bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential regulation of eye specification in Drosophila by Polycomb Group (PcG) epigenetic repressors
Authors: Brown Haley E. , Weasner Brandon P. , Kumar Justin P.
Published: August 2024

During metazoan development, chromatin plasticity and genetic regulation are tightly connected to establish proper tissue fate and patterning. Drosophila imaginal discs are excellent models to study these processes as both genetic and mechanical injury can redirect their fate during regeneration. Reducing expression of Polycomb (Pc) results in the ectopic activation of the wing selector gene vestigial (vg) which in turn interacts with its DNA-binding partner Scalloped (Sd), this forces the eye to transform into a wing. Reductions of other PcG members alone does not phenocopy this transformation. However, knocking down Sex combs on midleg (Scm) or Scm related gene containing four mbt domains (Sfmbt) alongside the Pax6 gene twin of eyeless (toy) enables the eye-to-wing transformation. Using high throughput sequencing we show that toy-Sfmbt and toy-Scm knockdowns alter expression of wing selector and Hox genes, respectively. These findings provide new insights into how the fate of the eye is specified.
Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function
Authors: Hampton Thomas H. , Barnaby Roxanna , Roche Carolyn , Nymon Amanda , Fukutani Kiyoshi Ferreira , MacKenzie Todd A. , Stanton Bruce A.
Published: August 2024

The combination of elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) reverses the primary defect in Cystic Fibrosis (CF) by improving CFTR mediated Cl-and HCO3-secretion by airway epithelial cells (AEC), leading to improved lung function and less frequent exacerbations and hospitalizations. However, studies have shown that CFTR modulators like ivacaftor, a component of ETI, has numerous effects on CF cells beyond improved CFTR channel function. Because little is known about the effect of ETI on CF AEC gene expression we exposed primary human AEC to ETI for 48 hours and interrogated the transcriptome by RNA-seq and qPCR. ETI increased defensin gene expression (DEFB1) an observation consistent with reports of decreased bacterial burden in the lungs of people with CF (pwCF). ETI also decreasedMMP10andMMP12gene expression, suggesting that ETI may reduce proteolytic induced lung destruction in CF. ETI also reduced the expression of the stress response gene heme oxygenase (HMOX1). qPCR analysis confirmedDEFB1, HMOX1, MMP10andMMP12gene expression results observed by RNA-seq. Gene pathway analysis revealed that ETI decreased inflammatory signaling, cellular proliferation and MHC Class II antigen presentation. Collectively, these findings suggest that the clinical observation that ETI reduces lung infections in pwCF is related in part to drug induced increases inDEFB1, and that ETI may reduce lung damage by reducingMMP10andMMP12gene expression, which is predicted to reduce matrix metalloprotease activity. Moreover, pathway analysis also identified several genes responsible for the ETI induced reduction in inflammation observed in people with CF.

New and Noteworthy

Gene expression responses by CF AEC exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to increase resistance to bacterial infection by increasing levels of beta defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10, and reduce airway inflammation by repressing proinflammatory cytokine secretion by AEC cells.
Structural basis for carbohydrate recognition by the Gal/GalNAc lectin ofEntamoeba histolytica
Authors: Gérard Samuel F. , Redfield Christina , Higgins Matthew K.
Published: August 2024

Intestinal amoebiasis is caused byEntamoeba histolytica,one of the deadliest human-infective parasites. Central to its pathogenicity is its binding to mucosal carbohydrates, which precedes tissue damage by trogocytosis. Carbohydrate binding is mediated by a single adhesin, the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin, which is the leading vaccine candidate for amoebiasis. We present the structure of the native heterodimeric lectin, revealing an ordered core containing the light chain and the N-terminal region of the heavy chain. Structures in the presence of ligand show that the Gal/GalNAc binding site is in the light chain. An elongated arm emerges from the heavy chain, which adopts multiple positions. This arm contains the epitope for a monoclonal antibody which blocks binding to target cells, located unexpectedly away from the Gal/GalNAc binding site. This study reveals the molecular basis for sugar binding by theEntamoeba histolyticaGal/GalNAc lectin and will guide future vaccine development against amoebiasis.
Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic
Authors: Ding Elizabeth , Pinho-Schwermann Maximillian , Zhang Shengliang , Purcell Connor , El-Deiry Wafik S.
Published: August 2024

