bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetically encoded affinity reagents (GEARs): A toolkit for visualizing and manipulating endogenous protein functionin vivo
Authors: Boswell Curtis W. , Hoppe Caroline , Sherrard Alice , Miao Liyun , Kojima Mina L. , Krishna Srikar , Musaev Damir , Zhao Ning , Stasevich Timothy J. , Giraldez Antonio J.
Published: November 2023

Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional, universal, and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use nanobodies and single chain variable fragments (scFv), which recognize small epitopes, enabling fluorescent visualization and selective degradation of protein targets in vivo. Furthermore, we delineate a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad multifunctionality. We use GEARs to examine the native behaviour of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.
Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging
Authors: Mizushima Kenta , Kumamoto Yasuaki , Tamura Shoko , Yamanaka Masahito , Mochizuki Kentaro , Li Menglu , Egoshi Syusuke , Dodo Kosuke , Harada Yoshinori , Smith Nicholas I. , Sodeoka Mikiko , Tanaka Hideo , Fujita Katsumasa
Published: November 2023

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.
The Impact of SIV-Induced Immunodeficiency on Clinical Manifestation, Immune Response, and Viral Dynamics in SARS-CoV-2 Coinfection
Authors: Melton Alexandra , Rowe Lori A , Penney Toni , Krzykwa Clara , Goff Kelly , Scheuermann Sarah , Melton Hunter J , Williams Kelsey , Golden Nadia , Green Kristyn Moore , Smith Brandon , Russell-Lodrigue Kasi , Dufour Jason P , Doyle-Meyers Lara A , Schiro Faith , Aye Pyone P , Lifson Jeffery D , Beddingfield Brandon J , Blair Robert V , Bohm Rudolf P , Kolls Jay K , Rappaport Jay , Hoxie James A , Maness Nicholas J
Published: November 2023

Persistent and uncontrolled SARS-CoV-2 replication in immunocompromised individuals has been observed and may be a contributing source of novel viral variants that continue to drive the pandemic. Importantly, the effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. Here we conducted a pilot study wherein two pigtail macaques (PTM) chronically infected with SIVmac239 were exposed to SARS-CoV-2 and monitored for six weeks for clinical disease, viral replication, and viral evolution, and compared to our previously published cohort of SIV-naïve PTM infected with SARS-CoV-2. At the time of SARS-CoV-2 infection, one PTM had minimal to no detectable CD4+ T cells in gut, blood, or bronchoalveolar lavage (BAL), while the other PTM harbored a small population of CD4+ T cells in all compartments. Clinical signs were not observed in either PTM; however, the more immunocompromised PTM exhibited a progressive increase in pulmonary infiltrating monocytes throughout SARS-CoV-2 infection. Single-cell RNA sequencing (scRNAseq) of the infiltrating monocytes revealed a less activated/inert phenotype. Neither SIV-infected PTM mounted detectable anti-SARS-CoV-2 T cell responses in blood or BAL, nor anti-SARS-CoV-2 neutralizing antibodies. Interestingly, despite the diminished cellular and humoral immune responses, SARS-CoV-2 viral kinetics and evolution were indistinguishable from SIV-naïve PTM in all sampled mucosal sites (nasal, oral, and rectal), with clearance of virus by 3-4 weeks post infection. SIV-induced immunodeficiency significantly impacted immune responses to SARS-CoV-2 but did not alter disease progression, viral kinetics or evolution in the PTM model. SIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants.
A putative rhamnogalacturonan-II CMP-β-Kdo transferase identified using CRISPR/Cas9 gene edited callus to circumvent embryo lethality
Authors: Zhang Yuan , Sharma Deepak , Liang Yan , Downs Nick , Dolman Fleur , Thorne Kristen , Black Ian M. , Pereira Jose Henrique , Adams Paul , Scheller Henrik V. , O’Neill Malcolm , Urbanowicz Breeanna , Mortimer Jenny C.
Published: November 2023

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method using CRISPR/Cas9-mediated gene to generate callus carrying loss-of-function mutations in theMPG2gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We show that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno- octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization.
Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5
Authors: Dawson John R.D. , Wadman Grant M. , Zhang Penglie , Tebben Andrew , Carter Percy H. , Gu Siyi , Shroka Thomas , Borrega-Roman Leire , Salanga Catherina L. , Handel Tracy M. , Kufareva Irina
Published: November 2023

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists.

