023

Current agricultural practices and change are the major cause of biodiversity loss. An important change associated with the intensification of agriculture in the last 50 years is the spatial homogenization of the landscape with substantial loss of such biodiversity-rich elements as seminatural linear features (hedgerows, field margins, grassy strips, etc.). In Europe, some management prescriptions serve to increase heterogeneity by the creation of these seminatural linear features which are not being used primarily for agricultural production. However, these elements are not equal in their support for biodiversity according to their structure and composition. The aim of this study is to determine the importance of landscape heterogeneity and specifically linear features on the spatial distribution of red-legged partridges, a small game species in decline in Europe. Through GPS-monitoring of adult birds, we first assess home range size throughout the year and during the breeding season, in relation to breeding status and to linear features (seminatural linear vegetation and tracks-roads for human traffic) density. Then, we focus on habitat selection during the breeding period in relation to linear features. We found that linear elements shape the use of space by red-legged partridges according to their reproductive status. Traffic routes and seminatural features structured by both herbaceous and woody cover, negatively influenced home range size. Further, breeding birds select linear elements with herbaceous cover while non-breeders select linear elements with woody cover, underlining the different needs of birds according to their breeding status. All birds selected areas near tracks, but non-breeders seemed to avoid roads. This study shows the importance for this species of the linear components that structure the agricultural landscape. We propose recommendations to promote the presence of the red-legged partridge in this agricultural environment but also of the biodiversity in general.
Context-Dependent Design of Induced-fit Enzymes using Deep Learning Generates Well Expressed, Thermally Stable and Active Enzymes
Authors: Zimmerman Lior , Alon Noga , Levin Itay , Koganitsky Anna , Shpigel Nufar , Brestel Chen , Lapidoth Gideon D.
Published: July 2023

The potential of engineered enzymes in practical applications is often constrained by limitations in their expression levels, thermal stability, and the diversity and magnitude of catalytic activities.De-novoenzyme design, though exciting, is challenged by the complex nature of enzymatic catalysis. An alternative promising approach involves expanding the capabilities of existing natural enzymes to enable functionality across new substrates and operational parameters. To this end we introduce CoSaNN (Conformation Sampling using Neural Network), a novel strategy for enzyme design that utilizes advances in deep learning for structure prediction and sequence optimization. By controlling enzyme conformations, we can expand the chemical space beyond the reach of simple mutagenesis. CoSaNN uses a context-dependent approach that accurately generates novel enzyme designs by considering non-linear relationships in both sequence and structure space. Additionally, we have further developed SolvIT, a graph neural network trained to predict protein solubility inE.Coli, as an additional optimization layer for producing highly expressed enzymes. Through this approach, we have engineered novel enzymes exhibiting superior expression levels, with 54% of our designs expressed in E.Coli, and increased thermal stability with more than 30% of our designs having a higher Tm than the template enzyme. Furthermore, our research underscores the transformative potential of AI in protein design, adeptly capturing high order interactions and preserving allosteric mechanisms in extensively modified enzymes. These advancements pave the way for the creation of diverse, functional, and robust enzymes, thereby opening new avenues for targeted biotechnological applications.
Structural mapping of patient-associatedKCNMA1gene variants
Authors: Moldenhauer Hans J. , Tammen Kelly , Meredith Andrea L.
Published: July 2023

