bluuebunny/crossref_metadata_2025 huggingface dataset:
The MAP2Ks can use ADP to phosphorylate and activate their substrate MAPKsin vitro
Authors: Juyoux Pauline , von Velsen Jill , Pellegrini Erika , Bowler Matthew W.
Published: February 2024

Kinases are a diverse group of enzymes that use ATP to phosphorylate a variety of substrates. Protein kinases evolved in eukaryotes as important mediators of cell signalling that target specific amino acid side chains to modulate downstream protein function. Among them, the MAPKs (mitogen-activated protein kinases) are a family of intracellular protein kinases that form signalling cascades responding to a number of stimuli, that control fundamental mechanisms such as proliferation, differentiation, inflammation and cell death. Signals propagate through consecutive kinases which eventually phosphorylate and activate a MAPK. Here, we show that the dual specificity threonine/tyrosine MAP kinase kinases (MAP2Ks or MEKs) are able to phosphorylate and activate their substrate MAPKs using ADP as well as ATPin vitro. As the pathways are involved in the stress response, we speculate that it would represent an advantage to be able to maintain signalling under conditions such as hypoxia, that occur under a number of cell stresses, including cancer and atherosclerosis, where the available pool of ATP could be depleted.

Highlights

The MAP2K dual-specificity protein kinases can phosphorylate their target MAPKs using ADPin vitro

The reaction with ADP is less efficient than with ATP

First example of an enzyme that can use both ATP and ADP

ADP phosphorylation might be a potential mechanism to maintain signal integrity when cell energy resources are constrained, as during ischemia
Degradation of IKAROS prevents epigenetic progression of T cell exhaustion in a novel antigen-specific assay
Authors: Tay Tristan , Bommakanti Gayathri , Jaensch Elizabeth , Gorthi Aparna , Reddy Iswarya Karapa , Hu Yan , Zhang Ruochi , Doshi Aatman , Tan Sin Lih , Brucklacher-Waldert Verena , Prickett Laura , Kurasawa James , Overstreet Michael Glen , Criscione Steven , Buenrostro Jason Daniel , Mele Deanna A.
Published: February 2024

In cancer, chronic antigen stimulation drives effector T cells to exhaustion, limiting the efficacy of T cell therapies. Recent studies have demonstrated that epigenetic rewiring governs the transition of T cells from effector to exhausted states and makes a subset of exhausted T cells non-responsive to PD1 checkpoint blockade. Here, we describe an antigen-specific assay for T cell exhaustion that generates T cells that are phenotypically and transcriptionally similar to those found in human tumors. We performed a screen of human epigenetic regulators, identifying and validating IKAROS as a driver of T cell exhaustion. We found that the IKAROS degrader iberdomide prevents exhaustion by blocking chromatin remodeling at T cell effector enhancers and preserving binding of AP-1, NF-κB, and NFAT. Thus, our study uncovered a role for IKAROS as a driver of T cell exhaustion through epigenetic modulation, providing a rationale for the potential use of iberdomide in solid tumors to prevent T cell exhaustion.

Highlights

Novelin vitroassay generates antigen-specific exhausted T cells from human T cells

IKAROS (IKZF1) is a key driver of T cell exhaustion

Iberdomide (IKZF1/3 degrader) prevents the progression of exhaustion

TF footprinting reveals IKAROS silences effector genes by inhibiting AP-1, NF-κB and NFAT binding
O6-Alkylguanine-DNA Alkyltransferase Maintains Genomic Integrity During Peroxynitrite-Mediated DNA Damage by Forming DNA-Protein Crosslinks
Authors: Chakraborty Shayantani , Mukherjee Gargi , Chakrabarty Anindita , Chowdhury Goutam
Published: February 2024

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases and may be fatal. Peroxynitrite (PN) is a major reactive nitrogen species (RNS) generated during inflammation. It produces various DNA lesions including labile 8-nitroguanine which spontaneously converts into abasic sites resulting in DNA strand breakage. Here, we report the discovery of a previously unrecognized function of the human repair proteinO6-alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate can spontaneously react with 8-nitroguanine in DNA to form a stable DNA-protein crosslink (DPC). Interestingly, the process of DPC formation provides protection from PN-mediated genome instability. The Cys145-mutant of hAGT failed to form DPC and provide protection against inflammation-associated, PN-mediated cytotoxicity. Gel shift, dot blot and UV-Vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs may seem detrimental, there are multiple systems in place in normal cells for their repair.
An experimental validation test of ecological coexistence theory to forecast extinction under rising temperatures
Authors: Terry J. Christopher D.
Published: February 2024

Interactions between species pose considerable challenges for forecasting the response of ecological communities to global changes. Coexistence theory could address this challenge by defining the conditions species can or cannot persist alongside competitors. However, although coexistence theory is increasingly deployed for projections, these frameworks have rarely been subjected to critical multigenerational validation tests. Here, using a highly replicated mesocosm experiment, I directly test if the modern coexistence theory approach can predict time-to-extirpation in the face of rising temperatures within the context of competition from a heat-tolerant species. Competition hastened expiration and the modelled point of coexistence breakdown overlapped with mean observations under both steady temperature increases and with additional environmental stochasticity. That said, although the theory identified the interactive effect between the stressors, predictive precision was low even in this simplified system. Nonetheless, these results support the careful use of coexistence modelling for forecasts and understanding drivers of change.
Stomatal patterning is differently regulated in adaxial and abaxial epidermis in Arabidopsis
Authors: Jalakas Pirko , Tulva Ingmar , Bērziņa Nele Malvīne , Hõrak Hanna
Published: February 2024

Stomatal pores in leaves mediate CO2uptake into the plant and water loss via transpiration. Most plants are hypostomatous with stomata present only in the lower leaf surface (abaxial epidermis). Many herbs, including the model plantArabidopsis thaliana, have substantial numbers of stomata also on the upper (adaxial) leaf surface. Studies of stomatal development have mostly focused on abaxial stomata and very little is known of adaxial stomatal formation. We addressed the role of leaf number in determination of stomatal density and stomatal ratio, and studied adaxial and abaxial stomatal patterns in mutants deficient in known abaxial stomatal development regulators. We found that stomatal density in some genetic backgrounds varies between different fully expanded leaves and recommend using defined leaves for analyses of stomatal patterning. Our results indicate that stomatal development is at least partly independently regulated in adaxial and abaxial epidermis, as i) plants deficient in ABA biosynthesis and perception have increased stomatal ratios, ii) theepf1epf2,tmmandsdd1mutants have reduced stomatal ratios, iii)erl2mutants have increased adaxial but not abaxial stomatal index, and iv) stomatal precursors preferentially occur in abaxial epidermis. Further studies of adaxial stomata can reveal new insights into stomatal form and function.
The evolution of reduced facilitation in a four-species bacterial community
Authors: Piccardi Philippe , Ulrich Eric , Garcia-Garcerà Marc , Martino Rita Di , Testa Samuele E. A. , Mitri Sara
Published: February 2024

Microbial evolution is typically studied in mono-cultures or in communities of competing species. But microbes do not always compete and how positive inter-species interactions drive evolution is less clear: Initially facilitative communities may either evolve increased mutualism, increased reliance on certain species according to the Black Queen Hypothesis (BQH), or weaker interactions and resource specialization. To distinguish between these outcomes, we evolved four species for 44 weeks either alone or together in a toxic pollutant. These species initially facilitated each other, promoting each other’s survival and pollutant degradation. After evolution, two species (Microbacterium liquefaciensandOchrobactrum anthropi) that initially relied fully on others to survive continued to do so, with no evidence for increased mutualism. Instead,Agrobacterium tumefaciensandComamonas testosteroni(Ct) whose ancestors interacted positively, evolved in community to interact more neutrally and grew less well than when they had evolved alone, suggesting that the community limited their adaptation. We detected several gene loss events inCtwhen evolving with others, but these events did not increase its reliance on other species, contrary to expectations under the BQH. We hypothesize instead that these gene loss events are a consequence of resource specialization. Finally, co-evolved communities degraded the pollutant worse than their ancestors. Together, our results support the evolution of weakened interactions and resource specialization, similar to what has been observed in competitive communities.
MaizeCODE reveals bi-directionally expressed enhancers that harbor molecular signatures of maize domestication
Authors: Cahn Jonathan , Regulski Michael , Lynn Jason , Ernst Evan , Alves Cristiane de Santis , Ramakrishnan Srividya , Chougule Kapeel , Wei Sharon , Lu Zhenyuan , Xu Xiaosa , Drenkow Jorg , Kramer Melissa , Seetharam Arun , Hufford Matthew B. , McCombie W. Richard , Ware Doreen , Jackson David , Schatz Michael C. , Gingeras Thomas R. , Martienssen Robert A.
Published: February 2024

Modern maize was domesticated fromTeosinte parviglumis, with subsequent introgressions fromTeosinte mexicana, yielding increased kernel row number, loss of the hard fruit case and dissociation from the cob upon maturity, as well as fewer tillers. Molecular approaches have identified several transcription factors involved in the development of these traits, yet revealed that a complex regulatory network is at play. MaizeCODE deploys ENCODE strategies to catalog regulatory regions in the maize genome, generating histone modification and transcription factor ChIP-seq in parallel with transcriptomics datasets in 5 tissues of 3 inbred lines which span the phenotypic diversity of maize, as well as the teosinte inbred TIL11. Integrated analysis of these datasets resulted in the identification of a comprehensive set of regulatory regions in each inbred, and notably of distal enhancers which were differentiated from gene bodies by their lack of H3K4me1. Many of these distal enhancers expressed non- coding enhancer RNAs bi-directionally, reminiscent of “super enhancers” in animal genomes. We show that pollen grains are the most differentiated tissue at the transcriptomic level, and share features with endosperm that may be related to McClintock’s chromosome breakage- fusion-bridge cycle. Conversely, ears have the least conservation between maize and teosinte, both in gene expression and within regulatory regions, reflecting conspicuous morphological differences selected during domestication. The identification of molecular signatures of domestication in transcriptional regulatory regions provides a framework for directed breeding strategies in maize.
Living in a multi-stressor world: nitrate pollution and thermal stress interact to affect amphibian larvae
Authors: Sinai Noa , Eterovick Paula C. , Kruger Natasha , Oetken Ben , Ruthsatz Katharina
Published: February 2024

The interaction of widespread stressors such as nitrate pollution and increasing temperatures associated with climate change are likely to affect aquatic ectotherms such as amphibians. The metamorphic and physiological traits of amphibian larvae during the critical onset of metamorphosis are particularly susceptible to these stressors. We conducted a common-garden experiment usingRana temporarialarvae subjected to four constant acclimation temperatures (18, 22, 26, 28 °C) crossed with three environmentally relevant nitrate concentrations (0, 50, 100 mg × L-1) to investigate the interactive and individual effects of these stressors on metamorphic (i.e., growth and development) and physiological traits (i.e., metabolism and heat tolerance) at the onset of metamorphosis. Larvae exposed to elevated nitrate concentrations and thermal stress displayed increased metabolic rates but decreased developmental rate, highlighting interactive effects of these stressors. However, nitrate pollution alone had no effect on either metamorphic or physiological traits, suggesting that detoxification processes were sufficient to maintain homeostasis but not in combination with increased acclimation temperatures. Furthermore, larvae exposed to nitrate displayed diminished abilities to exhibit temperature-induced plasticity in metamorphosis timing and heat tolerance, as well as reduced acclimation capacity in metabolic rate and heat tolerance to higher temperatures. These results highlight the importance of considering the exposure to multiple stressors when investigating how natural populations respond to global change.
Individualized temporal patterns dominate cortical upstate and sleep depth in driving human sleep spindle timing
Authors: Chen Shuqiang , He Mingjian , Brown Ritchie E. , Eden Uri T. , Prerau Michael J.
Published: February 2024

Sleep spindles are critical for memory consolidation and strongly linked to neurological disease and aging. Despite their significance, the relative influences of factors like sleep depth, cortical up/down states, and spindle temporal patterns on individual spindle production remain poorly understood. Moreover, spindle temporal patterns are typically ignored in favor of an average spindle rate. Here, we analyze spindle dynamics in 1008 participants from the Multi-Ethnic Study of Atherosclerosis using a point process framework. Results reveal fingerprint-like temporal patterns, characterized by a refractory period followed by a period of increased spindle activity, which are highly individualized yet consistent night-to-night. We observe increased timing variability with age and distinct gender/age differences. Strikingly, and in contrast to the prevailing notion, individualized spindle patterns are the dominant determinant of spindle timing, accounting for over 70% of the statistical deviance explained by all of the factors we assessed, surpassing the contribution of slow oscillation (SO) phase (∼14%) and sleep depth (∼16%). Furthermore, we show spindle/SO coupling dynamics with sleep depth are preserved across age, with a global negative shift towards the SO rising slope. These findings offer novel mechanistic insights into spindle dynamics with direct experimental implications and applications to individualized electroencephalography biomarker identification.
Distinct impacts of each anti-anti-sigma factor ortholog of the chlamydial Rsb partner switching mechanism on development inChlamydia trachomatis
Authors: Junker Shiomi , Singh Vandana , Al-Saadi Aamal G.M. , Wood Nicholas A. , Hamilton-Brehm Scott D. , Ouellette Scot P. , Fisher Derek J.
Published: February 2024

Partner Switching Mechanisms (PSM) are signal transduction systems comprised of a sensor phosphatase (RsbU), an anti-sigma factor (RsbW, kinase), an anti-anti-sigma factor (RsbV, the RsbW substrate), and a target sigma factor.Chlamydiaspp. are obligate intracellular bacterial pathogens of animals that undergo a developmental cycle transitioning between the infectious elementary body (EB) and replicative reticulate body (RB) within a host-cell derived vacuole (inclusion). Secondary differentiation events (RB to EB) are transcriptionally regulated, in part, by the house-keeping sigma factor (σ66) and two late-gene sigma factors (σ54and σ28). Prior research supports that the PSM inChlamydia trachomatisregulates availability of σ66. Pan-genome analysis revealed that PSM components are conserved across the phylum Chlamydiota, withChlamydiaspp. possessing an atypical arrangement of two anti-anti-sigma factors, RsbV1 and RsbV2. Bioinformatic analyses support RsbV2 as the homolog to the pan-genome conserved RsbV with RsbV1 as an outlier. This, combined within vitrodata, indicates that RsbV1 and RsbV2 are structurally and biochemically distinct. Reduced levels or overexpression of RsbV1/RsbV2 did not significantly impactC. trachomatisgrowth or development. In contrast, overexpression of a non-phosphorylatable RsbV2 S55A mutant, but not overexpression of an RsbV1 S56A mutant, resulted in a 3 log reduction in infectious EB production without reduction in genomic DNA (total bacteria) or inclusion size, suggesting a block in secondary differentiation. The block was corroborated by reduced production of σ54/28-regulated late proteins and via transmission electron microscopy.

Importance

C. trachomatisis the leading cause of reportable bacterial sexually transmitted infections (STIs) and causes the eye infection trachoma, a neglected tropical disease. Broad-spectrum antibiotics used for treatment can lead to microbiome dysbiosis and increased antibiotic resistance development in other bacteria, and treatment failure for chlamydial STIs is a recognized clinical problem. Here, we show that disruption of a partner switching mechanism (PSM) significantly reduces infectious progeny production via blockage of RB to EB differentiation. We also reveal a novel PSM expansion largely restricted to the species infecting animals, suggesting a role in pathogen evolution. Collectively, our results highlight the chlamydial PSM as a key regulator of development and as a potential target for the development of novel therapeutics to treat infections.
Positioning of negative feedback loops within immune signaling pathways influences host fitness through noise in AMP expression
Authors: Asgari Danial , Tate Ann T
Published: February 2024

Signaling pathways depend on negative feedback loops (NFLs) to regulate internal noise. Across diverse organisms, signaling pathways are regulated by NFLs that function at different cellular locations. These range from NFLs functioning upstream near signal-receiving receptors to those downstream within the nucleus. Multi-level regulation of signaling pathways by NFLs is ubiquitous, however, we do not know how it influences noise regulation and ultimately the host fitness. Here, we quantify noise in the expression of antimicrobial peptides (AMPs) upon induction of immune signaling using stochastic models. We hypothesize that noise regulation in the expression of immune genes is crucial for mounting nuanced responses to diverse environmental challenges. By altering the strength of NFLs that function at different cellular locations, we measured the effect of noise on fitness across various environmental conditions. We discovered that upstream NFLs reduce noise whereas downstream NFLs increase noise in the expression of AMPs. The noisy expression of AMPs by downstream NFLs increases host fitness during repeated exposure to pathogens. Conversely, upstream NFLs reduce fitness variation across genotypes possibly giving rise to bet-hedging. This study shows the significance of multi-level regulation by NFLs and contributes to our understanding of noise regulation in diverse signaling pathways.
Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection
Authors: Aggarwal Somya , Walker Forrest C. , Weagley James S. , McCune Broc T. , Wu Xiaofen , Schriefer Lawrence A. , Makimaa Heyde , Lawrence Dylan , Sridhar Pratyush , Baldridge Megan T.
Published: February 2024

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractablein vivomodel for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3’ position of the NS4 gene and used this pool as our viral inoculum forin vivoinfections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.
Plasmalogen oxidation induces the generation of excited molecules and electrophilic lipid species
Authors: Faria Rodrigo L. , Prado Fernanda M. , Junqueira Helena C. , Fabiano Karen C. , Diniz Larissa R. , Baptista Mauricio S. , Di Mascio Paolo , Miyamoto Sayuri
Published: February 2024

Plasmalogens are glycerophospholipids with a vinyl-ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species (ROS), our study reveals the generation reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) we demonstrate that singlet molecular oxygen [O2(1Δg)] reacts with the vinyl ether bond, producing hydroperoxyl acetal as major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates lead to the formation of excited triplet carbonyls, O2(1Δg), and electrophilic phospholipid and fatty aldehyde species, as secondary reactive products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoantracene as a triplet enhancer. Moreover, O2(1Δg) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phopspholipid and fatty aldehydes by LC-MS analysis using two probes that specifically reacts with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen oxidized products.

Significance Statement

Plasmalogens, the most abundant subclass of ether lipids in mammalian cells, have traditionally been regarded as antioxidants. However, our study reveals a new perspective, shedding light on the generation of chemiexcited and reactive lipid species during plasmalogen photooxidation. We provide direct evidence revealing the production of excited triplet carbonyls and singlet molecular oxygen as secondary reactive products originating from dioxetane and hydroperoxyacetal intermediates. Importantly, we also demonstrate the generation of electrophilic alpha-beta unsaturated phospholipids and fatty aldehydes through plasmalogen oxidation. These findings highlight the production of excited states and reactive lipid species resulting from plasmalogen oxidation, which can potentially induce oxidative modifications in biological systems.
5-Aza-Cytidine Enhances Terminal Polyadenylation Site Usage for Full-Length Transcripts in Cells
Authors: Ogunsola Samuel , Liu Ling , Das Urmi , Xie Jiuyong
Published: February 2024

As an inhibitor of DNA methyltransferases (DNMT) and anti-cancer drug, the many effects of 5-aza-cytidine (5-azaC) on gene expression remains unknown. Here we show that 5-azaC treatment of cultured pituitary tumour cells increases the relative usage of the genomic terminal exons (GTE) across the transcriptome. This effect is largely achieved by switching mRNA polyadenylation (poly(A)) from proximal sites to the GTE, which have a more optimal poly(A) signal consensus motif. Consistently, 5-azaC upregulates mRNA anti-terminators Scaf4 and Scaf8 while downregulating the early termination enhancer E2f2. In MOLM-13 leukaemia cells, 5-azaC similarly promotes full-length transcript production, regulating alternative polyadenylation factors in both common and cell-specific ways. Unexpectedly, PCF11, known to promote proximal poly(A) site usage, is upregulated in both cell lines, reminiscent of a homeostatic effort by the tumour/cancer cells to maintain their shortened transcripts during the 5-azaC treatment. This study highlights a previously unknown aspect of 5-azaC’s effect on gene expression as a DNMT inhibitor and anti-cancer drug: to directionally promote the terminal polyadenylation site usage to switch from the shortened to full-length transcripts of cancer cells and consequently the alternative usage of strings of 3’ exons.
Placental cytotrophoblast microvillar stabilization is required for cell-cell fusion
Authors: Duan Wendy K. , Shaha Sumaiyah Z. , Patel Khushali J. , Domingo Ivan K. , Riddell Meghan R.
Published: February 2024

The placenta is an essential organ of pregnancy required for maternal-fetal transport and communication. The surface of the placenta facing the maternal blood is formed by a single giant multinucleate cell: the syncytiotrophoblast. The syncytiotrophoblast is formed and maintained via fusion of progenitor cytotrophoblasts. Cell-cell fusion is a tightly regulated process, and in non-trophoblastic cells is accompanied by stereotypical alterations in cell shape by cells that have attained fusion-competence. The most prominent feature is the formation of actin-based membrane protrusions, but whether stereotypic morphological changes occur in fusion-competent cytotrophoblasts has not been characterized. Using a human placental explant model, we characterized cell shape factors associated with the attainment of cytotrophoblast fusion competence. We found that fusion-competent cytotrophoblasts are hypertrophic, elongated cells, that form microvilli at the apical membrane. The actin-membrane cross linker protein ezrin was found to have highly polarized expression within cytotrophoblast microvilli. Inhibition of ezrin activation destabilized cytotrophoblast microvilli and prevented cytotrophoblast fusion. Thus, we propose that the polarized activation of ezrin within apical microvilli and actin-mediated changes in membrane dynamics are necessary for cytotrophoblast fusion.

Summary statement

Fusion-competent cytotrophoblasts undergo dynamic changes in cell morphology including the acquisition of apically localized microvilli. Microvillar stabilization facilitates effective fusion and differentiation.
Deep phenotypic profiling of neuroactive drugs in larval zebrafish
Authors: Gendelev Leo , Taylor Jack , Myers-Turnbull Douglas , Chen Steven , McCarroll Matthew N. , Arkin Michelle R. , Kokel David , Keiser Michael J.
Published: February 2024

Behavioral larval zebrafish screens leverage a high-throughput small molecule discovery format to find neuroactive molecules relevant to mammalian physiology. We screened a library of 650 central nervous system active compounds in high replicate to train a deep metric learning model on zebrafish behavioral profiles. The machine learning initially exploited subtle artifacts in the phenotypic screen, necessitating a complete experimental re-run with rigorous well-wise randomization. These large matched phenotypic screening datasets (initial and well-randomized) provided a unique opportunity to quantify and understand shortcut learning in a full-scale, real-world drug discovery dataset. The final deep metric learning model substantially outperforms correlation distance–the canonical way of computing distances between profiles–and generalizes to an orthogonal dataset of novel druglike compounds. We validated predictions by prospectivein vitroradio-ligand binding assays against human protein targets, achieving a hit rate of 58% despite crossing species and chemical scaffold boundaries. These newly discovered neuroactive compounds exhibited diverse chemical scaffolds, demonstrating that zebrafish phenotypic screens combined with metric learning achieve robust scaffold hopping capabilities.
How Fungi Biosynthesize 3-Nitropropanoic Acid: the Simplest yet Lethal Mycotoxin
Authors: Johnson Colin W. , Ohashi Masao , Tang Yi
Published: February 2024

We uncovered the biosynthetic pathway of the lethal mycotoxin 3-nitropropanoic acid (3-NPA) from koji moldAspergillus oryzaethrough genome mining, genetic inactivation and biochemical characterization. The biosynthetic gene cluster (BGC) of 3-NPA, which encodes an amine oxidase and a decarboxylase, is conserved in many fungi used in food processing, although most of the strains have not been reported to produce 3-NPA. Our discovery will lead to efforts that improve the safety profiles of these indispensable microorganisms in making food, alcoholic beverages, and seasoning.
A synergistic culture dependent and independent approach reveals a conserved wheat seed mycobiome
Authors: Becker Lindsey E. , Marshall David , Cubeta Marc A.
Published: February 2024

The occurrence of pathogenic fungal taxa associated with wheat (Triticum aestivumL.) seeds is well studied, but less is known about non-pathogenic taxa of the wheat seed mycobiome. The goal of our research is to characterize wheat seed fungal endophyte diversity with a synergistic culture dependent and independent experimental approach. Four publicly available winter wheat cultivars developed in the southeastern United States with varying phenotypic and disease resistance traits were examined over a period of two years: Catawba, Hilliard, Shirley, and USG 3640. Our culture dependent methods involving two nutrient media generated 645 fungal isolates representing twelve genera sampled from multiple cultivars. Metabarcoding analysis identified a broader range of fungal taxa and a greater number of unique sequences than culture dependent methods. When examining fungal diversity across cultivars and years, richness decreased in 2021 for both culture dependent and independent approaches. However, wheat seed fungal community structure was stable across cultivars and years. Our results highlight the importance of combining culture independent and dependent methods to capture and establish a diverse endophytic fungal catalog associated with the wheat seed and highlight areas where future culture dependent efforts can focus their efforts.
The physiology of bud dormancy and cell cycle status in grapevine
Authors: Hermawaty Dina , Clode Peta L. , Considine John A. , Signorelli Santiago , Considine Michael J.
Published: February 2024

Evaluating the cell cycle status during dormancy of multicellular organisms is problematic. This is particularly so for woody perennial buds, where dormant and quiescent states are diffuse, and the organ may remain visibly unchanged for six to nine months of the year. In this study, we investigate cell cycle status of dormant grapevine buds by measuring mitotic index using an optimised method developed for grapevine bud tissue. The experimental material showed a dynamic range in the depth of dormancy, declining from 200 days in March to less than 60 days in May and 30 days in August, measured as the time to reach 50% bud burst in forcing conditions. Despite these differences, flow cytometry analysis showed that most nuclei isolated from these buds were arrested at the G1 phase. Ultrastructure analysis of the cells in the region of the shoot apical meristem confirmed that the mitotic activities of buds remained low at all time points, together with the development of starch grains and the relative absence of organelle development.

HIGHLIGHT

The cell cycle and ultrastructure data suggest interesting evidence correspond to the growth resumption capacity of grapevine cv. Cabernet Sauvignon buds, i.e., absence of mitosis activities regardless of dormancy depth and starch accumulation irrespective of chilling accumulation.
Spatial kinetics and immune control of murine cytomegalovirus infection in the salivary glands
Authors: Byrne Catherine , Márquez Ana Citlali , Cai Bing , Coombs Daniel , Gantt Soren
Published: February 2024

Human cytomegalovirus (HCMV) is the most common congenital infection. Several HCMV vaccines are in development, but none have yet been approved. An understanding of the kinetics of CMV replication and transmission may inform the rational design of vaccines to prevent this infection. The salivary glands (SG) are an important site of sustained CMV replication following primary infection and during viral reactivation from latency. As such, the strength of the immune response in the SG likely influences viral dissemination within and between hosts. To study the relationship between the immune response and viral replication in the SG, and viral dissemination from the SG to other tissues, mice were infected with low doses of murine CMV (MCMV). Following intra-SG inoculation, we characterized the viral and immunological dynamics in the SG, blood, and spleen, and identified organ-specific immune correlates of protection. Using these data, we constructed compartmental mathematical models of MCMV infection. Model fitting to data and analysis indicate the importance of cellular immune responses in different organs and point to a threshold of infection within the SG necessary for the establishment and spread of infection.

Author Summary

Cytomegalovirus (CMV) is the most common congenital infection and causes an enormous burden of childhood disease. To gain insight into the immune requirements for controlling infection, we used a mouse model to reproduce characteristics of natural CMV infection, employing a low viral inoculum, and delivering the virus to the salivary glands (SG), a key site of CMV replication. Our results provide detailed data on the spatial and temporal spread of infection throughout the body and identify key immune correlates of the control of viral replication. By translating these findings into mechanistic mathematical models, we revealed the importance of organ-specific immune responses, particularly the requirement of TNF-αand IFN-γto control infection within the salivary glands. Furthermore, our mathematical modeling allowed us to compare known characteristics of human CMV infection related to infection establishment and spread to those predicted in mice, underscoring the suitability of the MCMV model to study its human homologue. These insights provide guidance for developing targeted vaccines to prevent CMV infection and disease.
Acceptability of fixed-dose combination treatments for hypertension in Kenya: a qualitative study using the Theoretical Framework of Acceptability
Authors: Mbuthia Daniel , Willis Ruth , Gichagua Mary , Nzinga Jacinta , Mugo Peter , Murphy Adrianna
Published: February 2024

Background

Fixed-dose combinations (FDCs) – 2-3 anti-hypertensive medications in a single pill - have the potential to improve hypertension treatment and outcomes. Yet, they are not widely implemented. Factors undermining implementation remain unknown, particularly in sub– Saharan Africa, where hypertension is a major cause of disease burden and is poorly treated.

Methods

We used in-depth semi-structured interviews to explore the acceptability of FDCs among patients, caregivers, and healthcare workers. We interviewed a total of 58 participants across four purposively selected health facilities in one county in Kenya. Data were analyzed using abductive thematic analysis approach, and emergent themes categorized according to the Theoretical Framework of Acceptability.

Results

Overall, FDCs are potentially acceptable to all participant groups. Acceptability is supported by a perception of FDCs as a means of reducing treatment burden (for patients and healthcare workers) and improving treatment adherence, and patients’ deferral to and trust in health workers. Acceptability among health workers may be undermined by variable levels of FDC coherence, concerns among health workers about FDCs as an “inflexible” treatment that does not allow dose titration or identifying causes of side effects, and concerns about consistent availability and affordability of FDCs in Kenya.

Conclusion

FDCs are a potentially acceptable treatment approach for hypertension in Kenya. Efforts to improve acceptability and thus implementation of FDCs in Kenya should consider improving understanding of treatment and strengthening the capacity of all health worker cadres to appropriately prescribe, inform about, and support adherence to FDCs. These efforts must align with work to address upstream health system factors such as poor availability and affordability.
Cardiovascular risk management in the elderly with type 2 diabetes prior to death: A Danish nationwide register study of discontinuation patterns
Authors: Kosjerina Vanja , Scheuer Stine H. , Carstensen Bendix , Jørgensen Marit E. , Brock Birgitte , Christensen Hanne R , Andersen Gregers S , Rungby Jørgen
Published: February 2024

Background

Cardioprotective medication usage among elderly individuals with type 2 diabetes (T2D) is prevalent, however, the degree and timing of discontinuation is unknown. This study aims to describe the extent, timing, and secular changes of discontinuation of cardioprotective medication in elderly with T2D.

Methods

In this register-based cohort study all individuals with T2D, deceased between 2006-2018 at an age of 80 years or older, were identified through Danish nation-wide registers. We followed the population backward in time, from death to last intake of antihypertensive, lipid-lowering, and antithrombotic medication. Poisson regression models were used to model rates of discontinuation prior to death while binomial models were used to estimate the proportion on medication at time of death.

Results

We identified 52,523 individuals (55% women) with a mean (SD) age at T2D diagnosis of 77.3 (7.8) years and median (Q1-Q3) age at death of 86.5 [83.3-90.3] years. The proportion on any antihypertensive and antithrombotic medication was high (approximately 78% [95% CI 77-78] and 48% [95% CI 47-49%] at time of death, respectively) and increased slightly with increasing calendar year of death. Discontinuations occurred predominantly in the last year of life but were initiated earlier for individuals who died in recent calendar years. However, for angiotensin-converting enzyme, thiazides, and acetylsalicylic acid we found a more continuous discontinuation in the decade prior to death in the recent calendar years of death. The proportion on statins increased markedly with calendar year of death, peaking at 34% [95% CI 33-35%] in 2016 with a subsequent decrease to 29% [95% CI 28-30%] in 2018. Discontinuation patterns shifted from predominantly occurring in the last years of life to continues discontinuation in the decade before death.

Conclusion

Our results suggest an intensification with cardioprotective medication among elderly with type 2 diabetes and a more pronounced discontinuation during the last years of life. This may be a result of increased focus on individualized-treatment-regimens.
Speech therapy combined with Cerebrolysin in enhancing aphasia recovery after acute ischemic stroke: ESCAS pilot study
Authors: Homberg Volker , Jianu Dragoș Cătălin , Stan Adina , Strilciuc Ștefan , Chelaru Vlad-Florin , Karliński Michał , Brainin Michael , Heiss Wolf Dieter , Mureșanu Dafin , Enderby Pamela M
Published: February 2024

Background

Stroke-induced aphasia significantly impacts communication and quality of life. Despite the standard treatment being speech and language therapy (SLT), outcomes vary, highlighting the need for additional therapies. Cerebrolysin, a neuroprotective and neurotrophic agent, has shown potential in stroke management. This study examines the effectiveness of combining Cerebrolysin with SLT in treating post-stroke aphasia.

Methods

The ESCAS trial, a prospective, randomized-controlled, double-blinded phase 4 study, was conducted in two Romanian stroke centers. Participants included those with left middle cerebral artery territory ischemic stroke and Broca or mixed non-fluent aphasia, enrolled 3-5 days post-stroke. Inclusion criteria were right-handedness and Romanian as mother tongue; exclusion criteria were prior strokes, severe comprehension deficits, contraindications to MRI, and pre-existing neurodegenerative or psychiatric diseases. Participants received Cerebrolysin or a placebo combined with SLT in ten-day cycles over three intervals.

Results

Out of 132 enrolled patients, 123 were included in the Intention To Treat analysis, and 120 in the Per Protocol analysis. The Cerebrolysin group showed significant improvement in Western Aphasia Battery scores (p < 0.001) and National Institutes of Health Stroke Scale scores (p < 0.001). Modified Rankin Scale and Barthel index scores also improved, with notable differences at the final study visit (Day 90). Safety analysis raised no concerns.

Conclusions

Cerebrolysin combined with SLT offers promising potential for enhancing recovery in post-stroke aphasia. Significant improvements were observed in language and neurological deficits, underscoring the importance of adjunctive therapies in aphasia rehabilitation. Further research with larger cohorts is needed to fully establish the efficacy of this combination therapy.

Clinical Trial Registration

https://www.isrctn.com/ISRCTN54581790
A Machine Learning Framework to Identify the Correlates of Disease Severity in Acute Arbovirus Infection
Authors: Herder Vanessa , Caporale Marco , MacLean Oscar A , Pintus Davide , Huang Xinyi , Nomikou Kyriaki , Palmalux Natasha , Nichols Jenna , Scivoli Rosario , Boutell Chris , Taggart Aislynn , Allan Jay , Malik Haris , Ilia Georgios , Gu Quan , Ronchi Gaetano Federico , Furnon Wilhelm , Zientara Stephan , Bréard Emmanuel , Antonucci Daniela , Capista Sara , Giansante Daniele , Cocco Antonio , Mercante Maria Teresa , Di Ventura Mauro , Filipe Ana Da Silva , Puggioni Giantonella , Sevilla Noemi , Stewart Meredith E. , Ligios Ciriaco , Palmarini Massimo
Published: February 2024

Most viral diseases display a variable clinical outcome due to differences in virus strain virulence and/or individual host susceptibility to infection. Understanding the biological mechanisms differentiating a viral infection displaying severe clinical manifestations from its milder forms can provide the intellectual framework toward therapies and early prognostic markers. This is especially true in arbovirus infections, where most clinical cases are present as mild febrile illness. Here, we used a naturally occurring vector-borne viral disease of ruminants, bluetongue, as an experimental system to uncover the fundamental mechanisms of virus-host interactions resulting in distinct clinical outcomes. As with most viral diseases, clinical symptoms in bluetongue can vary dramatically. We reproduced experimentally distinct clinical forms of bluetongue infection in sheep using three bluetongue virus (BTV) strains (BTV-1IT2006, BTV-1IT2013and BTV-8FRA2017). Infected animals displayed clinical signs varying from clinically unapparent, to mild and severe disease. We collected and integrated clinical, haematological, virological, and histopathological data resulting in the analyses of 332 individual parameters from each infected and uninfected control animal. We subsequently used machine learning to identify the key viral and host processes associated with disease pathogenesis. We identified five different fundamental processes affecting the severity of bluetongue: (i) virus load and replication in target organs, (ii) modulation of the host type-I IFN response, (iii) pro-inflammatory responses, (iv) vascular damage, and (v) immunosuppression. Overall, our study using an agnostic machine learning approach, can be used to prioritise the different pathogenetic mechanisms affecting the disease outcome of an arbovirus infection.
Canonical AREs are tumor suppressive regulatory elements in the prostate
Authors: Augello Michael A. , Chen Xuanrong , Liu Deli , Lin Kevin , Hakansson Alex , Sjöström Martin , Khani Francesca , Deonarine Lesa D. , Liu Yang , Travascio-Green Jaida , Wu Jiansheng , Loda Massimo , Feng Felix Y. , Robinson Brian D. , Davicioni Elai , Sboner Andrea , Barbieri Christopher E.
Published: February 2024

The androgen receptor (AR) is the central determinant of prostate tissue identity and differentiation, controlling normal, growth-suppressive prostate-specific gene expression1. It is also a key driver of prostate tumorigenesis, becoming “hijacked” to drive oncogenic transcription2-5. However, the regulatory elements determining the execution of the growth suppressive AR transcriptional program, and whether this can be reactivated in prostate cancer (PCa) cells remains unclear. Canonical androgen response element (ARE) motifs are the classic DNA binding element for AR6. Here, we used a genome-wide strategy to modulate regulatory elements containing AREs to define distinct AR transcriptional programs. We find that activation of these AREs is specifically associated with differentiation and growth suppressive transcription, and this can be reactivated to cause death in AR+PCa cells. In contrast, repression of AREs is well tolerated by PCa cells, but deleterious to normal prostate cells. Finally, gene expression signatures driven by ARE activity are associated with improved prognosis and luminal phenotypes in human PCa patients. This study demonstrates that canonical AREs are responsible for a normal, growth-suppressive, lineage-specific transcriptional program, that this can be reengaged in PCa cells for potential therapeutic benefit, and genes controlled by this mechanism are clinically relevant in human PCa patients.