In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63and V2446.36are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.
The polyamines spermine and spermidine inhibit or induce programmed cell death inArabidopsis thaliana in vitroandin vivoin a dose dependent manner
Authors: Burke Rory , Nicotra Daniele , Phelan Jim , McCabe Paul F. , Kacprzyk Joanna
Published: November 2023

Polyamines are ubiquitous biomolecules with a number of established functions in eukaryotic cells. In plant cells, polyamines have previously been linked to abiotic and biotic stress tolerance, as well as to the modulation of programmed cell death (PCD), with contrasting reports on their pro-PCD and pro-survival effects. Here, we used two well established platforms for the study of plant PCD;Arabidopsis thalianasuspension cultures cells and the root hair assay, to examine the roles of the polyamines spermine and spermidine in the regulation of PCD. We demonstrate that both polyamines can trigger PCD when applied exogenously at higher doses, whereas at lower concentrations they inhibit PCD induced by both biotic and abiotic stimuli. Furthermore, we show that concentrations of polyamines resulting in inhibition of PCD generated a transient ROS burst in our experimental system, and activated the expression of oxidative stress- and pathogen response-associated genes. Finally, we examined PCD responses in existingArabidopsispolyamine synthesis mutants, and identified a subtle PCD phenotype inArabidopsisseedlings deficient in thermo-spermine. The presented data show that polyamines can have a role in PCD regulation, however that role is dose-dependent and consequently they may act as either inhibitors, or inducers, of PCD inArabidopsis.
Idiosyncrasies unveiled: examining the pace, patterns and predictors of biotic diversification in peninsular India
Authors: Roy Pragyadeep , Joshi Jahnavi
Published: November 2023

The Peninsular Indian Plate (PIP), an ancient Gondwanan landmass, the oldest region for diversification with complex geoclimatic history in Tropical Asia, harbours highly diverse and endemic biota with distinct biogeographic affinities. However, our understanding of the diversification dynamics of its biota in a quantitative framework is limited. Given this, we used time-calibrated molecular phylogenies and birth-death models to examine the diversification tempo, mode, and drivers across 34 well-studied endemic lineages (∼670 species). Lineage identity and biogeographic affinity differentially influenced the diversification rates, where plants had the highest and soil arthropods had the lowest diversification rates. Also, younger Asian groups diversified faster than older Gondwanan taxa. The gradual accumulation was supported in 19 lineages, suggesting that the historical stability of their habitat was an important driver, although the region has had a dynamic geoclimatic past. Miocene aridification, monsoon intensification, changes in paleotemperature, and species diversity explained diversification patterns among the other 15 lineages. The diversification rates predicted the species diversity better than clade age across lineages. Our results highlight the role of regional biogeographic, geoclimatic processes, and phylogenetic history on diversification dynamics, which is critical for predicting how PIP biota may respond to global change amid significant anthropogenic threats.
Comprehensive Survey of Conserved RNA Secondary Structures in Full-Genome Alignment of Hepatitis C Virus
Authors: Triebel Sandra , Lamkiewicz Kevin , Ontiveros Nancy , Sweeney Blake , Stadler Peter F. , Petrov Anton I. , Niepmann Michael , Marz Manja
Published: November 2023

Hepatitis C virus (HCV) is a plus-stranded RNA virus that often chronically infects liver hepatocytes and causes liver cirrhosis and cancer. These viruses replicate their genomes employing error-prone replicases. Thereby, they routinely generate a large “cloud” of RNA genomes which - by trial and error - comprehensively explore the sequence space available for functional RNA genomes that maintain the ability for efficient replication and immune escape. In this context, it is important to identify which RNA secondary structures in the sequence space of the HCV genome are conserved, likely due to functional requirements.

Here, we provide the first genome-wide multiple sequence alignment (MSA) with the prediction of RNA secondary structures throughout all representative full-length HCV genomes. We selected 57 representative genomes by clustering all complete HCV genomes from the BV-BRC database based on k-mer distributions and dimension reduction and adding RefSeq sequences. We include annotations of previously recognized features for easy comparison to other studies.

Our results indicate that mainly the core coding region, the C-terminal NS5A region, and the NS5B region contain secondary structure elements that are conserved beyond coding sequence requirements, indicating functionality on the RNA level. In contrast, the genome regions in between contain less highly conserved structures. The results provide a complete description of all conserved RNA secondary structures and make clear that functionally important RNA secondary structures are present in certain HCV genome regions but are largely absent from other regions. Full-genome alignments of all branches ofHepacivirus Care provided in the supplement.
Simultaneous Profiling of Chromatin Accessibility and DNA Methylation in Complete Plant Genomes Using Long-Read Sequencing
Authors: Leduque Basile , Edera Alejandro , Vitte Clementine , Quadrana Leandro
Published: November 2023

Epigenetic regulations, including chromatin accessibility, nucleosome positioning, and DNA methylation intricately shape genome function. However, current chromatin profiling techniques relying on short-read sequencing technologies fail to characterise highly repetitive genomic regions and cannot detect multiple chromatin features simultaneously. Here, we performed Simultaneous Accessibility and DNA Methylation Sequencing (SAM-seq) of purified plant nuclei. Thanks to the use of long-read nanopore sequencing, SAM-seq enables high-resolution profiling of m6A-tagged chromatin accessibility together with endogenous cytosine methylation in plants. Analysis of naked genomic DNA revealed significant sequence preference biases of m6A-MTases, controllable through a normalisation step. By applying SAM-seq to Arabidopsis and maize nuclei we obtained fine-grained accessibility and DNA methylation landscapes genome-wide. We uncovered crosstalk between chromatin accessibility and DNA methylation within nucleosomes of genes, TEs, and centromeric repeats. SAM-seq also detects DNA footprints over cis-regulatory regions. Furthermore, using the single-molecule information provided by SAM-seq we identified extensive cellular heterogeneity at chromatin domains with antagonistic chromatin marks, suggesting that bivalency reflects cell-specific regulations. SAM-seq is a powerful approach to simultaneously study multiple epigenetic features over unique and repetitive sequences, opening new opportunities for the investigation of epigenetic mechanisms.
Quantification of water exchange across the blood-brain barrier using non-contrast MR fingerprinting
Authors: Thomson Emma L. , Powell Elizabeth , Wheeler-Kingshott Claudia A. M. Gandini , Parker Geoff J. M.
Published: November 2023