Prostate cancer (PCa) neuroendocrine tumor (NET)-like cells with low or absent androgen receptor (AR) signaling cause hormone therapy resistance and poor prognosis. Small cell lung carcinoma (SCLC), a high-grade NET, presents with metastasis early and has poor survival. ONC201/TIC10 is a first-in-class cancer therapeutic with clinical activity in diffuse gliomas and neuroendocrine tumors. We hypothesized that markers of neuroendocrine differentiation, activation of the integrated stress response (ISR) and the TRAIL pathway, as well as the expression of ClpP, contribute to neuroendocrine tumor cell death and sensitivity to ONC201. We show that PCa and SCLC cell lines (N=6) are sensitive to ONC201, regardless of the extent of neuroendocrine differentiation. Endogenous levels of some NET markers (CgA, FoxO1, ENO2, PGP9.5, SOX2) are present in a spectrum in PCa and SCLC cell lines. Overexpression of neural transcription factor BRN2 in DU145 PCa cells does not increase expression of NET differentiation markers FoxO1, ENO2, PGP9.5, and CgA at 48 hours. However, the transient BRN2 overexpression showed slight decreases in some NET markers on the spectrum while maintaining sensitivity of PCa cells to ONC201 before any phenotypic change related to NET differentiation. Our results show that ONC201 has preclinical activity against PCa including those without NET markers or in PCa cells with transient overexpression of neural transcription factor BRN2. Our results have relevance to activity of ONC201 in PCa where most castrate-resistant androgen-independent cancers are not therapy resistant due to NET differentiation. Importantly, NET differentiation does not promote resistance to ONC201 supporting further clinical investigations across the spectrum of PCa.
Dietary zinc restriction mimics protein restriction and extends lifespan inDrosophila
Authors: Kosakamoto Hina , Aikawa Hide , Kitazawa Souto , Sakuma Chisako , Okada Rina , Miura Masayuki , Obata Fumiaki
Published: August 2024

Dietary restriction extends lifespan in model organisms, mainly through dietary amino acids. Compared to macronutrients, the effect of dietary micronutrients on organismal lifespan has not been intensively investigated. Here, using a synthetic diet, we test whether restriction of each micronutrient, including vitamins and minerals, affects lifespan and fecundity in adultDrosophila. While restriction of many of these micronutrients have either negative or no impact on lifespan, zinc (Zn) restriction alone can increase it. Dietary Zn restriction (ZnR) decreases fecundity, increases starvation resistance, and promotes preference for feeding amino acids, in adult females, phenocopying dietary amino acid restriction. Our study demonstrates that dietary intake of trace elements has profound impacts on physiology and lifespan, and that limiting dietary zinc may be a strategy to improve the healthspan of animals.
A Hybrid Type I and II Polyketide Synthases Yields Distinct Aromatic Polyketides
Authors: Zhao Li Ya , Shi Jing , Xu Zhao Yang , Sun Jia Lin , Yan Zhang Yuan , Tong Zhi Wu , Tan Ren Xiang , Jiao Rui Hua , Ge Hui Ming
Published: August 2024

Bacterial aromatic polyketides are compounds with multiple aromatic rings synthesized by bacterial type II polyketide synthases (PKSs), some of which have been developed into clinical drugs. Compounds containing aromatic polyketides synthesized by a hybrid type I and type II PKSs are extremely rare. Here, we report the discovery of a gene cluster encoding both modular type I PKS, type II PKS and KAS III through extensive bioinformatics analysis, leading to the characterization of the hybrid polyketide, spirocycline A. The structure of spirocycline A is unprecedented among all aromatic polyketides, featuring a unique starter unit, four spirocycles, and forming a dimer. Biosynthetic studies indicate that the starter unit of this molecule is synthesized by type I PKS in collaboration with twotrans-acting ketoreductase (KR) and enoylreductase (ER). It is then transferred by KAS III to the type II PKS system, which then synthesizes the tricyclic aromatic polyketide backbone. The subsequent formation of the spirocycle and dimerization is carried out by four redox enzymes encoded in the gene cluster. Overall, the discovery of spirocycline A provides a new approach for identifying novel aromatic polyketides and offers potential enzymatic tools for the bioengineering of these hybrid polyketides.

Table of Contents
AI-Generated Hallmarks of Aging and Cancer: A Computational Approach Using Causal Emergence and Dependency Networks
Authors: Xiong Jianghui
Published: August 2024

This study introduces “hallmarks engineering,” a computational approach to generate quantifiable hallmarks of aging and cancer. We evaluated these hallmarks using genome-wide DNA methylation data from ten age-related diseases. Causal emergence analysis revealed that hallmark-level features show stronger disease associations than individual genes, with improvements up to 9.7 orders of magnitude. Hallmark-based models achieved comparable predictive performance with fewer predictors compared to regular pathway-based models. Dependency network analysis uncovered regulatory networks with power-law distributions and identified top-level “super-regulators” such as genomic stability. Notably, the inclusion of neurodegenerative and cancer hallmarks enhanced representation for their respective disease categories. Our findings suggest that top-down modeling using computationally generated hallmarks may reveal common mechanisms across multiple diseases, offering a promising approach for modeling multimorbidity.
Adaptive Responses to PARP Inhibition Predict Response to Olaparib and Durvalumab: Multi-omic Analysis of Serial Biopsies in the AMTEC Trial
Authors: Mitri Zahi I. , Creason Allison L. , Stommel Jayne M. , Bottomly Daniel , Ozmen Tugba Y. , Rames Matthew J. , Ozmen Furkan , Jeong Boyoung , Lukashchuk Natalia , Ashton Jack , Lim Jeong Youn , Sivagnanam Shamilene , Betra Konjit , Lee Jinho , Labrie Marilyne ,   SMMART Clinical Trials Program, Coussens Lisa M. , Corless Christopher L. , McWeeney Shannon K. , Mills Gordon B.
Published: August 2024

