bluuebunny/crossref_metadata_2025 huggingface dataset:
Effectiveness of using implementation frameworks to facilitate the implementation of a stroke management guideline in the Traditional Chinese Medicine hospitals in China: protocol for a factorial randomized controlled trial
Authors: Xu Roman , He Wenjun , Hao Chun , Zhao Qing , Guo Pengfei , Zhang Lanping , Chen Zhuo , Cai Yiyuan , Lv Sensen , Cai Yefeng , Liu Lingrui , Shi Yuning
Published: June 2024

Background: Traditional Chinese medicine (TCM) is commonly used alongside western medicine for stroke management in China. However, there is significant variation in TCM practice, and the utilization of evidence-based clinical practice guidelines is inadequate.This study aims to evaluate the effectiveness of three popular frameworks (Consolidated Framework for Implementation Research, Theoretical Domains Framework, and Normalization Process Theory) in improving implementation outcomes for the integrated TCM and western medicine clinical practice guideline for stroke management. Methods: The study will employ a hybrid type III design, utilizing a factorial randomized controlled approach. Implementation facilitators will be trained and randomly assigned to participating organizations. Forty-five TCM hospitals will be randomly assigned to one of eight experiment conditions, allowing for the evaluation of each framework's main effect, two-way interactions, and three-way interaction. The outcomes will be assessed using the RE-AIM framework, including reach, effectiveness, adoption, implementation, and maintenance. Hierarchical logistic regression models will be used to test the hypotheses. Qualitative methods, such as interviews and focus groups, will provide contextual information, and a Cost-effectiveness analysis (CEA) will be conducted. Discussion: This proposed hybrid type III study will integrate implementation science(frameworks), epidemiology (factorial design), and health economic approaches (CEA) to advance our empirical understanding of the strengths and weaknesses of implementation frameworks, implementing the TCM guideline promotion practices in hospitals and its health economic evaluation. The study answers the question that which framework combination works well in facilitating the implementation of clinical intervention. The results of the trial will offer valuable insights into how best to implement evidence-based practices (TCM guidelines) in hospitals under the consideration of economic evaluation.
Neural Dynamics of Object Manifold Alignment in the Ventral Stream
Authors: Wang Binxu , Ponce Carlos R.
Published: June 2024

Visual neurons respond across a vast landscape of images, comprising objects, textures, and places. Natural images can be parameterized using deep generative networks, raising the question of whether latent factors learned by some networks control images in ways that better align with visual neurons. We studied neurons in areas V1, V4 and posterior IT, optimizing images using a closed-loop evolutionary algorithm. We used two generative image models: (1) DeePSim, which parameterizes local image patterns, and (2) BigGAN which parameterizes object identity and nuisance variables. We found that neurons could guide image optimization on both pattern- and object-based image manifolds across areas; V1 aligned best with the DeePSim image space, whereas PIT aligned well with both DeePSim and BigGAN spaces. While initially PIT neurons responded well to the textural manifold, their responses to objects also emerged over time, suggesting that object-like responses required further processing. We identified similar local features common to both textural and object images, but not optimal global configuration. We conclude that visual cortex neurons are aligned to a representational space not yet captured by current artificial model of the visual system.
ESR2 orchestrates cytokinin dynamics leading to developmental reprogramming and green callus formation
Authors: Durán-Medina Yolanda , Díaz-Ramírez David , Herrera-Ubaldo Humberto , Di Marzo Maurizio , Gómez Felipe Andrea , Cruz-Valderrama J. Erik , Vázquez Carlos A. , Guerrero-Largo Herenia , Colombo Lucia , Novak Ondrej , de Folter Stefan , Marsch-Martínez Nayelli
Published: June 2024

Callus formation and shoot regeneration are natural plant abilities triggered by stress and damage. They are also key components of tissue culture, which for many species is crucial for gene editing, transformation, propagation, and other technologies, and their study provides valuable insights into plant development. The transcription factor ENHANCER OF SHOOT REGENERATION 2 (ESR2/DRNL/BOL/SOB) promotes green callus formation in roots and shoot regeneration when overactive, while the phythormone cytokinin plays a prominent role in both phenomena. Yet, the positive action of ESR2 on the cytokinin pathway had not been previously described. We explored the effects of ESR2 and found that cytokinin content and the expression of the cytokinin biosynthesis geneISOPENTENYLTRANSFERASE 5(IPT5)increase in plants where ESR2 activity is induced. ESR2 also regulates the cytokinin signaling repressorARABIDOPSIS HISTIDINE PHOSPHOTRANSFER 6(AHP6),and surprisingly, the ESR2-stimulated green calli formation requires bothIPT5andAHP6. Therefore, ESR2 promotes both cytokinin biosynthesis and cytokinin signaling inhibition and requires a paradoxical combination of these two for green callus induction. This finding provides a foundation to better understand the processes involved in tissue reprogramming towards callus formation and the role of ESR2 in shoot regeneration and the development of new organs.
Rapid spread of African Swine Fever across Borneo
Authors: Daniel Olivia Z , Heon Sui Peng , Donnelly Christl A , Bernard Henry , Orme C. David L , Ewers Robert M
Published: June 2024

African Swine Fever (ASF) reached the island of Borneo at the end of 2020. The first mortalities occurred in wild bearded pigs (Sus barbatus) in Sabah, north-east Borneo. The virus then began to spread across the island but, confounded by COVID 19 lockdowns the spread was difficult to monitor on the ground. The Babi Hutan Project was launched in April 2021 to gather data on pig sightings using citizen science. Here we bring together the data from this project and other online sources to show how the virus encompassed almost the entire island within a one-year period. The speed of spread appears to increase with time following an exponential model: we estimate an average speed of spread of 0.89km/day after 100 days since the first observation and at 4.28km/day after 400 days. We include recommendations of next steps for the bearded pigs of Borneo.
Lactucasuper-pangenome reduces bias towards reference genes in lettuce research
Authors: van Workum Dirk-Jan M. , Mehrem Sarah L. , Snoek Basten L. , Alderkamp Marrit C. , Lapin Dmitry , Mulder Flip F.M. , Van den Ackerveken Guido , de Ridder Dick , Schranz M. Eric , Smit Sandra
Published: June 2024

Breeding of lettuce (Lactuca sativaL.), the most important leafy vegetable worldwide, for enhanced disease resistance and resilience relies on multiple wild relatives to provide the necessary genetic diversity. In this study, we constructed a super-pangenome based on fourLactucaspecies (representing the primary, secondary and tertiary gene pools) and comprising 474 accessions. We include 68 newly sequenced accessions to improve cultivar coverage and add important foundational breeding lines. With the super-pangenome we find substantial presence/absence variation (PAV) and copy-number variation (CNV). Functional enrichment analyses of core and variable genes show that transcriptional regulators are conserved whereas disease resistance genes are variable. PAV-genome-wide association studies (GWAS) and CNV-GWAS are largely congruent with single-nucleotide polymorphism (SNP)-GWAS. Importantly, they also identify several major novel quantitative trait loci (QTL) for resistance againstBremia lactucaein variable regions not present in the reference lettuce genome. The usability of the super-pangenome is demonstrated by identifying the likely origin of non-reference resistance loci from the wild relativesLactuca serriola, Lactuca salignaandLactuca virosa. The provided methodology and data provide a strong basis for research into PAVs, CNVs and other variation underlying important biological traits of lettuce and other crops.
Design of linear and cyclic peptide binders of different lengths from protein sequence information
Authors: Li Qiuzhen , Vlachos Efstathios Nikolaos , Bryant Patrick
Published: June 2024

Structure prediction technology has revolutionised the field of protein design, but key questions such as how to design new functions remain. Many proteins exert their functions through interactions with other proteins, and a significant challenge is designing these interactions effectively. While most efforts have focused on larger, more stable proteins, shorter peptides offer advantages such as lower manufacturing costs, reduced steric hindrance, and the ability to traverse cell membranes when cyclized. However, less structural data is available for peptides and their flexibility makes them harder to design. Here, we present a method to design both novel linear and cyclic peptide binders of varying lengths based solely on a protein target sequence. Our approach does not specify a binding site or the length of the binder, making the procedure completely blind. We demonstrate that linear and cyclic peptide binders of different lengths can be designed with nM affinity in a single shot, and adversarial designs can be avoided through orthogonalin silicoevaluation, tripling the success rate. Our protocol,EvoBind2is freely availablehttps://github.com/patrickbryant1/EvoBind.
Neocortical long-range inhibition promotes cortical synchrony and sleep
Authors: Ratliff Jacob M , Terral Geoffrey , Lutzu Stefano , Heiss Jaime , Mota Julie , Stith Bianca , Vazquez Lechuga Arenski , Ramakrishnan Charu , Fenno Lief E , Daigle Tanya , Deisseroth Karl , Zeng Hongkui , Ngai John , Tasic Bosiljka , Sjulson Lucas , Rudolph Stephanie , Kilduff Thomas S. , Batista-Brito Renata
Published: June 2024

Behavioral states such as sleep and wake are highly correlated with specific patterns of rhythmic activity in the cortex. During low arousal states such as slow wave sleep, the cortex is synchronized and dominated by low frequency rhythms coordinated across multiple regions. Although recent evidence suggests that GABAergic inhibitory neurons are key players in cortical state modulation, thein vivocircuit mechanisms coordinating synchronized activity among local and distant neocortical networks are not well understood. Here, we show that somatostatin and chondrolectin co-expressing cells (Sst-Chodl cells), a sparse and unique class of neocortical inhibitory neurons, are selectively active during low arousal states and are largely silent during periods of high arousal. In contrast to other neocortical inhibitory neurons, we show these neurons have long-range axons that project across neocortical areas. Activation of Sst-Chodl cells is sufficient to promote synchronized cortical states characteristic of low arousal, with increased spike co-firing and low frequency brain rhythms, and to alter behavioral states by promoting sleep. Contrary to the prevailing belief that sleep is exclusively driven by subcortical mechanisms, our findings reveal that these long-range inhibitory neurons not only track changes in behavioral state but are sufficient to induce both sleep-like cortical states and sleep behavior, establishing a crucial circuit component in regulating behavioral states.
Elucidating DNA-binding protein dynamics inSalmonellaTyphimurium within macrophages using a breakthrough low-input ChIP-exo approach
Authors: Park Joon Young , Jang Minchang , Choi Eunna , Lee Sang-Mok , Bang Ina , Woo Jihoon , Kim Seoyeon , Lee Eun-Jin , Kim Donghyuk
Published: June 2024

Genome-wide identification of binding profiles for DNA-binding proteins inSalmonellaTyphimurium within macrophages is crucial for understanding virulence gene expression and cellular processes. However, this task remains challenging due to the limited amount of intracellular bacterial cells. Here, we present ChIP-mini, a low-input ChIP-exo utilizing 5,000-fold reduced number of initial bacterial cells and analysis pipeline (DiffExo), to identify genome-wide binding dynamics of DNA-binding proteins in host-infected pathogens. Applying ChIP-mini to intracellularS.Typhimurium, we identified 642 and 1,837 binding sites of H-NS and RpoD, respectively, with near-base pair resolution, elucidating their roles in transcriptional initiation. Post-infection, we observed 21 significant reductions in H-NS binding at intergenic regions, exposing the promoter region of virulence genes, such that those inSalmonellapathogenicity islands-2, 3 and effectors, facilitating RpoD binding for transcription initiation. Furthermore, we identified 24 novel RpoD binding sites and 19 significantly increased RpoD bindings at the transcription start sites of virulence genes. These findings substantially enhance our understanding of how H-NS and RpoD simultaneously coordinate the transcription initiation of virulence genes within macrophages. Collectively, this optimized method demonstrates a tool that can be broadly adapted to elucidate transcriptional regulatory networks of host-infected pathogens, revealing critical interactions between host and microbe.
Integrative multi-environmental genomic prediction in apple
Authors: Jung Michaela , Quesada-Traver Carles , Roth Morgane , Aranzana Maria José , Muranty Hélène , Rymenants Marijn , Guerra Walter , Holzknecht Elias , Pradas Nicole , Lozano Lidia , Didelot Frédérique , Laurens François , Yates Steven , Studer Bruno , Broggini Giovanni AL , Patocchi Andrea
Published: June 2024

Genomic prediction for multiple environments can aid the selection of genotypes suited to specific soil and climate conditions. Methodological advances allow effective integration of phenotypic, genomic (additive, non-additive), and large-scale environmental (enviromic) data into multi-environmental genomic prediction models. These models can also account for genotype-by-environment interaction, utilize alternative relationship matrices (kernels), or substitute statistical approaches with deep learning. However, the application of multi-environmental genomic prediction in apple remained limited, likely due to the challenge of building multi-environmental datasets and structurally complex models. Here, we applied efficient statistical and deep learning models for multi-environmental genomic prediction of eleven apple traits with contrasting genetic architectures by integrating genomic- and enviromic-based model components. Incorporating genotype-by-environment interaction effects into statistical models improved predictive ability by up to 0.08 for nine traits compared to the benchmark model. This outcome, based on Gaussian and Deep kernels, shows these alternatives can effectively substitute the standard G-BLUP. Including non-additive effects slightly improved predictive ability by up to 0.03 for two traits, but enviromic-based effects resulted in no improvement. The deep learning approach achieved the highest predictive ability for three traits with simpler genetic architectures, outperforming the benchmark by up to 0.10. Our results demonstrate that the tested statistical models capture genotype-by-environment interactions particularly well, and the deep learning models efficiently integrate data from diverse sources. This study will foster the adoption of multi-environmental genomic prediction to select apple cultivars adapted to diverse environmental conditions, providing an opportunity to address climate change impacts.
Dissecting the genetic regulation of lateral root development in tomato under salt stress
Authors: Rahmati Ishka Maryam , Sussman Hayley , Zhao Jiantao , Craft Eric , Yu Li’ang , Nelson Andrew , Pineros Miguel , Tester Mark , Kawa Dorota , Fei Zhangjun , Julkowska Magdalena M.
Published: June 2024

Tomatoes are crucial for global food security and agricultural economies. With over 50% of arable land projected to become saline by 2050, understanding tomato responses to saline environments is essential. Soil salinization, prevalent in arid regions, leads to salt stress. Roots are primary salinity sensors and integrate stress responses like nutrient deficiency. Understanding the molecular processes maintaining root growth is vital for crop resilience. While salt stress impacts on tomato seed germination, shoot growth, and fruit yield are documented, root development remains underexplored. Most studies have focused on a limited number of cultivars and their physiological responses, not the genetic basis of salt resilience. Wild tomato relatives, such asSolanum pimpinellifolium, offer traits that are beneficial for breeding resilient crops. However, few studies have examined the genetics of salinity tolerance inS. pimpinellifolium. This study characterizes salt stress-induced changes in root system architecture (RSA) using a natural diversity panel of 220 wild tomato accessions. We identified natural variation in lateral root development and several candidate loci through Genome-Wide Association Study (GWAS). Bulk segregant analysis (BSA) revealed 22 candidate genes overlapping with GWAS findings. Exploring root transcriptome reprogramming in two accessions with differing lateral root responses highlighted genotype-specific changes. Integrating GWAS, BSA, and RNA sequencing data identified four key genes underlying differential lateral root development under salt stress. These findings offer novel genetic targets for improving salt resilience in tomatoes, providing opportunities for future research and breeding programs to enhance crop sustainability and productivity.
Within-host environmental heterogeneity is associated with phenotypic but not genomic diversity inWolbachiaendosymbionts
Authors: Pigeault Romain , Dussert Yann , Jorge Raphaël , Panza Marie , Raimond Maryline , Delaunay Carine , Ogereau David , Moumen Bouziane , Peccoud Jean , Cordaux Richard
Published: June 2024

The mechanisms underlying within-host diversification in parasite populations are still poorly understood. Yet, diversification in parasite phenotype and genotype may have important clinical and epidemiological consequences. Our aim was to determine if the constraints associated with the infection of different host tissues lead to the emergence and co-existence of multiple parasite sub-populations with different phenotypes. We tested this hypothesis by injecting the most widespread bacterial endosymbiont,Wolbachia, isolated from three tissues of the common pill-bugArmadillidium vulgareinto uninfected individuals, and by monitoring the growth rate and virulence of each bacterial sub-population in the new hosts. Our results highlighted that within-host environmental heterogeneity led to diverse phenotypes inWolbachia. This variability did not appear to result from genomic variation, but from phenotypic plasticity. Indeed, we did not find recurrent tissue-specific genomic variation among infected individuals. Whole genome resequencing of theWolbachiasub-populations and pedigree data from the animals used in this study demonstrated however the coexistence of twoWolbachiavariants within our host lineage for almost 20 years. Our results further supported the stability of theWolbachiagenome with respect to small-scale genomic variation, in agreement with reports on other symbiotic systems.
Spinal interneuron population dynamics underlying flexible pattern generation
Authors: Wimalasena Lahiru N. , Pandarinath Chethan , Yong Nicholas Au
Published: June 2024

The mammalian spinal locomotor network is composed of diverse populations of interneurons that collectively orchestrate and execute a range of locomotor behaviors. Despite the identification of many classes of spinal interneurons constituting the locomotor network, it remains unclear how the network’s collective activity computes and modifies locomotor output on a step-by-step basis. To investigate this, we analyzed lumbar interneuron population recordings and multi-muscle electromyography from spinalized cats performing air stepping and used artificial intelligence methods to uncover state space trajectories of spinal interneuron population activity on single step cycles and at millisecond timescales. Our analyses of interneuron population trajectories revealed that traversal of specific state space regions held millisecond-timescale correspondence to the timing adjustments of extensor-flexor alternation. Similarly, we found that small variations in the path of state space trajectories were tightly linked to single-step, microvolt-scale adjustments in the magnitude of muscle output.

One sentence summary

Features of spinal interneuron state space trajectories capture variations in the timing and magnitude of muscle activations across individual step cycles, with precision on the scales of milliseconds and microvolts respectively.
Deficiency in DNAH12 causes male infertility by impairing DNAH1 and DNALI1 recruitment in humans and mice
Authors: Yang Menglei , Hussain Hafiz Muhammad Jafar , Khan Manan , Muhammad Zubair , Zhou Jianteng , Ma Ao , Huang Xiongheng , Ye Jingwei , Chen Min , Zhi Aoran , Liu Tao , Khan Ranjha , Ali Asim , Shah Wasim , Zeb Aurang , Ahmad Nisar , Zhang Huan , Xu Bo , Ma Hui , Shi Qinghua , Shi Baolu
Published: June 2024

Asthenoteratozoospermia, a prevalent cause of male infertility, lacks a well-defined etiology. DNAH12 is a special dynein featured by the absence of a microtubule-binding domain, however, its functions in spermatogenesis remain largely unknown. Through comprehensive genetic analyses involving whole-exome sequencing and subsequent Sanger sequencing on infertile patients and fertile controls from six distinct families, we unveiled six biallelic mutations inDNAH12that co-segregate recessively with male infertility in the studied families. Transmission electron microscopy (TEM) revealed pronounced axonemal abnormalities, including inner dynein arms (IDAs) impairment and central pair (CP) loss in sperm flagella of the patients. Mouse models (Dnah12−/−andDnah12mut/mut) were generated and recapitulated the reproductive defects in the patients. Noteworthy, DNAH12 deficiency did not show effects on cilium organization and function. Mechanistically, DNAH12 was confirmed to interact with two other IDA components DNALI1 and DNAH1, while disruption of DNAH12 leads to failed recruitment of DNALI1 and DNAH1 to IDAs and compromised sperm development. Furthermore, DNAH12 also interacts with radial spoke head proteins RSPH1, RSPH9, and DNAJB13 to regulate CP stability. Moreover, the infertility ofDnah12−/−mice could be overcome by intracytoplasmic sperm injection (ICSI) treatment. Collectively, DNAH12 plays a crucial role in the proper organization of axoneme in sperm flagella, but not cilia, by recruiting DNAH1 and DNALI1 in both humans and mice. These findings expand our comprehension of dynein component assembly in flagella and cilia and provide a valuable marker for genetic counseling and diagnosis of asthenoteratozoospermia in clinical practice.
MVP-VSASL: measuring MicroVascular Pulsatility using Velocity-Selective Arterial Spin Labeling
Authors: Chen Conan , Barnes Ryan A. , Bangen Katherine J. , Han Fei , Pfeuffer Josef , Wong Eric C. , Liu Thomas T. , Bolar Divya S.
Published: June 2024

1

Purpose

By leveraging the small-vessel specificity of velocity-selective arterial spin labeling (VSASL), we present a novel technique for measuring cerebral MicroVascular Pulsatility named MVP-VSASL.

Theory and Methods

We present a theoretical model relating the pulsatile, cerebral blood flow-driven VSASL signal to the microvascular pulsatility index (PI), a widely used metric for quantifying cardiac-dependent fluctuations. The model describes the dependence of PI on bolus durationτ(an adjustable VSASL sequence parameter) and provides guidance for selecting a value ofτthat maximizes the SNR of the PI measurement. The model predictions were assessed in humans using data acquired with retrospectively cardiac-gated VSASL sequences over a broad range ofτvalues. In vivo measurements were also used to demonstrate the feasibility of whole-brain voxel-wise PI mapping, assess intrasession repeatability of the PI measurement, and illustrate the potential of this method to explore an association with age.

Results

The theoretical model showed excellent agreement to the empirical data in a gray matter region of interest (average R2value of 0.898±0.107 across six subjects). We further showed excellent intrasession repeatability of the pulsatility measurement (ICC = 0.960,p<0.001) and the potential to characterize associations with age (r= 0.554,p= 0.021).

Conclusion

We have introduced a novel, VSASL-based cerebral microvascular pulsatility technique, which may facilitate investigation of cognitive disorders where damage to the microvasculature has been implicated.
Impact of increasing CD4 count threshold eligibility for antiretroviral therapy initiation on advanced HIV disease and tuberculosis prevalence and incidence in South Africa: an interrupted time series analysis
Authors: Asare Kwabena , Lewis Lara , van der Molen Johan , Sookrajh Yukteshwar , Khubone Thokozani , Ngwenya Thulani , Ntokozo Mkhize Siyabonga , Lessells Richard J , Naidoo Kogieleum , Sosibo Phelelani , Bottomley Christian , Garrett Nigel , Dorward Jienchi
Published: June 2024

Introduction

We investigated the impact of increasing CD4 count eligibility for antiretroviral-therapy (ART) initiation on advanced HIV and tuberculosis (TB) prevalence and incidence among people living with HIV (PLHIV) in South Africa.

Methods

We conducted an interrupted time series analysis with de-identified data of PLHIV aged ≥15 initiating ART between April-2012 and February-2020 at 65 primary healthcare clinics in KwaZulu-Natal, South Africa. Outcomes included monthly proportions of new ART initiators presenting with advanced HIV (CD4 count <200 cells/µl) and TB disease. We created a cohort of monthly ART initiators without TB and evaluated the cumulative incidence of TB within 12 months follow-up. We used segmented binomial regression models to estimate relative risks (RR) of outcomes, allowing for a step and slope change after expanding the ART initiation CD4 count eligibility from <350 to <500 cells/µl in January- 2015 and following Universal-Test-and-Treat (UTT) implementation in September-2016.

Results

Among 187,544 participants, median age was 32 (27-39), and 125,065 (66.7%) were female. After January-2015, risk of advanced HIV at initiation decreased by 24.5% (RR=0.745, 95%CI 0.690-0.800) and further reduced by 26.2% following UTT implementation (RR=0.738, 95%CI 0.688-0.788). Risk of TB at initiation also decreased by 28.7% after January-2015 (RR=0.713, 95%CI 0.644-0.782) and further decreased by 17.6% after UTT implementation (RR=0.824, 95%CI 0.703-0.945) but remained stable among initiators with advanced HIV. Among the incidence cohort, the risk of new TB decreased by 31.9% (RR=0.681, 95%CI 0.441-0.921) following UTT implementation. Among the incidence cohort with advanced HIV, there was weak evidence of a decrease in risk of new TB (RR=0.755, 95%CI 0.489-1.021), but it gradually decreased per month (slope change per month 9.7%, RR=0.903, 95%CI 0.872-0.934) following UTT implementation.

Conclusions

Our data supports the added benefit of decreased TB co-burden with expanded ART access. Early diagnosis and immediate linkage to care should be prioritised among PLHIV.
Coaching ribosome biogenesis from the nuclear periphery
Authors: Zhuang Yinyin , Guo Xiangfu , Razorenova Olga V. , Miles Christopher E. , Zhao Wenting , Shi Xiaoyu
Published: June 2024

Severe invagination of the nuclear envelope is a hallmark of cancers, aging, neurodegeneration, and infections. However, the outcomes of nuclear invagination remain unclear. This work identified a new function of nuclear invagination: regulating ribosome biogenesis. With expansion microscopy, we observed frequent physical contact between nuclear invaginations and nucleoli. Surprisingly, the higher the invagination curvature, the more ribosomal RNA and pre-ribosomes are made in the contacted nucleolus. By growing cells on nanopillars that generate nuclear invaginations with desired curvatures, we can increase and decrease ribosome biogenesis. Based on this causation, we repressed the ribosome levels in breast cancer and progeria cells by growing cells on low-curvature nanopillars, indicating that overactivated ribosome biogenesis can be rescued by reshaping nuclei. Mechanistically, high-curvature nuclear invaginations reduce heterochromatin and enrich nuclear pore complexes, which promote ribosome biogenesis. We anticipate that our findings will serve as a foundation for further studies on nuclear deformation.

Highlights

Nuclear invaginations regulate ribosome biogenesis by physically contacting nucleoli.

High-curvature nuclear tunnels increase ribosome biogenesis.

Nanopillars reduce ribosome biogenesis by transforming high-curvature nuclear invaginations to low-curvature ones.
Metabolic and transcriptomic analyses of nectaries reveal differences in the mechanism of nectar production between monocots (Ananas comosus) and dicots (Nicotiana tabacum)
Authors: Göttlinger Thomas , Pirritano Marcello , Simon Martin , Fuß Janina , Lohaus Gertrud
Published: June 2024

Background

Nectar is offered by numerous flowering plants to attract pollinators. To date, the production and secretion of nectar have been analyzed mainly in eudicots, particularly rosids such asArabidopsis. However, due to the enormous diversity of flowering plants, further research on other plant species, especially monocots, is needed.Ananas comosus(monocot) is an economically important species and ideal for such analyses because it produce easily accessible nectar in sufficient quantities. In addition, the analyses were also carried out withNicotiana tabacum(dicot, asterids) for comparison.

Results

We performed transcriptome sequencing (RNA-Seq) analyses of the nectaries ofAnanas comosusandNicotiana tabacum, to test whether mechanisms described for nectar production and secretion inArabidopsisare also present in these plant species. The focus of these analyses is on carbohydrate metabolism and transport (e.g., sucrose-phosphate synthases, invertases, sucrose synthases, SWEETs and further sugar transporters). In addition, the metabolites were analyzed in the nectar, nectaries and leaves of both plant species to address the question whether concentration gradients for different metabolites exist between the nectaries and nectar.

The nectar ofN. tabacumcontains large amounts of glucose, fructose and sucrose, and the sucrose concentration in the nectar appears to be similar to the sucrose concentration in the nectaries. Nectar production and secretion in this species closely resembles corresponding processes in some other dicots, including sucrose synthesis in nectaries and sucrose secretion by SWEET9.

The nectar ofA. comosusalso contains large amounts of glucose, fructose and sucrose and in this species the sucrose concentration in the nectar appears to be higher than the sucrose concentration in the nectaries. Furthermore, orthologs of SWEET9 appear to be generally absent inA. comosusand other monocots. Therefore, sucrose export by SWEETs from the nectaries into the nectar can be excluded, rather, other mechanisms, such as active sugar export or exocytosis, are more likely.

Conclusion

The mechanisms of nectar production and secretion inN. tabacumappear to be largely similar to those in other dicots, whereas in the monocotyledonous speciesA. comosus, different synthesis and transport processes are involved.
Skull bone marrow-derived immune cells infiltrate the damaged cortex and exhibit anti-inflammatory properties
Authors: Soliman Eman , Basso Erwin Kristobal Gudenschwager , Ju Jing , Willison Andrew , Theus Michelle H.
Published: June 2024

Identifying the origins and contributions of different immune cell populations following brain injury is crucial for understanding their roles in inflammation and tissue repair. This study investigated the infiltration and phenotypic characteristics of skull bone marrow-derived immune cells in the murine brain after TBI. We performed calvarium transplantation from GFP donor mice and subjected the recipients to controlled cortical impact (CCI) injury 14 days post-transplant. Confocal imaging at 3 days post-CCI revealed GFP+ calvarium-derived cells infiltrating the ipsilateral core lesional area, expressing CD45 and CD11b immune markers. These cells included neutrophil (Ly6G+) and monocyte (Ccr2+) identities. Calvarium-derived GFP+/Iba1+ monocyte/macrophages expressed the efferocytosis receptor MerTK and displayed engulfment of NeuN+ and caspase 3+ apoptotic cells. Phenotypic analysis showed that greater calvarium-derived monocyte/macrophages disproportionately express the anti-inflammatory arginase-1 marker than pro-inflammatory CD86. To differentiate the responses of blood- and calvarium-derived macrophages, we transplanted GFP calvarium skull bone into tdTomato bone marrow chimeric mice, then performed CCI injury 14 days post-transplant. Calvarium-derived GFP+ cells predominantly infiltrated the lesion boundary, while blood-derived TdTomato+ cells dispersed throughout the lesion and peri-lesion. Compared to calvarium-derived cells, more blood-derived cells expressed pro-inflammatory CD86 and displayed altered 3D morphologic traits. These findings uniquely demonstrate that skull bone-derived immune cells infiltrate the brain after injury and contribute to the neuroinflammatory milieu, representing a novel immune cell source that may be further investigated for their causal role in functional outcomes.
Network controllability analysis reveals the antiviral potential of Etravirine against Hepatitis E Virus infection
Authors: Ansari Shabnam , Sarmah Dipanka Tanu , Verma Rohit , Chandrasekar Kannan , Chatterjee Samrat , Surjit Milan
Published: June 2024

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in lower- and middle-income countries. HEV infection may lead to acute liver failure, chronic liver disease and high mortality in pregnant women. Antiviral therapy is not a standard treatment for HEV patients. Computational biology tools promise to revolutionize the antiviral drug discovery. Here, we analyzed the transcriptome data of HEV infected primary human hepatocyte (PHH)-cells through connectivity map database and applied control theory on functional network to identify antiviral targets against HEV. The above analyses predicted PKCβ, PKB/AKT and CK1ε as potential antiviral targets against HEV. The antiviral function of PKB/AKT and CK1ε was experimentally validated by using respective biochemical inhibitors in g3 (genotype 3)-HEV replicon and Huh7 cell-based model of g3 and g1-HEV infection. Further, knockdown of CK1ε showed a similar effect. These data confirmed that CK1ε is an antiviral target for HEV. At present, there are no FDA approved drugs targeting CK1ε. Etravirine is an FDA approved non-nucleoside reverse transcriptase inhibitor drug, used for the treatment of Human immunodeficiency virus type 1 (HIV-1) infected patients. Anin silicostudy predicted Etravirine to be a potent inhibitor of CK1ε. Our experiments revealed potent antiviral activity of Etravirine against HEV, which was mediated via its ability to inhibit the activity of CK1ε. Taken together, the current study demonstrates that PKB/AKT and CK1ε are bonafide antiviral targets for HEV and paves the way for repurposing Etravirine for the treatment of HEV infected patients.

Importance

Antiviral treatment is not the standard care for acute viral hepatitis E patients. Unbiased identification of antiviral targets or large-scale screening of antiviral compounds against the hepatitis E virus (HEV) has not been reported. Here, computational biology approach was followed to unbiasedly identify antiviral targets of HEV. Transcriptome data of HEV infected primary human hepatocyte (PHH) cells were analyzed to identify modulators of the network and generate directional networks. Network controllability analysis identified PKCβ, PKB/AKT and CK1ε as potential antiviral targets against HEV. Antiviral function of PKB/AKT and CK1ε was confirmed using cell-based models of genotype 1 (g1)- and g3-HEV infection. Further experiments demonstrated the antiviral activity of Etravirine against HEV, mediated via its ability to inhibit the CK1ε activity. Etravirine is an FDA approved non-nucleoside reverse transcriptase inhibitor, used for the treatment of Human immunodeficiency virus type-1 (HIV-1)-infected patients. This study reveals the potential of repurposing Etravirine for treatment of HEV patients and illustrate the importance of computational biology in antiviral drug discovery.
EPITHELIAL REMODELING AND MICROBIAL DYSBIOSIS IN THE LOWER RESPIRATORY TRACT OF VITAMIN A-DEFICIENT MOUSE LUNGS
Authors: Quiles Kiloni , Chen Felicia , Johnson William Evan
Published: June 2024

The World Health Organization identified vitamin A deficiency (VAD) as a major public health issue in low-income communities and developing countries, while additional studies have shown dietary VAD leads to various lung pathologies. Once believed to be sterile, research now shows that transient microbial communities exist within healthy lungs and are often dysregulated in patients suffering from malnourishment, respiratory infections, and disease. The inability to parse vitamin A-mediated mechanisms from other metabolic mechanisms in humans with pathogenic endotypes, as well as the lack of data investigating how VAD affects the lung microbiome, remains a significant gap in the field. To address this unmet need, we compared molecular, metatranscriptomic, and morphometric data to identify how dietary VAD affects the lung as well as the lung microbiome. Our research shows structural and functional alterations in host-microbe-diet interactions in VAD lungs compared to vitamin A-sufficient (VAS) lungs; these changes are associated with epithelial remodeling, a breakdown in mucociliary clearance, microbial imbalance, and altered microbial colonization patterns after 8 weeks of vitamin A deficient diet. These findings confirm vitamin A is critical for lung homeostasis and provide mechanistic insights that could be valuable for the prevention of respiratory infections and disease.
Bacteriophage Treatment against Carbapenem-resistantKlebsiella pneumoniae(KPC) in a Neutropenic Murine Model of Gastrointestinal Translocation and Renal infection
Authors: Zagaliotis Panagiotis , Michalik-Provasek Jordyn , Mavridou Eleftheria , Naing Ethan , Vizirianakis Ioannis S. , Chatzidimitriou Dimitrios , Gill Jason J. , Walsh Thomas J.
Published: June 2024

Background

Carbapenemase producingKlebsiella pneumoniae(KPC) are globally emerging pathogens which that cause life-threatening infections. Novel treatment alternatives are urgently needed.

Methods

We therefore investigated the effectiveness of three novel bacteriophages (Spivey, Pharr and Soft) in a neutropenic murine model of KPC gastrointestinal colonization, translocation, and disseminated infection. Bacteriophage efficacy was determined by residual bacterial burden of KPC in kidneys. Parallel studies were conducted of bacteriophage pharmacokinetics and resistance..

Results

Treatment of mice with 5×109PFU of phage cocktail via intraperitoneal injection was effective in significantly reducing renal KPC burden by 102CFU (p<0.01) when administered every 24 hours and 103CFU (p<0.01) every 12 hours. Moreover, a combination of bacteriophage and ceftazidime-avibactam produced a synergistic effect, resulting in a 105reduction in bacterial burden in caecum and kidney (p<0.001 in both tissues). Prophylactic administration of bacteriophages via oral gavage did not prevent KPC translocation to the kidneys. Bacteriophage decay determined by linear regression of the ln of mean concentrations demonstrated R2values in plasma of 0.941, kidney 0.976, and caecum 0.918, with half-lives of 2.5h < t1/2< 3.5 h. Furthermore, a phage-resistant mutant displayed increased sensitivity to serum killingin vitro, but did not show significant defects in renal infectionin vivo.

Conclusions

A combination of bacteriophages demonstrated significant efficacy alone and synergy with ceftazidime/avibactam in treatment of experimental disseminated KPC infection in neutropenic mice.
Precise measurement of molecular phenotypes with barcode-based CRISPRi systems
Authors: Lobel Joseph H. , Ingolia Nicholas T.
Published: June 2024

Genome-wide CRISPR-Cas9 screens have untangled regulatory networks and revealed the genetic underpinnings of diverse biological processes. Their success relies on experimental designs that interrogate specific molecular phenotypes and distinguish key regulators from background effects. Here, we realize these goals with a generalizable platform for CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) that dramatically improves the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely-matched control promoters, integrated together into the genome at single copy. To test our ability to capture post-transcriptional and post-translational regulation through sequencing, we screened for genes that affected nonsense-mediated mRNA decay and Doa10-mediated cytosolic protein decay. Our optimized CiBER-seq screens accurately capture the known components of well-studied RNA and protein quality control pathways with minimal background. These results demonstrate the precision and versatility of CiBER-seq for dissecting the genetic networks controlling cellular behaviors.
Structural basis of bulk lipid transfer by bridge-like lipid transfer protein LPD-3
Authors: Kang Yunsik , Lehmann Katherine S. , Vanegas Juan , Long Hannah , Jefferson Amanda , Freeman Marc , Clark Sarah
Published: June 2024

Bridge-like lipid transport proteins (BLTPs) are an evolutionarily conserved family of proteins that localize to membrane contact sites and are thought to mediate the bulk transfer of lipids from a donor membrane, typically the endoplasmic reticulum (ER), to an acceptor membrane, such as a that of the cell or an organelle1. Despite the fundamental importance of BLTPs for cellular function, the architecture, composition, and lipid transfer mechanisms remain poorly characterized. Here, we present the subunit composition and the cryo-electron microscopy structure of the native LPD-3 BLTP complex isolated from transgenicC. elegans. LPD-3 folds into an elongated, rod-shaped tunnel whose interior is filled with ordered lipid molecules that are coordinated by a track of ionizable residues that line one side of the tunnel. LPD-3 forms a complex with two previously uncharacterized proteins, here named “Intake” and “Spigot”, both of which interact with the N-terminal end of LPD-3 where lipids enter the tunnel. Intake has three transmembrane helices, one of which borders the entrance to the tunnel; Spigot has one transmembrane helix and extends 80 Å along the cytosolic surface of LPD-3. Experiments in multiple model systems indicate that Spigot plays a conserved role in ER-PM contact site formation. Our LPD-3 complex structural data, together with molecular dynamics simulations of the transmembrane region in a lipid bilayer, reveal protein-lipid interactions that suggest a model for how the native LPD-3-complex mediates bulk lipid transport and provide a foundation for mechanistic studies of BLTPs.
Conformational modulation of a mobile loop controls catalysis in the (βα)8-barrel enzyme of histidine biosynthesis HisF
Authors: Hupfeld Enrico , Schlee Sandra , Wurm Jan Philip , Rajendran Chitra , Yehorova Dariia , Vos Eva , Raju Dinesh Ravindra , Kamerlin Shina Caroline Lynn , Sprangers Remco , Sterner Reinhard
Published: June 2024

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site β1α1-loop (loop1) of the (βα)8-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase. Loop1 variants containing single mutations of conserved amino acids showed drastically reduced rates for the turnover of the substrates N'-[(5'-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PrFAR) and ammonia to the products imidazole glycerol phosphate (ImGP) and 5-aminoimidazole-4-carboxamide-ribotide (AICAR). A comprehensive mechanistic analysis including stopped-flow kinetics, X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations detected three conformations of loop1 (open, detached, closed) whose populations differed between wild-type HisF and functionally affected loop1 variants. Transient stopped-flow kinetic experiments demonstrated that wt-HisF binds PrFAR by an induced-fit mechanism whereas catalytically impaired loop1 variants bind PrFAR by a simple two-state mechanism. Our findings suggest that PrFAR-induced formation of the closed conformation of loop1 brings active site residues in a productive orientation for chemical turnover, which we show to be the rate-limiting step of HisF catalysis. After the cyclase reaction, the closed loop conformation is destabilized, which favors the formation of detached and open conformations and hence facilitates the release of the products ImGP and AICAR. Our data demonstrate how different conformations of active site loops contribute to different catalytic steps, a finding that is presumably of broad relevance for the reaction mechanisms of (βα)8-barrel enzymes and beyond.
People's expectations about the likely duration of common acute infections: a national survey of the Australian public
Authors: Boaitey Kwame Peprah , Bakhit Mina , Jones Mark , Hoffmann Tammy
Published: June 2024

Objectives To explore people's expectations about the likely duration of acute infections that are commonly managed in primary care, and if care is sought for these infections, reasons for doing so. Design A cross-sectional online survey. Participants A nationwide sample of 589 Australian residents, 18 years or older with representative quotas for age and gender, recruited via an online panel provider. Outcome measures For eight acute infections, participants' estimated duration of each, time until they would seek care, and reasons for seeking care. Results For four infections, participants mean estimates of duration were within an evidence-based range - common cold (7.2 days), sore throat (5.2 days), acute otitis media (6.2 days), and impetigo (8.3 days); and >70% of participants estimated a duration within the range. However, participants estimated mean duration was shorter than evidence-based estimates for acute cough (7.6 days), sinusitis (5.6 days), conjunctivitis (5.7 days), and uncomplicated urinary tract infections (5.4 days); and >60% of participants underestimated the duration. Of the 589 participants, 365 (62%) indicated they were unlikely to routinely seek care for self-limiting infections. Most common reasons for care-seeking were severe or worsening symptoms, a desire for quick recovery, and fear of progression to complications. After being shown typical durations, the proportion of participants who reported having no concerns waiting for spontaneous resolution while managing symptoms with over-the-counter medications ranged across the infections and was highest for common cold (68%) and lowest for UTI (31%). Conclusion Participants underestimated the duration of some infections compared to evidence-based estimates and were accurate in their estimates for other infections. Many stated that they would not be concerned about waiting for illnesses to self-resolve after learning the typical duration. Communicating the expected duration of common acute infections during routine consultations can help manage patients' expectations of recovery and need to seek care.