Purpose

A method is proposed to quantify cerebral blood volume (vb) and intravascular water residence time (τb) using magnetic resonance fingerprinting (MRF), applied using a spoiled gradient echo sequence, without the need for contrast agent.

Methods

An in silico study optimised an acquisition protocol to maximise the sensitivity of the measurement tovbandτbchanges. Its accuracy in the presence of variations inT1,t,T1,b, andB1was evaluated. The optimised protocol (scan time of 19 minutes) was then tested in a exploratory healthy volunteer study (10 volunteers, mean age 24 ± 3, 6 male) at 3 T with a repeat scan taken after repositioning to allow estimation of repeatability.

Results

Simulations show that assuming literature values forT1,bandT1,t, no variation inB1, while fitting onlyvbandτb, leads to large errors in quantification ofvbandτb, regardless of noise levels. However, simulations also show that matchingandτb, simultaneously is feasible at clinically achievable noise levels. Across the healthy volunteers, all parameter quantifications fell within the expected literature range. In addition, the maps show good agreement between hemispheres suggesting physiologically relevant information is being extracted. Expected differences between white and grey matterT1,t(p<0.0001) andvb(p<0.0001) are observed,T1,bandτbshow no significant differences, p=0.4 and p=0.6 repectively. Good repeatability was seen between repeat scans: mean ICC ofT1,t: 0.91,T1,b: 0.58,vb: 0.90, andvτb: 0.96.

Conclusion

We demonstrate that regional simultaneous quantification ofvb,τb,T1,b,T1,t, andB1using MRF is feasible in vivo.
Pain persists in mice lacking both Substance P and CGRPα signaling
Authors: MacDonald Donald Iain , Jayabalan Monessha , Seaman Jonathan , Balaji Rakshita , Nickolls Alec , Chesler Alexander
Published: November 2023

The neuropeptides Substance P and CGRPα have long been thought important for pain sensation. Both peptides and their receptors are expressed at high levels in pain-responsive neurons from the periphery to the brain making them attractive therapeutic targets. However, drugs targeting these pathways individually did not relieve pain in clinical trials. Since Substance P and CGRPα are extensively co-expressed we hypothesized that their simultaneous inhibition would be required for effective analgesia. We therefore generated Tac1 and Calca double knockout (DKO) mice and assessed their behavior using a wide range of pain-relevant assays. As expected, Substance P and CGRPα peptides were undetectable throughout the nervous system of DKO mice. To our surprise, these animals displayed largely intact responses to mechanical, thermal, chemical, and visceral pain stimuli, as well as itch. Moreover, chronic inflammatory pain and neurogenic inflammation were unaffected by loss of the two peptides. Finally, neuropathic pain evoked by nerve injury or chemotherapy treatment was also preserved in peptide-deficient mice. Thus, our results demonstrate that even in combination, Substance P and CGRPα are not required for the transmission of acute and chronic pain.
Time-restricted feeding ameliorates MCDD-induced steatohepatitis in mice
Authors: Jung Ik-Rak , Ahima Rexford S. , Kim Sangwon F.
Published: November 2023

Non-Alcoholic Steatohepatitis (NASH) is an inflammatory form of Non-Alcoholic Fatty Liver Disease (NAFLD), closely associated with disease progression, cirrhosis, liver failure, and hepatocellular carcinoma. Time-restricted feeding (TRF) has been shown to decrease body weight and adiposity and improve metabolic outcomes, however, the effect of TRF on NASH has not yet been fully understood. We had previously reported that inositol polyphosphate multikinase (IPMK) mediates hepatic insulin signaling. Importantly, we have found that TRF increases hepatic IPMK levels. Therefore, we investigated whether there is a causal link between TRF and IPMK in a mouse model of NASH, i.e., methionine and choline deficient diet (MCDD)-induced steatohepatitis. Here, we show that TRF alleviated markers of NASH, i.e., reduced hepatic steatosis, liver triglycerides (TG), serum alanine transaminase (ALT) and aspartate aminotransferase (AST), inflammation and fibrosis in MCDD mice. Interestingly, MCDD led to a significant reduction in IPMK levels, and the deletion of hepatic IPMK exacerbates the NASH phenotype induced by MCDD, accompanied by increased gene expression of pro-inflammatory chemokines. Conversely, TRF restored IPMK levels and significantly reduced gene expression of proinflammatory cytokines and chemokines. Our results demonstrate that TRF attenuates MCDD-induced NASH via IPMK-mediated changes in hepatic steatosis and inflammation.
De novo lipogenesis pathway is a vulnerability in IDH1 mutant glioma
Authors: Elahi Lubayna S , Condro Michael C , Kawaguchi Riki , Qin Yue , Alvarado Alvaro G. , Gruender Brandon , Qi Haocheng , Li Tie , Lai Albert , Castro Maria G. , Lowenstein Pedro R. , Garrett Matthew C , Kornblum Harley I.
Published: November 2023