KCNMA1-linked channelopathy is a neurological disorder characterized by seizures, motor abnormalities, and neurodevelopmental disabilities. The disease mechanisms are predicted to result from alterations inKCNMA1-encoded BK K+channel activity; however, only a subset of the patient-associated variants have been functionally studied. The localization of these variants within the tertiary structure or evaluation by pathogenicity algorithms has not been systematically assessed. In this study, 82 nonsynonymous patient-associatedKCNMA1variants were mapped within the BK channel protein. Fifty-three variants localized within cryo-EM resolved structures, including 21 classified as either gain-of-function (GOF) or loss-of-function (LOF) in BK channel activity. Clusters of LOF variants were identified in the pore, the AC region (RCK1), and near the Ca2+bowl (RCK2), overlapping with sites of pharmacological or endogenous modulation. However, no clustering was found for GOF variants. To further understand variants of uncertain significance (VUS), assessments by multiple standard pathogenicity algorithms were compared, and new thresholds for sensitivity and specificity were established from confirmed GOF and LOF variants. An ensemble algorithm was constructed (KCNMA1 Meta Score), consisting of a weighted summation of this trained dataset combined with a structural component derived from the Ca2+bound and unbound BK channels. KMS assessment differed from the highest performing individual algorithm (REVEL) at 10 VUS residues, and a subset were studied further by electrophysiology in HEK293 cells. M578T, E656A, and D965V (KMS+;REVEL-) were confirmed to alter BK channel properties in voltage-clamp recordings, and D800Y (KMS-;REVEL+) was assessed as benign under the test conditions. However, KMS failed to accurately assess K457E. These combined results reveal the distribution of potentially disease-causingKCNMA1variants within BK channel functional domains and pathogenicity evaluation for VUS, suggesting strategies for improving channel-level predictions in future studies by building on ensemble algorithms such as KMS.
A Subset of HIV-1 Controllers Lack Cortical Actin Disruption Indicative of ARP2/3 Inhibition
Authors: O’Brien Robert L. Furler , Kovacs Colin
Published: July 2023

A small fraction of people living with HIV-1 suppress viral replication naturally and exhibit delayed or absent disease progression without antiretroviral therapy, yet the underlying mechanisms of viral control remain elusive. Despite the known role of HIV-1 in disrupting the actin cytoskeleton and altering cell migration and morphology within tissues, the molecular underpinnings that link viral actin disruption to disease progression have yet been linked to disease progression. We have previously shown through ultrastructural and time-lapse imaging that HIV-1 mediated actin disruption mirrors ARP2/3 inhibition within primary CD4+T cells of normal progressors and uninfected controls. Infected CD4+T cells from these two cohorts routinely exhibit two unique phenotypes when migrating. The first morphological difference is a sharp elongated and pointed lamellipodial tip, “Rhino” phenotype, distinct from the broad leading edge of uninfected cells. The second morphological difference is a non-apoptotic polarized blebbing at the lamellipodia of infected cells. These two pathological morphologies can be recapitulated in uninfected cells with chemical inhibitors of the ARP2/3 complex and are temporally linked based on the differentiation status of the T cell. These effects are dampened, but not totally eradicated, in the absence of the HIV-1 Nef protein. In contrast to normal progressors, infected cells from two out of the three HIV-1 controllers tested in this study did not exhibit these cellular pathologies. The profound impact of ARP2/3 inhibition on immunopathogenesis within genetic and infectious diseases provides context into how HIV-1 may cause cellular and systemic immune dysfunction in normal progressors. The mechanically destabilized cellular cortex may also provide a selective protection for viral genome-intact and long-lived defective reservoirs from cell-mediated killing by host CD8+T cells and NK cells. This mechanical instability is absent in some HIV-1 controllers. Restoring ARP2/3 function and cortical actin integrity in people living with HIV-1 infection is a new avenue of investigation to eradicate HIV-1 infected cells from the body.
TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner
Authors: Taber Alexis , Konecny Andrew , Scott-Browne James , Prlic Martin
Published: July 2023

Transforming growth factor β (TGF-β) directly acts on naïve, effector and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer, however the dose-dependent effects of TGF-β on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of Tmemreactivation. We found that TGF-β could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or pro-inflammatory signals. In contrast, even high doses of TGF-β had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmemmay not always receive TGF-β signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-β signals is of importance. We found that exposure to TGF-β prior to as well as after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-β altered ∼10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-β-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-β is not simply suppressing CD8 Tmem, but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner.