bluuebunny/crossref_metadata_2025 huggingface dataset:
Draft Genome Sequences of 11 Xanthomonas Strains associated with bacterial spot disease in Turkey
Authors: Subedi Aastha , Kara Serhat , Aysan Yesim , Minsavage Gerald V. , Timilsina Sujan , Roberts Pamela D. , Goss Erica M. , Jones Jeffrey B.
Published: April 2023

Bacterial spot is an economically significant disease in tomato and pepper-producing countries globally. We report the whole genome sequence of 11 Xanthomonas strains associated with bacterial spot disease on pepper, tomato, and eggplant in the Southeastern Anatolia Region, Turkey.  This genomic information can be used as a reference to study the genetic diversity of these species and contribute to illuminating pathogen evolution with respect to host specificity.
Novosphingobium album sp. nov., Novosphingobium organovorum sp. nov. and Novosphingobium mangrovi sp. nov. with the organophosphorus pesticides degrading ability isolated from mangrove sediments
Authors: Hu Wenjin , Li Zhe , Ou Haisheng , Wang Xiaochun , Wang Qiaozhen , Tao Zhanhua , Huang Shushi , Huang Yuanlin , Wang Guiwen , Pan Xinli
Published: April 2023

Members of the genus

Novosphingobium

were frequently isolated from polluted environments and possess great bioremediation potential. Here, three species, designated B2637T, B2580T and B1949T, were isolated from mangrove sediments and might represent novel species in the genus

Novosphingobium

based on a polyphasic taxonomy study. Phylogenomic analysis revealed that strains B2580T, B1949T and B2637T clustered with

Novosphingobium naphthalenivorans

NBRC 102051T, ‘

N. profundi

’ F72 and

N. decolorationis

502str22T, respectively. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between isolates and their closely related species were less than 94 and 54 %, respectively, all below the threshold of species discrimination. The sizes of the genomes of isolates B2580T, B2637T and B1949T ranged from 4.4 to 4.6 Mb, containing 63.3–66.4 % G+C content. Analysis of their genomic sequences identified genes related to pesticide degradation, heavy-metal resistance, nitrogen fixation, antibiotic resistance and sulphur metabolism, revealing the biotechnology potential of these isolates. Except for B2637T, B1949T and B2580T were able to grow in the presence of quinalphos. Results from these polyphasic taxonomic analyses support the affiliation of these strains to three novel species within the genus

Novosphingobium

, for which we propose the name Novosphingobium album sp. nov. B2580T (=KCTC 72967T=MCCC 1K04555T), Novosphingobium organovorum sp. nov. B1949T (=KCTC 92158T=MCCC 1K03763T) and Novosphingobium mangrovi sp. nov. B2637T (KCTC 72969T=MCCC 1K04460T).
On the dynamic contact angle of capillary-driven microflows in open channels
Authors: Tokihiro Jodie C. , McManamen Anika M. , Phan David N. , Thongpang Sanitta , Blake Terence D. , Theberge Ashleigh B. , Berthier Jean
Published: April 2023

The true value of the contact angle between a liquid and a solid is a thorny problem in capillary microfluidics. The Lucas-Washburn-Rideal (LWR) law assumes a constant contact angle during fluid penetration. However, recent experimental studies have shown lower liquid velocities than predicted by the LWR equation, which are attributed to a velocity-dependent dynamic contact angle that is larger than its static value. Inspection of fluid penetration in closed channels has confirmed that a dynamic angle is needed in the LWR equation.

In this work, the dynamic contact angle in an open channel configuration is investigated using experimental data obtained with a range of liquids, aqueous and organic, and a PMMA substrate. We demonstrate that a dynamic contact angle must be used to explain the early stages of fluid penetration, i.e., at the start of the viscous regime, when flow velocities are sufficiently high. Moreover, the open channel configuration, with its free surface, enhances the effect of the dynamic contact angle, making its inclusion even more important. We found that for the liquids in our study, the molecular-kinetic theory (MKT) is the most accurate in predicting the effect of the dynamic contact angle on liquid penetration in open channels.
Leakage beyond the primary infarction: A temporal analysis of cerebrovascular dysregulation at sites of hippocampal secondary neurodegeneration following cortical photothrombotic stroke
Authors: Hood Rebecca J. , Sanchez-Bezanilla Sonia , Beard Daniel J. , Rust Ruslan , Turner Renée J. , Stuckey Shannon M. , Collins-Praino Lyndsey E. , Walker Frederick R. , Nilsson Michael , Ong Lin Kooi
Published: April 2023

We have previously demonstrated that a cortical stroke causes persistent impairment of hippocampal-dependent cognitive tasks concomitant with secondary neurodegenerative processes such as amyloid-β accumulation in the hippocampus, a region remote from the primary infarct. Interestingly, there is emerging evidence suggesting that deposition of amyloid-β around cerebral vessels may lead to cerebrovascular structural changes, neurovascular dysfunction, and disruption of blood-brain barrier integrity. However, there is limited knowledge about the temporal changes of hippocampal cerebrovasculature after cortical stroke. In the current study, we aimed to characterise the spatiotemporal cerebrovascular changes after cortical stroke. This was done using the photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Cerebrovascular morphology as well as the colocalization of amyloid-β with vasculature and blood-brain-barrier integrity were assessed in the cortex and hippocampal regions at 7, 28 and 84 days post-stroke. Our findings showed transient cerebrovascular remodelling in the peri-infarct area up to 28 days post-stroke. Importantly, the cerebrovascular changes were extended beyond the peri-infarct region to the ipsilateral hippocampus and were sustained out to 84 days post-stroke. When investigating vessel diameter, we showed a decrease at 84 days in the peri-infarct and CA1 regions that was exacerbated in vessels with amyloid-β deposition. Lastly, we showed sustained vascular leakage in the peri-infarct and ipsilateral hippocampus, indicative of a compromised blood-brain-barrier. Our findings indicate that hippocampal vasculature may represent an important therapeutic target to mitigate the progression of post-stroke cognitive impairment.
Riboformer: A Deep Learning Framework for Predicting Context-Dependent Translation Dynamics
Authors: Shao Bin , Yan Jiawei , Zhang Jing , Buskirk Allen R.
Published: April 2023

Translation elongation is essential for maintaining cellular proteostasis, and alterations in the translational landscape are associated with a range of diseases. Ribosome profiling allows detailed measurement of translation at genome scale. However, it remains unclear how to disentangle biological variations from technical artifacts and identify sequence determinant of translation dysregulation. Here we present Riboformer, a deep learning-based framework for modeling context-dependent changes in translation dynamics. Riboformer leverages the transformer architecture to accurately predict ribosome densities at codon resolution. It corrects experimental artifacts in previously unseen datasets, reveals subtle differences in synonymous codon translation and uncovers a bottleneck in protein synthesis. Further, we show that Riboformer can be combined within silicomutagenesis analysis to identify sequence motifs that contribute to ribosome stalling across various biological contexts, including aging and viral infection. Our tool offers a context-aware and interpretable approach for standardizing ribosome profiling datasets and elucidating the regulatory basis of translation kinetics.
RaptorX-Single: single-sequence protein structure prediction by integrating protein language models
Authors: Jing Xiaoyang , Wu Fandi , Luo Xiao , Xu Jinbo
Published: April 2023

Protein structure prediction has been greatly improved by deep learning in the past few years. However, the most successful methods rely on multiple sequence alignment (MSA) of the sequence homologs of the protein under prediction. In nature a protein folds in the absence of its sequence homologs and thus, a MSA-free structure prediction method is desired. Here we develop a single sequence-based protein structure prediction method RaptorX-Single by integrating several protein language models and a structure generation module and then study its advantage over MSA-based prediction methods. Our experimental results indicate that in addition to running much faster than MSA-based methods such as AlphaFold2, RaptorX-Single outperforms AlphaFold2 and other MSA-free methods in predicting the structure of antibodies, proteins of very few sequence homologs and single mutation effects. RaptorX-Single also compares favorably to MSA-based AlphaFold2 when the protein under prediction has a large number of sequence homologs.
Cell based dATP delivery as a therapy for chronic heart failure
Authors: Mhatre Ketaki N , Mathieu Julie , Martinson Amy , Flint Galina , Blakley Leslie P. , Tabesh Arash , Reinecke Hans , Yang Xiulan , Guan Xuan , Murali Eesha , Klaiman Jordan M , Odom Guy L , Brown Mary Beth , Tian Rong , Hauschka Stephen D , Raftery Daniel , Moussavi-Harami Farid , Regnier Michael , Murry Charles E
Published: April 2023

Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2’-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host contractility offers a novel approach to treating the chronically failing heart.

One Sentence Summary

Transplanting gene-edited dATP-donor cardiomyocytes in chronically infarcted heart restores their cardiac function, improving both exercise tolerance and survival.
Assessment of Total Oocyte Transcripts Representation through Single Ooplasm Biopsy in Bovine with High Reliability
Authors: Ambrizi Dewison Ricardo , Nociti Ricardo Perecin , De Bem Tiago Henrique Camara , Paixao Joao Vitor Puttini , Therrien Jacinthe , De Matos Elisangela Chicaroni , Ferraz Jose Bento Sterman , Chiaratti Marcos Roberto , Sangalli Juliano , Da Silveira Juliano Coelho , Perecin Felipe , Smith Lawrence Charles , Meirelles Flavio Vieira
Published: April 2023

Understanding the entire transcriptional and epigenetic landscape is facilitated by the application of omics in a number of ways. Today, omic instruments are more affordable and easier to implement. In human research, for instance, single-omics are a reality and are used extensively to generate vast quantities of data. This method permits the comprehensive reconstruction of transcriptome and epigenetic markers removing bias from pooled samples. In tandem with the evolution of machines and protocols, algorithms and genome annotation have undergone continuous improvement. The genome annotation of domestic animals is inferior to that of humans, rodents, and less complex organisms. In the case of heifers, the reference is incomplete, with significant gaps and only a portion of the noncoding transcripts. The purpose of this study is to validate our compartmentalized single oocyte biopsy by comparing a small fraction of bovine oocytes, 1%, to the entire oocyte at the Metaphase II stage. In addition, we examined the use of four database sources (NCBI, ENSEMBL, UCSC, and NONCODE) to produce a merged non-redundant gene alignment and counting in order to enhance gene detection and normalization, resulting in a more accurate method to comprehend the entire landscape. This study is a continuation of our research titled “Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine” in which this method was used to retrospectively compare biopsy oocytes collected during the MII phase. With the addition of NONCODE information, gene normalization was significantly enhanced. In addition, our analysis identified 4560 noncoding genes from NONCODE references. ENSEMBL and NCBI have nearly the same number of annotated genes (16,423 vs. 17,804), but using ENSEMBL as a reference, 2356 genes were able to be normalized and identified. Proceeding to biopsy x oocyte analysis, we were able to detect a greater number of genes in oocytes than in biopsy, where the preponderance was from NONCODE sources (68). Despite these minor differences, the high correlation of expression between them (89%) was consistent and proved to be a valuable instrument for studying the oocyte without destroying it.
High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing
Authors: Li Weizhen , McLeod David , Ketzenberger John T. , Kowalik Grant , Russo Rebekah , Li Zhenyu , Kay Matthew W. , Entcheva Emilia
Published: April 2023

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a scalable experimental model relevant to human physiology. Oxygen consumption of hiPSC-CMs has not been studied in high-throughput (HT) format plates used in pre-clinical studies. Here, we provide comprehensive characterization and validation of a system for HT long-term optical measurements of peri-cellular oxygen in cardiac syncytia (human iPSC-CM and human cardiac fibroblasts), grown in glass-bottom 96-well plates. Laser-cut oxygen sensors having a ruthenium dye and an oxygen-insensitive reference dye were used. Ratiometric measurements (409nm excitation) reflected dynamic changes in oxygen, as validated with simultaneous Clark electrode measurements. Emission ratios (653nm vs. 510nm) were calibrated for percent oxygen using two-point calibration. Time-dependent changes in the Stern-Volmer parameter, Ksv, were observed during the initial 40 min of incubation, likely temperature-related. Effects of pH on oxygen measurements were negligible in the pH range of 4 to 8, with a small ratio reduction for pH>10. Time-dependent calibration was implemented, and light exposure time was optimized (0.6 to 0.8s) for oxygen measurements inside an incubator. Peri-cellular oxygen dropped to levels < 5% within 3 -10 hours for densely-plated hiPSC-CMs in glass-bottom 96-well plates. After the initial oxygen decrease, samples either settled to low steady-state or exhibited intermittent peri-cellular oxygen dynamics. Cardiac fibroblasts showed slower oxygen depletion and higher steady-state levels without oscillations, compared to hiPSC-CMs. Overall, the system has great utility for long-term HT monitoring of peri-cellular oxygen dynamics in vitro for tracking cellular oxygen consumption, metabolic perturbations, and characterization of the maturation of hiPSC-CMs.
Clade-Specific MPXV PCR Assays
Authors: Negrón Daniel Antonio , Tolli Nicholas , Guertin Stephanie , Wollen-Roberts Suzanne , Mitchell Shane , Haas Jared , Pratt Catherine , Jennings Katharine , Abramson Bradley , Brinkac Lauren , Ashford David
Published: April 2023

In an evolving infectious disease outbreak, there are two priorities: rapid and accurate detection of the causative agent and characterization of its spread. The polymerase chain reaction (PCR) assay is an effective and portable diagnostic method that can quickly provide information associated with virulence, transmissibility, and pathogenicity. Compared to genomic sequencing, PCR requires less infrastructure, funding, and training. However, the development of sensitive and specific primer sequences is costly, particularly those with subspecies resolution. The recentmpox (monkeypox) virusoutbreak underscores the need for the rapid development of clade-specific primers, particularly when there are differences in morbidity and mortality rates. Current mpox assays use primer sequences that also bind to the broaderOrthopoxvirusgenus, resulting in suspect diagnoses, delays in treatment, and poor allocation of scarce healthcare resources. Additionally, these orthopox-based primer sets cannot distinguish between different mpox clades and cannot illuminate intra-clade evolution over the course of the outbreak. Here, we present thein silicodesign andin vitrotesting of novel clade-specific mpox assays.

Importance

There is an ongoing global outbreak of Mpox disease, an illness with a characteristic blistering rash progression. The Mpox virus clusters into two clades with differing levels of virulence and mortality rates. Thus, proper sample identification is critical for surveillance and the public health response programs that rely on such data. Accordingly, the US government specifically lists Clade I under the Federal Select Agent Program. Current diagnostics may fail to identify the virus or its clade membership as the genome mutates. In this work, we demonstrate an end-to-end workflow to quickly evaluate existing PCR assays and design new ones for clade-based identification with respect to large sequence databases in service of preventing a catastrophic fog-of-war from forming during an outbreak.
A neural model of modified excitation/inhibition and feedback levels in schizophrenia
Authors: Zhu Jiating , Zikopoulos Basilis , Yazdanbakhsh Arash
Published: April 2023

The strength of certain visual illusions is weakened in individuals with schizophrenia. Such phenomena have been interpreted as the impaired integration of inhibitory and excitatory neural responses, and impaired top–down feedback mechanisms. To investigate whether and how these factors influence the perceived illusions in individuals with schizophrenia, we propose a two-layer network that can model visual receptive fields (RFs), their inhibitory and excitatory subfields, and the top-down feedback. Our neural model suggests that illusion perception changes in individuals with schizophrenia can be influenced by altered top-down mechanisms and the organization of the on-center off-surround receptive fields. Alteration of the RF inhibitory surround and/or the excitatory center can replicate the difference of illusion precepts between individuals with schizophrenia and normal controls. The results show that the simulated top-down feedback modulation enlarges the difference of the model illusion representations, replicating the difference between the two groups.
Precision pharmacological reversal of genotype-specific diet-induced metabolic syndrome in mice informed by transcriptional regulation
Authors: Wulfridge Phillip , Davidovich Adam , Salvador Anna C. , Manno Gabrielle C. , Tryggvadottir Rakel , Idrizi Adrian , Huda M. Nazmul , Bennett Brian J. , Adams L. Garry , Hansen Kasper D. , Threadgill David W. , Feinberg Andrew P.
Published: April 2023

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genomic DNA methylation analyses and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects reveals a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the Farnesoid X receptor pathway, and found that GW4064 exerts genotype-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis, as well as increased inflammatory-related gene expression changes in NOD. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention, and a mouse platform for guiding this approach.
Moonlight synchronous flights across three western palearctic swifts mirror size dependent prey preferences
Authors: Hufkens Koen , Meier Christoph M. , Evens Ruben , Paredes Josefa Arán , Karaardiç Hakan , Vercauteren Stef , Gysel Ann Van , Fox James W. , Pacheco Carlos Miguel , da Silva Luis P. , Fernandes Sandra , Henriques Pedro , Elias Gonçalo , Costa Luís T. , Poot Martin , Kearsley Lyndon
Published: April 2023

Recent studies have suggested the presence of moonlight mediated behaviour in avian aerial insectivores, such as swifts. At the same time swift species also show differences in prey (size) preferences. Here, we use the combined analysis of state-of-the-art activity logger data across three swift species, the Common, Pallid and Alpine swifts, to quantify flight height and activity responses to crepuscular and nocturnal light conditions. Our results show a significant response in flight heights to moonlight illuminance for Common and Pallid swifts, while a moonlight driven response is absent in Alpine swifts. Swift flight responses followed the size dependent altitude gradient of their insect prey. We show a weak relationship between night-time illuminance driven responses and twilight ascending behaviour, suggesting a decoupling of both crepuscular and night-time behaviour. We suggest that swifts optimise their flight behaviour to adapt to favourable night-time light conditions, driven by light responsive and size-dependent vertical insect stratification and weather conditions.
Slow gamma oscillations in the mouse olfactory bulb are correlated with sniffing in the dark period
Authors: Mochizuki-Koike Ryosuke , Okada Mami , Ikegaya Yuji , Matsumoto Nobuyoshi
Published: April 2023

Neural activity in the olfactory bulb is reflected in local field potentials (LFPs). Functionally, LFPs in the olfactory bulb are categorized into different frequency bands: 1-4 Hz, 6-12 Hz, 25-50 Hz, and 65-130 Hz, which respectively correspond to respiration, sniffing, slow gamma, and fast gamma oscillations. While gamma oscillations in the olfactory bulb are modulated by respiration and sniffing, it remains unknown how and whether the modulation of LFP oscillations is affected by the time of day. To address this question, we recorded LFPs in the olfactory bulb, hippocampus, and neocortex of unrestrained mice for up to 3 d. For each recording site, we calculated the correlation coefficients of normalized LFP powers between pairs of frequency bands in the three regions during the dark and light periods. We then compared these correlations with those generated by surrogate data to investigate whether the correlation was statistically significant. We found that the correlation between sniffing and slow gamma oscillations was higher in the dark period than in the light period. Our finding has the potential to shed light on the coding scheme of olfactory information that is dependent on the light/dark cycle.
Dorsal peduncular cortex activity modulates affective behavior and fear extinction in mice
Authors: Botterill Justin J. , Khlaifia Abdessattar , Appings Ryan , Wilkin Jennifer , Violi Francesca , Premachandran Hanista , Cruz-Sanchez Arely , Canella Anna Elisabete , Patel Ashutosh , Zaidi S. Danyal , Arruda-Carvalho Maithe
Published: April 2023

The medial prefrontal cortex (mPFC) is critical to cognitive and emotional function and underlies many neuropsychiatric disorders, including mood, fear and anxiety disorders. In rodents, disruption of mPFC activity affects anxiety- and depression-like behavior, with specialized contributions from its subdivisions. The rodent mPFC is divided into the dorsomedial prefrontal cortex (dmPFC), spanning the anterior cingulate cortex (ACC) and dorsal prelimbic cortex (PL), and the ventromedial prefrontal cortex (vmPFC), which includes the ventral PL, infralimbic cortex (IL), and in some studies the dorsal peduncular cortex (DP) and dorsal tenia tecta (DTT). The DP/DTT have recently been implicated in the regulation of stress- induced sympathetic responses via projections to the hypothalamus. While many studies implicate the PL and IL in anxiety-, depression-like and fear behavior, the contribution of the DP/DTT to affective and emotional behavior remains unknown. Here, we used chemogenetics and optogenetics to bidirectionally modulate DP/DTT activity and examine its effects on affective behaviors, fear and stress responses in C57BL/6J mice. Acute chemogenetic activation of DP/DTT significantly increased anxiety-like behavior in the open field and elevated plus maze tests, as well as passive coping in the tail suspension test. DP/DTT activation also led to an increase in serum corticosterone levels and facilitated auditory fear extinction learning and retrieval. Activation of DP/DTT projections to the dorsomedial hypothalamus (DMH) acutely decreased freezing at baseline and during extinction learning, but did not alter affective behavior. These findings point to the DP/DTT as a new regulator of affective behavior and fear extinction in mice.
Photosynthesis in the biomass model speciesLemna minordisplays plant-conserved and species-specific features
Authors: Liebers Monique , Hommel Elisabeth , Grübler Björn , Danehl Jakob , Offermann Sascha , Pfannschmidt Thomas
Published: April 2023

Lemnaceaeare aquatic freshwater plants with extraordinary high growth rates. We have studied selected physiological and molecular photosynthesis properties of the duckweedLemna minorand compared these to the terrestrial model speciesArabidopsis thaliana. LemnaandArabidopsisplants grown under identical light intensities displayed similar photosynthesis characteristics, however,Lemnaexhibited slighty better quenching efficiencies pointing to improved light utilization in the duckweed. Western-immuno-blot analyses of representative photosynthesis proteins suggest various post-translational modifications inLemnathat might be associated to this. Phospho-threonine phosphorylation patterns of thylakoid membranes uncovered differences between the two species. Testing the photosystem II antenna association ofLemna minorin dark and light by 77K chlorophyll fluorescence emission experiments, however, revealed a typical association as reported in terrestrial plants. High light stress experiments causing photoinhibition and subsequent recovery from it were not substantially different inLemnawhen compared toArabidopsis. We hypothesize that the molecular differences inLemnaphotosynthesis proteins are associated with evolutionary adaptations to the aquatic life style and ultimately with the high growth rates. We also developed a video imaging approach forLemnamultiplication at high magnification that will be useful to assess the impact of external factors onLemnaphotosynthesis and growth.
TheDrosophila drop-deadgene is required for eggshell integrity
Authors: Sheahan Tayler D. , Grewal Amanpreet , Korthauer Laura E. , Blumenthal Edward M.
Published: April 2023

The eggshell of the fruit flyDrosophila melanogasteris a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized during mid-to-late oogenesis by the somatic follicle cells that surround the developing oocyte. We previously reported that female flies mutant for the genedrop-dead(drd) are sterile, but the underlying cause of the sterility remained unknown. In this study, we examined the role ofdrdin eggshell synthesis. We show that eggs laid bydrdmutant females are fertilized but arrest early in embryogenesis, and that the innermost layer of the eggshell, the vitelline membrane, is abnormally permeable to dye in these eggs. In addition, the major vitelline membrane proteins fail to become crosslinked by nonreducible bonds, a process that normally occurs during egg activation following ovulation, as evidenced by their solubility and detection by Western blot in laid eggs. In contrast, the Cp36 protein, which is found in the outer chorion layers of the eggshell, becomes crosslinked normally. To link thedrdexpression pattern with these phenotypes, we show thatdrdis expressed in the ovarian follicle cells beginning in mid-oogenesis, and, importantly, that alldrdmutant eggshell phenotypes could be recapitulated by selective knockdown ofdrdexpression in the follicle cells. To determine whetherdrdexpression was required for the crosslinking itself, we performedin vitroactivation and crosslinking experiments. The vitelline membranes of control egg chambers could become crosslinked either by incubation in hyperosmotic medium, which activates the egg chambers, or by exogenous peroxidase and hydrogen peroxide. In contrast, neither treatment resulted in the crosslinking of the vitelline membrane indrdmutant egg chambers. These results indicate thatdrdexpression in the follicle cells is necessary for vitelline membrane proteins to serve as substrates for peroxidase-mediated cross-linking at the end of oogenesis.
Gut microbiota and metabolites drive chronic sickle cell disease pain
Authors: Sadler Katelyn E. , Atkinson Samantha N. , Ehlers Vanessa L. , Waltz Tyler B. , Hayward Michael , Rodríguez García Dianise M. , Salzman Nita H. , Stucky Cheryl L. , Brandow Amanda M.
Published: April 2023

Pain is a debilitating symptom and leading reason for hospitalization of individuals with sickle cell disease. Chronic sickle cell pain is poorly managed because the biological basis is not fully understood. Using transgenic sickle cell mice and fecal material transplant, we determined that the gut microbiome drives persistent sickle cell pain. In parallel patient and mouse analyses, we identified bilirubin as one metabolite that induces sickle cell pain by altering vagus nerve activity. Furthermore, we determined that decreased abundance of the gut bacteriaAkkermansia mucinophilais a critical driver of chronic sickle cell pain. These experiments demonstrate that the sickle cell gut microbiome drives chronic widespread pain and identify bacterial species and metabolites that should be targeted for chronic sickle cell disease pain management.

One-Sentence Summary

Gut microbes and metabolites drive chronic sickle cell disease pain by altering vagus nerve activity.
Imetelstat-Mediated Alterations in Fatty Acid Metabolism To Induce Ferroptosis As Therapeutic Strategy for Acute Myeloid Leukemia
Authors: Bruedigam Claudia , Porter Amy H. , Song Axia , Vroeg in de Wei Gerjanne , Stoll Thomas , Straube Jasmin , Cooper Leanne , Cheng Guidan , Kahl Vivian F. S. , Sobinoff Alexander P. , Ling Victoria Y. , Jebaraj Billy Michael Chelliah , Janardhanan Yashaswini , Haldar Rohit , Bray Laura J. , Bullinger Lars , Heidel Florian H. , Kennedy Glen A. , Hill Michelle M. , Pickett Hilda A. , Abdel-Wahab Omar , Hartel Gunter , Lane Steven W.
Published: April 2023

Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft (PDX) resource, and perform integrated genomics, transcriptomics, and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized Phase II-like preclinical trial in PDX, imetelstat effectively diminishes AML burden, and preferentially targets subgroups containing mutantNRASand oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing significant disease control in AML.
Epigenetic control of myogenic identity of human muscle stem cells in Duchenne Muscular Dystrophy
Authors: Massenet Jimmy , Weiss-Gayet Michèle , Bandukwala Hina , Magnan Mélanie , Hubas Arnaud , Nusbaum Patrick , Desguerre Isabelle , Gitiaux Cyril , Dilworth F Jeffrey , Chazaud Bénédicte
Published: April 2023

In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic defects that limit regenerative potential or are disrupted by their degenerative/regenerative environment is unclear. We investigated cell behavior and gene expression in human using MuSCs derived from DMD or healthy muscles. We found that proliferation, differentiation and fusion were not altered in DMD-MuSCs, but with time, they lost their myogenic identity twice as fast as healthy MuSCs. The rapid drift towards a fibroblast-like cell identity was observed at the clonal level, and resulted from the altered expression of epigenetic enzymes required to maintain the myogenic cell fate. Indeed, the re-expression ofCBX3,SMC3,H2AFVandH3F3Bprevented the MuSC identity drift. Amongst the epigenetic changes, a closing of chromatin at the gene encoding the transcription factorMEF2Bcaused a down-regulation of its expression and a loss of the myogenic fate. Thus, MEF2B is a key mediator of the myogenic identity in human MuSCs, that is altered in DMD pathology.
Social status in zebrafish modulates the behavioral response to 5-HT2C receptor agonists and antagonists
Authors: de Oliveira Larissa Nunes , Alves Nuno Felix Paiva , Soares Marta Candeias , Maximino Caio
Published: April 2023

The effects of previous social experiences on social behavior have been demonstrated across species both in cooperative and competitive contexts. In dominance-subordinate hierarchies, differences across social ranks have been observed in many different mechanisms. Dominance hierarchies interfere in defensive behavior, where subordinate animals present a greater defensive behavior, regarding potential threats (“anxiety-like behavior”), than dominant animals. The serotonergic system plays a key role in regulating and mediating threat responses, including 5-HT2 receptors in the types of proximal threat responses modulated by the stress of social defeat. We separated 148 adult zebrafish in pairs, and allowed to interact for five days; after that, the dominant-subordinate rank was determined, and animals were treated with a 5-HT2Creceptor agonist (MK-212) or antagonist (RS-102221) before being observed in the novel tank test. While MK-212 increased bottom-dwelling, erratic swimming, and freezing across all statuses, RS-102221 decreased these variables in dominants but increased them in subordinates. Moreover, the effects of MK-212 were larger in subordinates than in controls or dominants, suggesting a sensitization of the 5-HT2Creceptor.
HIV-1 infection of genetically engineered iPSC-derived central nervous system-engrafted microglia in a humanized mouse model
Authors: Min Alice K. , Javidfar Behnam , Missall Roy , Doanman Donald , Durens Madel , Vil Samantha St , Masih Zahra , Graziani Mara , Mordelt Annika , Marro Samuele , de Witte Lotje , Chen Benjamin K. , Swartz Talia H. , Akbarian Schahram
Published: April 2023

The central nervous system (CNS) is a major human immunodeficiency virus type 1 reservoir. Microglia are the primary target cell of HIV-1 infection in the CNS. Current models have not allowed the precise molecular pathways of acute and chronic CNS microglial infection to be tested with in vivo genetic methods. Here, we describe a novel humanized mouse model utilizing human-induced pluripotent stem cell-derived microglia to xenograft into murine hosts. These mice are additionally engrafted with human peripheral blood mononuclear cells that served as a medium to establish a peripheral infection that then spread to the CNS microglia xenograft, modeling a trans-blood-brain barrier route of acute CNS HIV-1 infection with human target cells. The approach is compatible with iPSC genetic engineering, including inserting targeted transgenic reporter cassettes to track the xenografted human cells, enabling the testing of novel treatment and viral tracking strategies in a comparatively simple and cost-effective wayvivomodel for neuroHIV.

Importance

Our mouse model is a powerful tool for investigating the genetic mechanisms governing CNS HIV-1 infection and latency in the CNS at a single-cell level. A major advantage of our model is that it uses iPSC-derived microglia, which enables human genetics, including gene function and therapeutic gene manipulation, to be exploredin vivo, which is more challenging to study with current hematopoietic stem cell-based models for neuroHIV. Our transgenic tracing of xenografted human cells will provide a quantitative medium to develop new molecular and epigenetic strategies for reducing the HIV-1 latent reservoir and to test the impact of therapeutic inflammation-targeting drug interventions on CNS HIV-1 latency.
Enhancedin VivoBlood Brain Barrier Transcytosis of Macromolecular Cargo Using an Engineered pH-sensitive Mouse Transferrin Receptor Binding Nanobody
Authors: Esparza Thomas J. , Su Shiran , Francescutti Caroline M. , Rodionova Elvira , Kim Joong Hee , Brody David L.
Published: April 2023

Background

The blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency.

Methods

A mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin forin vivofunctional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102Hand two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transportin vivousing quantitatively verified capillary depleted brain lysates andin situhistology.

Results

The most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused >8°C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M156,96,102His-13A7-13A7 in capillary depleted brain lysates peaked at 1 hour and were 60% retained at 8 hours. A control construct with no brain targets was only 15% retained at 8 hours. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 minutes to 2.6 hours. At 30-60 minutes, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries usingin situhistochemistry, whereas at 2-16 hours it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect.

Conclusion

The pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102Hmay be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.
WNK1 enforces macrophage lineage fidelity
Authors: Trzeciak Alissa J. , Rojas Waleska Saitz , Liu Zong-Lin , Krebs Adam S. , Wang Zhaoquan , Saavedra Pedro H. V. , Miranda Isabella C. , Lipshutz Allie , Xie Jian , Huang Chou-Long , Overholtzer Michael , Glickman Michael S. , Parkhurst Christopher N. , Vierbuchen Thomas , Lucas Christopher D. , Perry Justin S. A.
Published: April 2023

The appropriate development of macrophages, the body’s professional phagocyte, is essential for organismal development, especially in mammals. This dependence is exemplified by the observation that loss-of-function mutations in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities owing to an absence of macrophages. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for development of tissue-resident macrophages (TRMs). Myeloid-specific deletion ofWnk1resulted in a dramatic loss of TRMs, disrupted organ development, systemic neutrophilia, and mortality between 3 and 4 weeks of age. Strikingly, we found that myeloid progenitors or precursors lacking WNK1 not only failed to differentiate into macrophages, but instead differentiated into neutrophils. Mechanistically, the cognate CSF1R cytokine macrophage-colony stimulating factor (M-CSF) stimulates macropinocytosis by both mouse and human myeloid progenitors and precursor cells. Macropinocytosis, in turn, induces chloride flux and WNK1 phosphorylation. Importantly, blocking macropinocytosis, perturbing chloride flux during macropinocytosis, and inhibiting WNK1 chloride-sensing activity each skewed myeloid progenitor differentiation from macrophages into neutrophils. Thus, we have elucidated a role for WNK1 during macropinocytosis and discovered a novel function of macropinocytosis in myeloid progenitors and precursor cells to ensure macrophage lineage fidelity.

Highlights

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Myeloid-specific WNK1 loss causes failed macrophage development and premature death

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M-CSF-stimulated myeloid progenitors and precursors become neutrophils instead of macrophages

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M-CSF induces macropinocytosis by myeloid progenitors, which depends on WNK1

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Macropinocytosis enforces macrophage lineage commitment
Designer DNA NanoGripper
Authors: Zhou Lifeng , Xiong Yanyu , Cooper Laura , Shepherd Skye , Song Tingjie , Dwivedy Abhisek , Rong Lijun , Wang Tong , Cunningham Brian T. , Wang Xing
Published: April 2023

DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the “NanoGripper”, that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of ∼ 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza.

One-sentence summary

Design, synthesis, characterization, and functional showcase of a human-hand like designer DNA nanobot
Discovery of Type IV filament membrane alignment complex homologs inH. pylorithat promote soft-agar migration
Authors: Sagoo Jashwin , Abedrabbo Samar , Liu Xiaolin , Ottemann Karen M.
Published: April 2023

The stomach pathogenHelicobacter pyloriutilizes two scaffold proteins, CheW and CheV1, to build critical chemotaxis arrays. Chemotaxis helps bacteria establish and maintain infection. Mutants lacking either of these chemotaxis proteins have different soft agar phenotypes: deletion ofcheWcreates non-chemotactic strains, while deletion ofcheV1results in 50% loss of chemotaxis. In this work, we characterized thecheV1deletion mutant phenotype in detail.cheV1deletion mutants had poor soft-agar migration initially, but regained migration ability over time. This improved bacterial migration was stable, suggesting a genetic suppressor phenotype, termed Che+. Whole-genome sequencing analysis of four distinctcheV1Che+ strains revealed single nucleotide polymorphisms (SNPs) in a common gene, HPG27_252 (HP0273). These SNPs were predicted to truncate the encoded protein. To confirm the role of HPG27_252 in thecheV1phenotype, we created a targeted deletion of HPG27_252 and found that loss of HPG27_252 enhanced soft-agar migration. HPG27_252 and CheV1 appear to interact directly, based on bacterial two-hybrid analysis. HPG27_252 is predicted to encode a 179 amino acid, 21 kDa protein annotated as a hypothetical protein. Computational analysis revealed this protein to be a remote homolog of the PilO Type IV filament membrane alignment complex protein. AlthoughH. pyloriis not known to possess Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM, but does not possess other Type IV pili genes. Our data suggest the PilO homolog plays a role in regulatingH. pylorichemotaxis and motility, suggesting new ideas about evolutionary steps for controlling migration through semi-solid media.