Histone deacetylases (HDACs) have a wide range of targets and can rewire both the chromatin and lipidome of cancer cells. In this study, we show that valproic acid (VPA), a brain penetrant anti-epileptic and histone deacetylase inhibitor, inhibits the growth of IDH1 mutant tumors in vivo and in vitro, with at least some selectivity over IDH1 wild type tumors. Surprisingly, genes upregulated by VPA showed no change in chromatin accessibility at the promoter, but there was a correlation between VPA downregulated genes and diminished promoter chromatin accessibility. VPA inhibited the transcription of lipogenic genes and these lipogenic genes showed significant decrease in promoter chromatin accessibility only in the IDH1 MT glioma cell lines tested. VPA targeted a key lipogenic gene, fatty acid synthase (FASN), via inhibition of the mTOR pathway and both VPA and a selective FASN inhibitor TVB-2640 rewired the lipidome and promoted apoptosis in an IDH1 MT but not in an IDH1 WT glioma cell line. We further find HDACs are involved in the regulation of lipogenic genes and in particular HDAC6 is important for regulation of FASN in IDH1 MT glioma. Finally, we show that FASN knockdown alone and VPA in combination with FASN knockdown significantly improved the survival of mice in a IDH1 MT primary orthotopic xenograft model in vivo. We conclude that targeting fatty acid metabolism through HDAC inhibition and/or FASN inhibition may be a novel therapeutic option in IDH1 mutant gliomas.
Well-tempered metadynamics calculations of free energy surfaces of benzothiadiazine derivatives in aqueous solution
Authors: Hu Zheyao , Marti Jordi
Published: November 2023

KRAS oncogenes are the largest family of mutated RAS isoforms, participating in about 30% of all cancers. Due to their paramount medical importance, enormous effort is being devoted to the development of inhibitors using clinical tests, wet-lab experiments and drug design, being this a preliminary step in the process of creating new drugs, prior to synthesis and clinical testing. One central aspect in the development of new drugs is the characterization of all species that can be used for treatment. In this aim we propose a computational framework based on combined all-atom molecular dynamics and metadynamics simulations in order to accurately access the most stable conformational variants for several derivatives of a recently proposed small-molecule, called DBD15-21-22. Free energy calculations are essential to unveil mechanisms at the atomic scale like binding affinities or dynamics of stable states. Considering specific atom-atom distances and torsional angles as reliable reaction coordinates we have obtained free-energy landscapes by well-tempered metadynamics simulations, revealing local and global minima of the free-energy hypersurface. We have observed that a variety of stable states together with transitions states are clearly detected depending on the particular species, leading to predictions on the behaviour of such compounds in ionic aqueous solution.
Metabolic and imaging phenotypes associated withRB1loss in castrate resistant prostate cancer
Authors: Ahmad Fahim , White Margaret , Yamamoto Kazutoshi , Crooks Daniel R. , Agarwal Supreet , Yang Ye , Capaldo Brian , Raj Sonam , Alilin Aian Neil , Ton Anita , Adler Stephen , Seidel Jurgen , Olkowski Colleen , Cherukuri Murali Krishna , Choyke Peter L , Kelly Kathleen , Brender Jeffrey R.
Published: November 2023

Purpose

Advanced prostate cancer is treated with androgen receptor (AR) signaling inhibitors, which are initially effective, but the majority of patients eventually develop resistance and progress to castrate resistant prostate cancer (CRPC). Loss ofRB1in CRPC tumors is correlated with rapid progression and poor patient survival, and in combination withTP53loss, predisposes to the development of transitional neuroendocrine prostate cancer (NEPC). Although progressing CRPC is clinically associated with higher18FDG-PET SUVmaxvalues, it is unknown whether inactivation ofRB1and/orTP53is a driver of increased glucose import.

Experimental Design

A cohort of patient-derived xenograft (PDX)-derived CRPC organoids was screened to assess18FDG uptake in ARPC and NEPC, considering the influence of RB1 and TP53 status. Experimental loss ofRB1and/orTP53was induced in an androgen sensitive and a castrate resistant model, and metabolic changes were evaluated using18FDG-PET,13C-hyperpolarized magnetic resonance spectroscopy, Seahorse, andex vivoNMR.