In syngeneic murine breast cancer models, poly ADP-ribose polymerase inhibitor (PARPi) and anti-PD-L1 combinations induce deep, sustained responses independent ofBRCA1/2mutation status. We therefore investigated this combination in the AMTEC clinical trial, in which a one-month olaparib run-in was followed by combined olaparib and durvalumab in participants withBRCA1/2wild-type metastatic triple negative breast cancer. To characterize adaptive responses to olaparib monotherapy, paired biopsies taken before and during PARPi lead-in were deeply characterized by DNA, RNA, and protein multi-omic analyses, including spatially-resolved single cell proteomics for tumor and immune contexture. We identified multiple potential tumor-intrinsic and microenvironmental biomarkers from pre-treatment and on-olaparib biopsies that robustly predicted participant response to combined olaparib and durvalumab. Notably, the on-olaparib biopsy provided the greatest information content, suggesting that adaptation of malignant cells and the tumor ecosystem to PARPi can serve as a predictor of potential benefit from combined PARPi and anti-PD-L1 therapy.
Estimated morbimortality and costs attributable to child and adolescent obesity in Brazil from 2024 to 2060: a multistate life table study
Authors: Nilson Eduardo Augusto Fernandes , de Oliveira Ana Carolina Rocha , Gusmão Bruna , Barbosa Raphael Barreto da Conceição
Published: August 2024

Introduction

Childhood obesity is a major global public health issue globally and in Brazil. The impacts of childhood obesity include higher risk of disease during childhood and of obesity and non-communicable diseases in adulthood and represent an important epidemiological and economic burden to countries.

Methods

This study is based on the multistate life table modeling of different scenarios of change3s in the child and adolescent obesity on the estimated costs attributable to obesity and the epidemiological burden of obesity-related diseases.

Results

According to this study, if the current trends in childhood and adolescent obesity in Brazil continue, the prevalence will significantly increase across different age subgroups and for both sexes by 2060 and obesity among adults will nearly double, resulting in R$3.84 billion costs attributable to childhood and adolescent obesity to the Brazilian Unified Health System during this period. Alternatively, if obesity prevalence among children and adolescents is reduced and remains constant these direct costs could be reduced by R$1.05 to R$1.27 billion by 2060 and up to 244,600 incident cases and 70,800 deaths from obesity-related diseases could be prevented.

Conclusion

This study highlights that the costs of childhood obesity are not limited to the impacts on adult health and represent a relevant economic burden to the Brazilian National Health System and to families because of additional costs during childhood. Therefore, the prevention and control of childhood obesity is a public health priority that demands immediate and robust policies.
The NeuroBioBank Whole-Genome Catalog: Sequencing from human brain donors with central nervous system disorders
Authors: Hupalo Daniel , McCauley Jacob L. , Gomez Lissette , Griswold Anthony J. , Hoher Gabriela , Konidari Ioanna , Lorenzo Jose , Parker Griffin S. , Pascual Julianna , Sandford Amanda R. , Whitehead Patrice L. , Davis David A. , Garamszegi Susanna , Gultekin S. Humayun , Sun Xiaoyan , Vontell Regina T. , Chatigny Michael , Chernicky Darren , Constant Myrtha M. , Darling Isabelle G. , Ennulat David J. , Esposito John M. , Morris Kiely , Lawton Elisabeth S. , Morakabati Neda R. , Oduor Phyllis , Rodgers Allison P. , Sang Lorelle A. , Sullivan Kathleen M. , Tabit Catalina J. , Turpin Tori , Zeabi Aya , Zheng Tina , Berretta Sabina , Klengel Torsten , Oakley Derek H. , Ruzicka W. Brad , Blanchard Thomas , Ho Eric , Johnson Robert , LeFevre Alexandra , Bustamante Maxwell , Haroutunian Vahram , Katsel Pavel , Marino Christine , Panopoulos Stephen , Purohit Dushyant P. , Wysocki Michael , Glausier Jill R. , Lewis David .A. , Nagra Rashed M. , Alba Camille , Martin Julianna , Rice Elizabeth , Rosenberger John , Smith Grace , Sukumar Gauthaman , Tompkins Miranda , Wilkerson Matthew , Dalgard Clifton L. , Scott William K.
Published: August 2024

Central nervous system diseases are a prevailing cause of morbidity and mortality worldwide, and are influenced by environmental and biological factors including genetic risk. Here we generated genome-wide genetic data on a large cohort of brain tissue donors with in-depth clinical and neuropathological phenotyping, allowing for broad investigations into the risk and mechanisms of these neurological, neurodevelopmental, and psychiatric conditions. This resource consists of 9,663 donors with array-based genotyping and 9,543 donors with whole-genome sequencing completed. The clinical diagnoses of these donors include 148 central nervous system diseases clustered in 15 broad categories by ICD-10 coding. These donors were collected by six repositories comprising the NIH NeuroBioBank, with an average participant age of 60 years. While primarily older individuals of European descent, the cohort also contains younger donors and individuals from non-European backgrounds. Variants detected by Whole-Genome Sequencing (WGS) were called genome-wide and annotated to describe their functional impact, resulting in 171,121,209 unique mutations and 1,078,774 non-silent mutations. This whole-genome resource has been made available in the NIMH Data Archive (nda.nih.gov) and accompanying deep demographic and phenotypic descriptions are available at the NeuroBioBank Portal (neurobiobank.nih.gov). To illustrate an application of this resource, we replicated the strong association observed in previous studies between pathogenic CAG repeat expansions in theHTTgene with the clinical diagnosis of Huntington’s disease.
Tentative analysis of biomarkers associated with filariasis in moungo division, littoral, cameroon
Authors: Hzounda Fokou Jean Baptiste , Teudem Biyong Syntiche , Kouemo Francine , Akumwah Fru Awah , Reine Ambassa , Fannang Véronique Simone , Koube Juliette , Assob Jules Clement
Published: August 2024

Filariasis is a significant cause of morbidity and a public health concern in most tropical countries. These diseases are usually contracted in childhood and most often diagnosed in adulthood. This study aimed to identify biochemical markers associated with filariasis and the endobacteria present in microfilariae.

This was a cross-sectional analytical study within a hospital setting. Persons aged from six years and above were included in the study. They were then clustered into asymptomatic and symptomatic. Data was collected from February to June 2023. Blood, urine, and skin samples were collected and analysed by microscopy to identify microfilariae. The plasmatic and urinary markers were evaluated by spectrophotometry and protein fraction was monitored by electrophoresis. The data was analysed using SPSS 25 with the significant threshold set at 0.005.

A total of 55 persons were included in the study, representing 74,32% of the total population questioned. The sex ratio (F/M) was 2.66. The infection rate was 18.2%Onchocerca volvulus, 54.54%Mansonella perstans, 22.72%Loa loaand 4.54%Wuchereria bancrofti. Protein levels were 35% higher in the asymptomatic with no parasite, 80% in the asymptomatic with detected parasite and 91.67% in the symptomatic. The decrease in albumin fraction and increase in gamma fractions seem to be correlated with the presence of parasites in the blood.

These results suggest that protidemia and serum protein are significant indicators in the diagnosis of filariasis.
Minimum Days Estimation for Reliable Dietary Intake Information: Findings from a Digital Cohort
Authors: Singh Rohan , Verest Mathieu Théo Eric , Salathé Marcel
Published: August 2024

Accurate dietary assessment is crucial for understanding diet-health relationships, but variability in daily food intake poses challenges in capturing precise data. This study leveraged data from 958 participants of the “Food & You“ digital cohort to determine the minimum number of days required for reliable dietary intake estimation. Participants tracked meals using the AI-assisted MyFoodRepo app, providing a comprehensive dataset of over 315,000 dishes across 23,335 participant days. We employed multiple analytical approaches, including Linear Mixed Models (LMM), Intraclass Correlation Coefficient (ICC), and Coefficient of Variation (CV) methods. LMM analysis revealed significant day-of-week effects, with increased energy, carbohydrate, and alcohol intake on weekends, particularly pronounced in younger individuals and those with higher BMI. ICC and CV analyses demonstrated that the required number of days varies considerably among nutrients and food groups. Water, coffee, and total food quantity by weight could be reliably estimated (ICC>0.9) with just 1-2 days of data. Most macronutrients, including carbohydrates, protein, and fat, achieved good reliability (ICC>0.75 or r=0.8) with 3-4 days of data. Micronutrients and some food groups like meat and vegetables typically required 4-5 days for highly reliable estimation. Optimal day combinations often included both weekdays and weekend days. Our findings largely align with and refine FAO recommendations, suggesting that 3-4 days, typically non-consecutive and including a weekend day, are generally sufficient for reliable estimation of energy and macronutrient intake. However, our results provide more nuanced, nutrient-specific guidelines that can inform the design of future nutritional studies.
Application of chaos and systems theories to explore health workforce re-organisation following district splitting in Uganda
Authors: Mutebi Aloysius , Mukuru Moses , Kiwanuka Suzanne , Makumbi Fredrick , Rutebemberwa Elizeus
Published: August 2024

Background

District splitting (DS) began in 1962 and intensified in 1997 during implementation of decentralization. This legislative process focuses on geographical demarcation. The health system, organized along local government structures, was re-organized as a result of DS. However, this study explored how the health workforce, as a component of the local government health system, re-organized following district splitting.

Methods

Conducted between June and December 2018, the qualitative study involved 22 key informant interviews in eight districts, representing four broader regions (North, Western, Eastern and Central) of Uganda. Participants included district political leaders, local government staff, district service commission members, district health team officials, and district senior management. Data was collected using an interview guide and four themes were identified for deductive thematic analysis.

Results

Findings reveal that district splitting led to an involuntary division of the health system between old and new districts, triggering multidirectional reorganization of the health workforce. Health workers were systematically allocated to districts based on the geographical location of their health facilities during the splitting process. Subsequently, inter-district migration occurred through health workforce secondment or appointment, while intra-health system migration involved vertical movement of health workers within districts during recruitment. This reorganization resulted in workforce shortages in health facilities vacated by migrating health workers.

Conclusions

The study concludes that district splitting caused unintended consequences as a result of chaotic occurrences that led to reorganization of the health workforce in both new and old districts, and caused gaps in service delivery capabilities. Adopting a systems and chaos perspective, the study emphasizes the importance of considering frontline service delivery systems as subsystems of the overall district system when evaluating the impact of district splitting. Such an approach provides a comprehensive framework for understanding the disruptive effects of district splitting on health system organization and service delivery.
Quantitative Multi-pathway Assessment of Exposure to Fecal Contamination for Infants in Rural Ethiopia
Authors: Wang Yuke , Yang Yang , Slanzi Crystal M. , Li Xiaolong , Ojeda Amanda , Paro Fevi , Deblais Löıc , Yakubu Habib , Hassen Bahar Mummed , Game Halengo , Roba Kedir Teji , Schieber Elizabeth , Ibrahim Abdulmuen Mohammed , Wolyie Jeylan , Hassen Jemal Yusuf , Rajashekara Gireesh , McKune Sarah L. , Havelaar Arie H. , Moe Christine L. , Liang Song
Published: August 2024

In low- and middle-income countries (LMICs), enteric infections pose a significant threat to children’s health. However, understanding the specifics of when, where, and how young children in LMICs are exposed to enteric pathogens and the roles of animal reservoirs, environmental media, and human hosts play during exposure remains limited. This study systematically evaluates infants’ exposure toE. colithrough various pathways in the rural Haramaya woreda of Ethiopia. Between June 2021 and June 2022, we conducted over 1300 hours of structured behavioral observations on 79 infants when aged 4–8 months (Timepoint 1) and 11–15 months (Timepoint 2). Enumerators recorded the infant’s behavior related to exposure, including when and where it occurred, using the Countee © application. Concurrently, we collected 1338 environmental samples from key contact interfaces between infants, other people, and the environment to test forE. coli. We used a competing hazard model for duration-based behaviors, an inhomogeneous Poisson point process model for frequency-based behaviors, and a left-censored lognormal distribution model forE. colicontamination levels. The behavioral and environmental information was then integrated into an agent-based exposure model framework to quantify the exposure toE. colithrough different pathways.

The infant behavior, which altered the relative importance of different exposure pathways, changed as children grew older. Notably, we observed increased rates of touching behavior (e.g., touching fomites) and soil-pica, increased consumption of solid food, and more time spent on the bare ground at Timepoint 2. The major sources of exposure toE. coliwere food and breastfeeding at Timepoint 1 and food and soil at Timepoint 2. This study provides insights for interventions to minimize infants’ risk of exposure to fecal contamination and subsequent risk of enteric infections, including improved food handling practices, enhanced personal hygiene for breastfeeding caregivers, and education on the risk of soil-pica.
Can Place-Based Modifications Make a Difference to Local Health Inequalities in Urban Essex: An Evaluation Protocol
Authors: Cusimano K. , Freeman P. , Pettican A. , Brinkley A. J.
Published: August 2024

Stemming from a complex picture of compositional, contextual and wider determinants, health inequalities are presented at the level in which people reside (i.e., their place). Examples of this exist within Essex, England, where despite seeming affluence, pockets of high multiple deprivation exist. Programmes delivered across the system representing each place may provide a solution to these complex challenges. For this reason, Epping Forest District Council commissioned the evaluation of a programme representing two place-based projects within their district (i.e., Limes Farm and Oakwood Hill). This paper provides the evaluation protocol for this programme. Broadly, the evaluation seeks to investigate the design, implementation, mechanisms and effectiveness of both projects. Our evaluation is underpinned by the Medical Research Council (MRC) guidelines for the design, evaluation and implementation for complex interventions, and takes inspiration from a realist approach. We aim to understandwhereeach project works,whodoes the projects work for,whatimpact do the projects have, andhowandwhydoes the projects work. This will be achieved through a mixed-methods approach which utilises a cohort study, ripple-effects mapping, focus groups, and secondary data analysis. Quantitative data will be analysed using descriptive, general linear and multi- level models, while qualitative data will be understood using visualisation (ripple-effects maps) and reflexive thematic analysis. Data will be triangulated to create programme theory configurations, which explain the outcomes which stemming from the programme, and how these are shaped by mechanisms within a given context. We anticipate our novel and robust approach to contribute to policy surrounding the adoption and implementation of place-based approaches.
A bifunctional antibody conjugate marks the location of DNA binding proteins on deproteinized DNA fibers
Authors: Pokharel Durga , Shaik Althaf , Gali Himabindu , Ling Chen , Bellani Marina A. , Seidman Michael M
Published: August 2024

Immunofluorescent foci of DNA Damage Response (DDR) proteins serve as surrogates for DNA damage and are frequently interpreted as denoting specific lesions. For example, Double Strand Breaks (DSBs) are potent inducers of the DDR, whose best-known factor is the phosphorylated histone variant H2AX (γ-H2AX). The association with DSBs is so well established that the reverse interpretation that γ-H2AX invariably implies DSBs is routine. However, this conclusion is inferential and has been challenged. The resolution of this question has been hampered by the lack of methods for distinguishing the location of DDR proteins relative to DSBs caused by sequence indifferent agents. Here, we describe an approach for marking the location of DDR factors in relation to DSBs on DNA fibers. We synthesized a two-arm “Y” conjugate containing biotin and trimethylpsoralen (TMP) coupled to a secondary antibody. After exposure to a DNA breaker, permeabilized mammalian cells were incubated with a primary antibody against the DDR factor followed by binding of the secondary antibody in the conjugate to the primary antibody. Exposure to longwave UV light covalently linked the psoralen to the DNA. DNA fibers were spread, and the immunofluorescence of the biotin tag denoted the location of the target protein.

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Graphical abstract
Metabolic engineering of stomatal precursor cells enhances photosynthetic water-use efficiency and vegetative growth under water-deficit conditions inArabidopsis thaliana
Authors: Bouvier Jacques W. , Kelly Steven
Published: August 2024

Stomata are epidermal pores that control the exchange of gaseous CO2and H2O between plants and their environment. Modulating stomatal density can alter this exchange, and thus presents a target for engineering improved crop productivity and climate resilience. Here, we show that stomatal density inArabidopsis thalianacan be decreased by the expression of a water-forming NAD(P)H oxidase targeted to stomatal precursor cells. We demonstrate that this reduction in stomatal density occurs irrespective of whether the expressed enzyme is targeted to the cytosol, chloroplast stroma, or chloroplast intermembrane space of these cells. We reveal that this decrease in stomatal density occurs in the absence of any measurable impact on stomatal dynamics, or the efficiency or thermal sensitivity of photosynthesis. Consequently, overexpression plants exhibit a higher intrinsic water use efficiency due to an increase in CO2fixed per unit water transpired. Finally, we demonstrate that this enhanced water-use efficiency translates to an improvement in vegetative growth and biomass accumulation under water-deficit conditions. Together, these results thus provide a novel approach for enhancing plant productivity through metabolic engineering of stomatal density.
Exploiting XAI maps to improve MS lesion segmentation and detection in MRI
Authors: Spagnolo Federico , Molchanova Nataliia , Ocampo Pineda Mario , Melie-Garcia Lester , Bach Cuadra Meritxell , Granziera Cristina , Andrearczyk Vincent , Depeursinge Adrien
Published: August 2024

To date, several methods have been developed to explain deep learning algorithms for classification tasks. Recently, an adaptation of two of such methods has been proposed to generate instance-level explainable maps in a semantic segmentation scenario, such as multiple sclerosis (MS) lesion segmentation. In the mentioned work, a 3D U-Net was trained and tested for MS lesion segmentation, yielding an F1 score of 0.7006, and a positive predictive value (PPV) of 0.6265. The distribution of values in explainable maps exposed some differences between maps of true and false positive (TP/FP) examples. Inspired by those results, we explore in this paper the use of characteristics of lesion-specific saliency maps to refine segmentation and detection scores. We generate around 21000 maps from as many TP/FP lesions in a batch of 72 patients (training set) and 4868 from the 37 patients in the test set. 93 radiomic features extracted from the first set of maps were used to train a logistic regression model and classify TP versus FP. On the test set, F1 score and PPV were improved by a large margin when compared to the initial model, reaching 0.7450 and 0.7817, with 95% confidence intervals of [0.7358, 0.7547] and [0.7679, 0.7962], respectively. These results suggest that saliency maps can be used to refine prediction scores, boosting a model’s performances.
Developmental control of E-cadherin junctions by mechanical contractility inDrosophilaembryos
Authors: Zhang Na , Liu Wangfang , Lu Huiying , Häring Matthias , Wolf Fred , Großhans Jörg , Lv Zhiyi , Kong Deqing
Published: August 2024

Adherens junctions are very plastic structures that change their composition and structure depending on the state of the epithelial tissue, involving mechanical stress and tissue dynamics. InDrosophilaembryos, adherens junctions mature only during gastrulation following their formation a few minutes before during cellularization. Although the developmental maturation is obvious during gastrulation, the factors and conditions controlling it have remained unclear. Here, we assay the levels, distribution pattern, and mobility of E-cadherin duringDrosophilagastrulation. Indicating a maturation, we find an increase in total levels at junctions and a drop in the mobile fraction of E-cadherin molecules. Both developmental changes depend on Myo-II contractility. Consistent with stereotypic Myo-II regulation, we find that interference with Rho signaling leads to corresponding changes in E-cadherin levels and mobility, at least in part. Besides Rho signaling, Src42A contributes to junction maturation, as tested by both loss-function and gain-of-function situations. Together, we demonstrate developmental maturation of adherens junctions and identify Rho signaling and Src42A as upstream regulatory pathways.
Sodalis praecaptivussubsp.spalangiaesubsp. nov., a nascent bacterial endosymbiont isolated from the parasitoid wasp,Spalangia cameroni
Authors: Teh Li Szhen , James Ian , Dolgova Anna , Chiel Elad , Dale Colin
Published: August 2024

An endosymbiotic bacterium of the genusSodalis,designated as strain HZT, was cultured from the parasitoid waspSpalangia cameroni, that develops on pupae of various host flies. The bacterium was detected inS. cameronithat developed on house flies,Musca domestica, in a poultry facility in Hazon, northern Israel. Following culture, this bacterium displayed no surface motility on LB agar, is rod-shaped and irregular in size, approximating 10-30 nm in diameter and 5-20 μm in length. Phylogenetic analyses revealed that strain HZTis closely related toSodalis praecaptivusstrain HST, a free-living species of the genusSodalisthat includes many insect endosymbionts. Although these bacteria maintain >98% sequence identity in shared genes, genomic characterization revealed that strain HZThas undergone substantial reductive evolution, such that it lacks many gene functions that are maintained inS. praecaptivusstrain HST. Based on the results of phylogenetic, genomic and chemotaxonomic analyses, we propose that this endosymbiont should be classified in a new subspecies asSodalis praecaptivussubsp. spalangiaesubsp. nov. The type strain for this new subspecies is HZT(=ATCC TSD-398T=NCIMB 15482T). The subspeciesSodalis praecaptivussubsp.praecaptivusstrain HSTis created automatically with the type strain ATCC BAA-2554T(=DSMZ 27494T).
Orchestrating Improbable Chemistries: Structural Snapshots of B12-Dependent Methionine Synthase’s Catalytic Choreography
Authors: Mendoza Johnny , Yamada Kazuhiro , Castillo Carmen , Wilhelm Catherine A. , Koutmos Markos
Published: August 2024

Cobalamin (vitamin B12) and its derivatives play an essential role in biological methylation, with cobalamin-dependent methionine synthase (MS) serving as a canonical example. MS catalyzes multiple methyl transfers within a single, dynamic multi-domain architecture that has proven challenging to study, hampering efforts to elucidate its catalytic mechanism(s). Utilizing a thermostable MS homolog and non-native cobalamin cofactors, we have captured crystal structures of transient conformational states of MS, including those directly involved in folate demethylation and homocysteine methylation. These snapshots reveal the mechanistic significance of five-coordinate, His-off methylcobalamin in homocysteine methylation and highlight the crucial role of the folate-binding domain and interdomain linkers in orchestrating the intricate structural rearrangements required for catalysis. This expanded conformational ensemble, including the unexpected capture of novel ‘Cap-on’ conformations, underscores the remarkable plasticity of MS, exceeding previous estimations. Our findings provide crucial insights into the catalytic mechanism of MS, laying the foundation for harnessing cobalamin’s biocatalytic potential and elucidating how nature exploits protein dynamics to facilitate complex transformations.
Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells
Authors: Banik Debasis , Ward Christopher , Zhang Ziwei , Heraghty Daniel , Suresh Prasanna , Li Bing , Kedia Shekhar , Davis Simon J. , Roy James P. , Chapman Michael A. , Mahata Bidesh , Klenerman David
Published: August 2024

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting CAR primary T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We find that CRC cells’ glycocalyx is much thicker than the CAR T-cell and likely contributes to immune-escape. Oscillating calcium flux, a signature of non-sustained triggering and decreased killing, was observed when CAR T-cells interacted with CRC, which increased with increasing cell-seeding time. This was because CEACAM5 became increasingly unavailable on the CRC cell monolayer, as revealed by fluorescence imaging. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T-cell calcium fluxes, increasing the killing of CRC cells. Our results reveal why CAR T-cells targeting CEACAM5 are ineffective and suggest possible routes for improved therapy.

One Sentence Summary

CEACAM5 unavailability and the thick CRC cell glycocalyx are major barriers to CAR T-cells, which local proteolysis helps to overcome.
Development of a Robust Gel-Free 3D Culture System for Generating Spheroids from Axolotl Blastema Cells
Authors: Aladağ Zeynep , Vatandaşlar Emre , Vilain Sven , Öztürk Gürkan
Published: August 2024

Regenerative biology is a pivotal field aimed at understanding and harnessing the ability of organisms to restore damaged tissues and organs. The axolotl (Ambystoma mexicanum) is a key model organism renowned for its exceptional regenerative capabilities, particularly in limb regeneration. However, currentin vitroresearch is hampered by the lack of well-defined axolotl blastema cell lines and unreliable primary culture protocols. To address these challenges, we present a novel, robust, gel-free 3D culture system for axolotl blastema cells. This system overcomes limitations of 2D culture methods by enabling the formation of spheroid structures that closely mimic thein vivoenvironment. Using this protocol, we observed that spheroids derived from axolotl blastema tissues retained key regenerative markers, including PRRX1, and exhibited stable expression of crucial blastema markers through extended culture periods. Our approach facilitates the study of cellular and molecular mechanisms underlying limb regeneration in axolotls and provides a valuable tool for drug testing and regenerative research. The development of this 3D culture system represents a significant advancement in regenerative biology, offering a more consistent and reliable model for exploring the regenerative potential of axolotl cells.
Enhanced Salt Tolerance inSynechocystissp. PCC 6803 Through Adaptive Evolution: Mechanisms and Applications for Environmental Bioremediation
Authors: Zhu Xiaofei , Zou Rongsong , Liu Dailin , Liu Jing , Wu Xuejing , Chen Lei , Sun Tao , Zhang Weiwen
Published: August 2024

Salt stress is common in natural environments, where elevated salt levels in brackish water and saline soil can hinder the growth of organisms, thereby exacerbating environmental challenges. Developing salt-tolerant organisms not only uncovers novel mechanisms of salt tolerance but also lays the groundwork for managing and utilizing saline environments. Cyanobacteria, which are widely distributed in hydrosphere and soil, serve as ideal models for studying salt stress. In this study, the model cyanobacteriumSynechocystissp. PCC 6803 was selected, whose salt (NaCl) tolerance improved from 4.0% to 6.5% (m/v) through adaptive laboratory evolution. Genome re-sequencing and mutant analysis identified six key genes associated with salt tolerance. Notably, the deletion ofslr1670, which encodes glycerol glucoside hydrolase, improved the strain’s salt tolerance. In addition,slr1753encodes a membrane protein that may enhance salt tolerance by facilitating ion transport to the extracellular space. Further analysis revealed that overexpression ofslr1753significantly accumulates Na+on the cell surface, enabling effective seawater treatment using the engineered strain, resulting in a 6.35% reduction of Na+in the seawater. Moreover, the adapted bacteria can be used for the remediation of saline soil samples, leading to a 184.2% and 43.8% increase in the germination rate and average height ofBrassica rapa chinensis, respectively, along with a 25.3% rise in total organic carbon content and reductions in both total salt content by 1.82% and pH by 1.91% in soil. This study provides novel insights into salt tolerance mechanisms and the bioremediation of high-salinity environments.

Graphical abstract
Inferring super-resolved spatial metabolomics from microscopy
Authors: Rappez Luca , Haase Kristina
Published: August 2024

Current spatial metabolomics techniques have transformed our understanding of cellular metabolism, yet accessible methods are limited in spatial resolution due to sensitivity constraints. MetaLens, a deep generative approach, disrupts this trade-off by quantitatively propagating cellular-resolutionin situimaging mass spectrometry readouts to subcellular scales through integration with high-resolution light microscopy. MetaLens identifies subcellular metabolic domains with distinct molecular composition, enabling accessible label-free subcellular metabolomic analysis from microscopy.
A combination of systemic mannitol administration and mannitol-modified polyester nanoparticles facilitate gene delivery to the brain through caveolae-mediated endocytosis
Authors: Betsy Reshma G , Miglani Chirag , Karmakar Arundhati , Gupta Sarika , Pal Asish , Ganguli Munia
Published: August 2024

Overcoming the blood-brain barrier (BBB) remains a significant challenge for nucleic acid delivery to the brain. We have explored a combination of mannitol-modified poly (β-amino ester) (PBAE) nanoparticles and systemic mannitol injection for crossing the BBB. We incorporated mannitol in the PBAE polymer for caveolae targeting and also selected monomers that may help avoid delivery to the liver. We also induced caveolae at the BBB through systemic mannitol injection in order to create an opportunity for the caveolae-targeting nanoparticles (M30 D90)containing plasmid DNA to cross the BBB. When a clinically relevant dose was administered intravenously in this caveolae induction model, M30 D90 demonstrated significant transgene expression of a reporter plasmid in the brain, with selective uptake by neuronal cells and minimal liver accumulation. We also demonstrate that both caveolae modulation using systemic mannitol administration and caveolae targeting using designed nanoparticles are necessary for efficient delivery to the brain. This delivery platform offers a simple, scalable, and controlled delivery solution and holds promise for treating central nervous system diseases with functional targets.
RcsF-independent mechanisms of signaling within the Rcs Phosphorelay
Authors: Petchiappan Anushya , Majdalani Nadim , Wall Erin , Gottesman Susan
Published: August 2024

The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducting signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF and characterized the underlying regulatory mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs signaling can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator inE. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for signaling; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that RcsC acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms.

Author summary

The Rcs phosphorelay signaling cascade regulates the expression of genes related to capsule synthesis, biofilm formation, virulence, and cell division in Enterobacteria and is critical for cell membrane integrity and response to beta-lactam antibiotics and antimicrobial peptides. RcsF is the sole known sensor, but other proteins have been reported to activate this pathway in the absence of RcsF. We have discovered a novel RcsF-independent Rcs activator and found that each of three RcsF-independent proteins activate the system differently. Most significantly, we find that the histidine kinase RcsC can be involved in signal sensing independently of RcsF. Our study sheds light into the complex mechanisms of Rcs activation and adds to our knowledge of non-orthodox signaling systems across organisms.