Results

Knockdown of eitherRB1orTP53increased glycolysis and TCA cycle intermediates, while knockdown of both created a new phenotype where glucose was diverted to the pentose phosphate pathway and into glycogen synthesis. These large-scale metabolic changes were not reflected in18FDG uptake, which was not increased upon knockdown of either gene.13C-hyperpolarized magnetic resonance spectroscopy, on the other hand, showed significant differences in lactate dehydrogenase flux upon loss ofRB1. The metabolic heterogeneity revealed here suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment.
The anti-B7-H3 blocking antibody MJ18 does not recognize B7-H3 in murine tumor models
Authors: Nammor Talah , Frizzell Jenna , Lavoie Roxane R. , Lucien Fabrice
Published: November 2023

The immune checkpoint molecule B7-H3 is regarded as one of the most promising therapeutic targets for the treatment of human cancers. B7-H3 is highly expressed in many cancers and its expression has been associated to impaired antitumor immunity and poor patient prognosis. In immunocompetent mouse tumor models, genetic deletion of B7-H3 in tumor cells enhances antitumor immune response leading to tumor shrinkage. The underlying mechanisms of B7-H3 inhibitory function remain largely uncharacterized and the identity of potential cognate(s) receptor(s) of B7-H3 is still to be defined. To better understand B7-H3 functionin vivo, several studies have employed MJ18, a monoclonal antibody reported to bind murine B7-H3 and blocks its immune-inhibitory function. In this brief research report, we show that 1) MJ18 does not bind B7-H3, 2) MJ18 binds the Fc receptor FcγRIIB on surface of murine splenocytes, and 3) MJ18 does not induce tumor regression in a mouse model responsive to B7-H3 knockout. Given the high profile of B7-H3 as therapeutic target for human cancers, our work emphasizes that murine B7-H3 studies using the MJ18 antibody should be interpreted with caution. Finally, we hope that our study will motivate the scientific community to establish much-needed validated research tools to study B7-H3 biology in mouse models.
Use of dye sensitizers for increased photoacoustic mechanosensation
Authors: Rafeedi Tarek , Becerra Laura , Root Nicholas , Qi Baiyan , Fu Lei , Esparza Guillermo , Qie Yi , Sasi Lekshmi , Rouw Romke , Jokerst Jesse , Lipomi Darren
Published: November 2023

The photoacoustic effect refers to the generation of pressure waves in matter stimulated by light[1]. In the context of radiology (i.e., photoacoustic imaging) waves generated by pulsed laser light are detected by an ultrasound transducer[2–4]. It has been shown that photoacoustic waves produce a mechanical, tactile sensation in humans on bare skin[5]. In a series of psychophysical experiments, performed with both medical grade and off-the-shelf pulsed light systems, participants could detect, categorically describe, and discern the direction of travel of pulsed optical stimuli with the use of a dye as an optical absorber on the skin. To a large extent, the sensations were perceived as localized vibration on the glabrous surface of the fingers, when sensitized with the thin film of dye. This form of sensory stimulation demonstrates an enhanced non-contact, non-optogenetic, in situ activation of the mechanosensory system. This modality of sensation may provide a tool that leads to new insights in psychology, neuroscience, mechanobiology, and the health sciences. Finally, it has many advantageous characteristics for human interaction with artificial environments, as optical signals can be projected onto the skin across distances.
Comprehensive mapping of sensory and sympathetic innervation of the developing kidney
Authors: N’Guetta Pierre-Emmanuel Y. , McLarnon Sarah R. , Tassou Adrien , Geron Matan , Shirvan Sepenta , Hill Rose Z. , Scherrer Grégory , O’Brien Lori L.
Published: November 2023

The kidney functions as a finely tuned sensor to balance body fluid composition and filter out waste through complex coordinated mechanisms. This versatility requires tight neural control, with innervating efferent nerves playing a crucial role in regulating blood flow, glomerular filtration rate, water and sodium reabsorption, and renin release. In turn sensory afferents provide feedback to the central nervous system for the modulation of cardiovascular function. However, the cells targeted by sensory afferents and the physiological sensing mechanisms remain poorly characterized. Moreover, how the kidney is innervated during development to establish these functions remains elusive. Here, we utilized a combination of light-sheet and confocal microscopy to generate anatomical maps of kidney sensory and sympathetic nerves throughout development and resolve the establishment of functional crosstalk. Our analyses revealed that kidney innervation initiates at embryonic day (E)13.5 as the nerves associate with vascular smooth muscle cells and follow arterial differentiation. By E17.5 axonal projections associate with kidney structures such as glomeruli and tubules and the network continues to expand postnatally. These nerves are synapsin I-positive, highlighting ongoing axonogenesis and the potential for functional crosstalk. We show that sensory and sympathetic nerves innervate the kidney concomitantly and classify the sensory fibers as calcitonin gene related peptide (CGRP)+, substance P+, TRPV1+, and PIEZO2+, establishing the presence of PIEZO2 mechanosensory fibers in the kidney. Using retrograde tracing, we identified the primary dorsal root ganglia, T10-L2, from which PIEZO2+sensory afferents project to the kidney. Taken together our findings elucidate the temporality of kidney innervation and resolve the identity of kidney sympathetic and sensory nerves.
A novel antagonist of the CCL5/CCR5 axis suppresses the tumor growth and metastasis of triple-negative breast cancer by CCR5-YAP1 regulation
Authors: Chen Ling , Xu Guiying , Song Xiaoxu , Zhang Lianbo , Chen Chuyu , Xiang Gang , Wang Shuxuan , Zhang Zijian , Wu Fang , Yang Xuanming , Zhang Lei , Ma Xiaojing , Yu Jing
Published: November 2023

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) with a high mortality rate, and few effective therapeutic strategies are available. CCL5/CCR5 is an appealing immunotherapeutic target for TNBC. However, its signaling mechanism is poorly understood and its direct antagonists have not been reported. Here, we developed a high-throughput screening (HTS) assay for discovering its antagonists. Verteporfin was identified as a more selective and potent antagonist than the known CCR5 antagonist maraviroc. Without photodynamic therapy, verteporfin demonstrated significant inhibition on TNBC tumor growth through immune regulation, remarkable suppression of lung metastasis by cell-intrinsic mechanism, and a significant extension of overall survivalin vivo. Mechanistically, CCR5 was found to be essential for expression of the key hippo effector YAP1. It promotedYAP1transcription via HIF-1α and exerted further control over the migration of CD8+T, NK, and MDSC immune cells through chemokines CXCL16 and CXCL8 which were identified from RNA-seq. Moreover, the CCR5-YAP1 axis played a vital role in promoting metastasis by modulating β-catenin and core epithelial-mesenchymal transition transcription factors ZEB1 and ZEB2. It is noteworthy that the regulatory relationship between CCR5 and YAP1 was observed across various BC subtypes, TNBC patients, and showed potential relevance in fifteen additional cancer types. Overall, this study introduced an easy-to-use HTS assay that streamlines the discovery of CCL5/CCR5 axis antagonists. Verteporfin was identified as a specific molecular probe of this axis with great potentials as a therapeutic agent for treating sixteen malignant diseases characterized by heightened CCR5 and YAP1 levels.
Refining the taxonomy of the orderHyphomicrobiales(Rhizobiales) based on whole genome comparisons of over 130 genus type strains
Authors: diCenzo George C. , Yang Yuqi , Young J. Peter W. , Kuzmanović Nemanja
Published: November 2023

The alphaproteobacterial orderHyphomicrobialesconsists of 38 families comprising 155 validly published genera as of June 2023. The orderHyphomicrobialeswas first described in 1957 and underwent important revisions in 2020. However, several inconsistencies in the taxonomy of this order remain, and there is a need for a consistent framework for defining families within the order. We propose a common genome-based framework for defining families within the orderHyphomicrobiales, suggesting that families represent monophyletic groups in core-genome phylogenies that share pairwise average amino acid identity values above ∼75% when calculated from a core set of 59 proteins. Applying this framework, we propose the formation of four new families and to reassign the generaSalaquimonas,Rhodoblastus, andRhodoligotrophosintoSalaquimonadaceaefam. nov.,Rhodoblastaceaefam. nov., andRhodoligotrophaceaefam. nov., respectively, and the generaAlbibacter,Chenggangzhangella,Hansschlegelia, andMethylopilaintoMethylopilaceaefam. nov.. We further propose to unify the familiesBartonellaceae,Brucellaceae,Phyllobacteriaceae, andNotoacmeibacteraceaeasBartonellaceae; the familiesSegnochrobactraceaeandPseudoxanthobacteraceaeasSegnochrobactraceae; the familiesLichenihabitantaceaeandLichenibacteriaceaeasLichenihabitantaceae; and the familiesBreoghaniaceaeandStappiaceaeasStappiaceae. Lastly, we propose to reassign several genera to existing families. Specifically, we propose to reassign the genusPseudohoefleato the familyRhizobiaceae; the generaOricola,Roseitalea, andOceaniradiusto the familyAhrensiaceae; the genusLimoniibacterto the emended familyBartonellaceae; the genusFaunimonasto the familyAfifellaceae; and the genusPseudochelatococcusto the familyChelatococcaceae. Our data also support the recent proposal to reassign the genusProsthecomicrobiumto the familyKaistiaceae.
Transcriptome analyses describe the consequences of persistent HIF-1 over-activation inCaenorhabditis elegans
Authors: Feng Dingxia , Qu Long
Published: November 2023

Metazoan animals rely on oxygen for survival, but during normal development and homeostasis, animals are often challenged by hypoxia (low oxygen). In metazoans, many of the critical hypoxia responses are mediated by the evolutionarily conserved hypoxia-inducible transcription factors (HIFs). The stability and activity of HIF complexes are strictly regulated. In the model organismC. elegans, HIF-1 stability and activity are negatively regulated by VHL-1, EGL-9, RHY-1 and SWAN-1. Importantly,C. elegansmutants carrying strong loss-of-function mutations in these genes are viable, and this provides opportunities to interrogate the molecular consequences of persistent HIF-1 over-activation. We find that the genome-wide gene expression patterns are compellingly similar in these mutants, supporting models in which RHY-1, SWAN-1 and EGL-9 function in common pathway(s) to regulate HIF-1 activity. These studies illuminate the diversified biological roles played by HIF-1, including metabolism, hypoxia and other stress responses, reproduction and development. Genes regulated by persistent HIF-1 over-activation overlap with genes responsive to pathogens, and they overlap with genes regulated by DAF-16. As crucial stress regulators, HIF-1 and DAF-16 converge on key stress-responsive genes and function synergistically to enable hypoxia survival.
Species traits and community structure can drive large-scale spatial propagation of effects in ecosystems
Authors: Garcia-Callejas David , Lavorel Sandra , Peltzer Duane , Ovaskainen Otso , Tylianakis Jason M
Published: November 2023

Species can directly and indirectly affect others across communities and habitats, yet the spatial scale over which such effects spread remains unclear. This uncertainty arises partly because the species traits and landscape structures allowing indirect effects to propagate may differ across scales. Here, we use a topological network metric, communicability, to explore the spatial propagation of effects in a large-scale plant-frugivore network projected across the territory of Aotearoa New Zealand. We show that generalism and species prevalence, and complementary morphological traits such as fruit and body size, are important predictors of species' capacity to propagate effects, but their importance differed across scales. Furthermore, native bird species (but not exotics) showed a positive relationship between body size and their potential to propagate effects. Habitat composition was the most important landscape factor in our study, generating hotspots of effect propagation around forested areas, whereas landscapes containing a variety of habitats acted as a buffer against propagation. Overall, our results indicate that species displaying specific sets of traits, including ubiquity, interaction generalism, and a combination of large body size and native status, are the most likely to propagate large-scale ecological impacts in the plant-frugivore communities studied, yet landscape properties may moderate this spread.
Comparing natural language processing representations of disease sequences for prediction in the electronic healthcare record
Authors: Beaney Thomas , Jha Sneha , Alaa Asem , Smith Alexander , Clarke Jonathan , Woodcock Thomas , Majeed Azeem , Aylin Paul , Barahona Mauricio
Published: November 2023

Natural language processing (NLP) is increasingly being applied to obtain unsupervised representations of electronic healthcare record (EHR) data, but their performance for the prediction of clinical endpoints remains unclear. Here we use primary care EHRs from 6,286,233 people with Multiple Long-Term Conditions in England to generate vector representations of sequences of disease development using two input strategies (212 disease categories versus 9,462 diagnostic codes) and different NLP algorithms (Latent Dirichlet Allocation, doc2vec and two transformer models designed for EHRs). We also develop a new transformer architecture, named EHR-BERT, which incorporates socio-demographic information. We then compare use of each of these representations to predict mortality, healthcare use and new disease diagnosis. We find that representations generated using disease categories perform similarly to those using diagnostic codes, suggesting models can equally manage smaller or larger vocabularies. Sequence-based algorithms perform consistently better than bag-of-words methods, with the highest performance for EHR-BERT.
Guided versus standard antiplatelet therapy in patients undergoing interventional intracranial aneurysm treatment using stents: protocol of a cluster randomized controlled cohort study
Authors: Zhou Yangyang , Li Wenqiang , Zhang Hongqi , Luo Bin , Wang Yang , Wang Jun , Zhang Pinyuan , Li Liang , Xiao Fushun , Mu Shiqing , Liu Jian , Zhao Yuanli , Yang Xinjian , Jiang Peng
Published: November 2023

Background

The goal of standard dual antiplatelet therapy (SDAT) in patients undergoing endovascular interventional treatment of intracranial aneurysms is to prevent thrombosis; however, some patients have a poor response to these drugs, which increases the risk of cerebral infarction. This study aims to examine whether adjusting antiplatelet therapy based on light transmission aggregometry (LTA) can reduce the incidence of ischemic events compared with SDAT.

Methods

We will conduct a cluster randomized controlled trial using 16 treatment teams from eight hospitals in mainland China, enrolling 590 patients with unruptured intracranial aneurysms treated using endovascular stent placement. The treatment teams serving as clusters will be randomly assigned to either the test or control group at a 1:1 ratio. Test group patients will receive an antiplatelet regimen guided by LTA. Control group patients will receive SDAT. Patients will be followed for 1 month after the treatment period. The primary outcome measure is cerebral ischemic events within 30 days of stent placement, including stent thrombosis, ischemic stroke, and transient ischemic attack. The safety measure is all bleeding events within 30 days of treatment.

Discussion

The trial aims to determine whether LTA-guided antiplatelet therapy reduces the incidence of ischemic events without increasing the risk of bleeding in patients with intracranial aneurysms treated with endovascular stenting. Completion of this clinical trial may provide an individualized safe and effective regimen for antiplatelet therapy.

Trial registration ClinicalTrials.gov,NCT05825391. Registered on April 11, 2023.

What is already known on this topic

Dual antiplatelet therapy is administered to reduce thrombotic events in neurointerventional therapy of intracranial aneurysms. Platelet function testing is used to evaluate the antiplatelet effect of aspirin and clopidogrel.

What this study adds

The trial aims to determine whether LTA-guided antiplatelet therapy reduces the incidence of ischemic events without increasing the risk of bleeding in patients with intracranial aneurysms treated with endovascular stenting.

How this study might affect research, practice or policy

For stent neurointerventional therapy of unruptured intracranial aneurysms, this study could provide a promising method for adjusting appropriate antiplatelet therapy.
The antiviral protein Shiftless blocks p-body formation during KSHV infection
Authors: Hatfield David , Rodriguez William , Mehrmann Timothy , Muller Mandy
Published: November 2023

P-bodies (PB) are non-membranous foci involved in determining mRNA fate by affecting translation and mRNA decay. In this study, we identify the anti-viral factor SHFL as a potent disassembly factor of PB. We show that PBs remain sparse in the presence of SHFL even in the context of oxidative stress, a major trigger for PB induction. Mutational approaches revealed that SHFL RNA binding activity is not required for its PB disassembly function. However, we have identified a new region of SHFL which bridges two distant domains as responsible for PB disassembly. Furthermore, we show that SHFL ability to disrupt PB formation is directly linked to its anti-viral activity during KSHV infection. While WT SHFL efficiently restricts KSHV lytic cycle, PB disruption defective mutants no longer lead to reactivation defects. SHFL-mediated PB disassembly also leads to increased expression of key anti-viral cytokines, further expanding SHFL dependent anti-viral state. Taken together, our observations suggest a role of SHFL in PB disassembly, which could have important anti-viral consequences during infection.
Molecular mechanisms of re-emerging chloramphenicol susceptibility in extended-spectrum beta-lactamase producing Enterobacterales
Authors: Graf Fabrice E , Goodman Richard N , Gallichan Sarah , Forrest Sally , Picton-Barlow Esther , Fraser Alice J , Phan Minh-Duy , Mphasa Madalitso , Hubbard Alasdair T M , Musicha Patrick , Schembri Mark A , Roberts Adam P , Edwards Thomas , Lewis Joseph M , Feasey Nicholas A
Published: November 2023

Infections with Enterobacterales (E) are increasingly difficult to treat due to antimicrobial resistance. After ceftriaxone replaced chloramphenicol (CHL) as empiric therapy for suspected sepsis in Malawi in 2004, ESBL-E rapidly emerged. Concurrently, resistance to CHL inEscherichia coliandKlebsiellaspp. decreased, raising the possibility of CHL re-introduction. However, many phenotypically susceptible isolates still carry CHL acetyltransferase (CAT) genes.

We used a combination of genomics, phenotypic susceptibility assays, experimental evolution and functional assays for CAT activity to understand the molecular mechanisms and stability of this re-emerging CHL susceptibility.

Of 840 Malawian isolates, 31% had discordant CHL susceptibility genotype-phenotype, and we selected 42 isolates for in-depth analysis. Stable degradation ofcatgenes by insertion sequences led to re-emergence of CHL susceptibility. Our study suggests CHL could be reintroduced as reserve agent for critically ill patients with ESBL-E infections in Malawi and similar settings and highlights the ongoing challenges in inferring antimicrobial resistance from sequence data.
Establishment of an intragastric surgical model using C57BL/6 mice to study the vaccine efficacy of OMV-based immunogens againstHelicobacter pylori
Authors: Das Sanjib , Halder Prolay , Banerjee Soumalya , Mukhopadhyay Asish Kumar , Dutta Shanta , Koley Hemanta
Published: November 2023

Chronic gastritis is one of the major symptoms of gastro-duodenal disorders typically induced byHelicobacter pylori(H. pylori).To date, no suitable model is available to study pathophysiology and therapeutic measures accurately. Here, we have presented a successful surgical infection model ofH. pylori-induced gastritis in C57BL/6 mice that resembles features similar to human infection. The proposed model does not require any preparatory treatment other than surgical intervention. C57BL/6 mice were injected with wild-type SS1 (Sydney strain 1, reference strain) directly into the stomach. Seven days post infection, infected animals showed alterations in cytokine responses along with inflammatory cell infiltration in the lamina propria, depicting a prominent inflammatory response due to infection. To understand the immunogenicity and protective efficacy, the mice were immunized with outer membrane vesicles (OMVs) isolated from an indigenous strain with putative virulence factors ofH. pylori[A61C (1),cag+/vacA s1m1]. In contrast to the nonimmunized cohort, the OMV-immunized cohort showed a gradual increase in serum immunoglobulin(s) levels on the 35thday after the first immunization. This conferred protective immunity against subsequent challenge with the reference strain (SS1). Direct inoculation ofH. pyloriinto the stomach influenced infection in a short time and, more importantly, in a dose-dependent manner, indicating the usefulness of the developed model for pathophysiology, therapeutic and prophylactic studies.