bluuebunny/crossref_metadata_2025 huggingface dataset:
SARS-CoV-2 infection studies in lung organoids identify TSPAN8 as novel mediator
Authors: Hysenaj Lisiena , Little Samantha , Kulhanek Kayla , Gbenedio Oghenekevwe M. , Rodriguez Lauren , Shen Alan , Lone Jean-Christophe , Lupin-Jimenez Leonard C. , Bonser Luke R. , Serwas Nina K. , Bahl Kriti , Mick Eran , Li Jack Z. , Ding Vivianne W. , Matsumoto Shotaro , Maishan Mazharul , Simoneau Camille , Fragiadakis Gabriela , Jablons David M. , Langelier Charles R. , Matthay Michael , Ott Melanie , Krummel Matthew , Combes Alexis J. , Sil Anita , Erle David J. , Kratz Johannes R. , Roose Jeroen P.
Published: June 2021

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2-positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.
Hierarchical Harmonization of Atom-Resolved Metabolic Reactions Across Metabolic Databases
Authors: Jin Huan , Moseley Hunter N.
Published: June 2021

Metabolic models have been proven to be useful tools in system biology and have been successfully applied to various research fields in a wide range of organisms. A relatively complete metabolic network is a prerequisite for deriving reliable metabolic models. The first step in constructing metabolic network is to harmonize compounds and reactions across different metabolic databases. However, effectively integrating data from various sources still remains a big challenge. Incomplete and inconsistent atomistic details in compound representations across databases is a very important limiting factor. Here, we optimized a subgraph isomorphism detection algorithm to validate generic compound pairs. Moreover, we defined a set of harmonization relationship types between compounds to deal with inconsistent chemical details while successfully capturing atom-level characteristics, enabling a more complete enabling compound harmonization across metabolic databases. In total, 15,704 compound pairs across KEGG (Kyoto Encyclopedia of Genes and Genomes) and MetaCyc databases were detected. Furthermore, utilizing the classification of compound pairs and EC (Enzyme Commission) numbers of reactions, we established hierarchical relationships between metabolic reactions, enabling the harmonization of 3,856 reaction pairs. In addition, we created and used atom-specific identifiers to evaluate the consistency of atom mappings within and between harmonized reactions, detecting some consistency issues between the reaction and compound descriptions in these metabolic databases.
SARS-CoV-2 Infects Syncytiotrophoblast and Activates Inflammatory Responses in the Placenta
Authors: Argueta Lissenya B. , Lacko Lauretta A. , Bram Yaron , Tada Takuya , Carrau Lucia , Zhang Tuo , Uhl Skyler , Lubor Brienne C. , Chandar Vasuretha , Gil Cristianel , Zhang Wei , Dodson Brittany , Bastiaans Jeroen , Prabhu Malavika , Salvatore Christine M. , Yang Yawei J. , Baergen Rebecca N. , tenOever Benjamin R. , Landau Nathaniel R. , Chen Shuibing , Schwartz Robert E. , Stuhlmann Heidi
Published: June 2021

SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested nasal swab positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 54 placentas. Two placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and in rare events endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.
Neural asymmetry during memory encoding and its association with markers of preclinical Alzheimer’s Disease
Authors: Li Jinghang , Mountz Elizabeth J. , Mizuno Akiko , Shah Ashti M , Weinstein Andrea , Cohen Ann D. , Klunk William E. , Snitz Beth E. , Aizenstein Howard J. , Karim Helmet T.
Published: June 2021

Background

Alzheimer’s Disease (AD) is the most common form of dementia and is characterized by cognitive dysfunction that impacts daily functioning. Beta-amyloid (Aβ) is a cytotoxic protein that deposits in the brain many years prior to the onset of cognitive dysfunction. The preclinical period is a stage of AD in which significant pathology is present without clinical symptoms. Aβ has been shown to deposit asymmetrically early in the AD trajectory, which has shown to have functional consequences (e.g., asymmetric hypometabolism). We aimed to investigate whether markers of AD and cognitive function were correlated with neural activation asymmetry during memory encoding tasks.

Methods

We recruited participants who were cognitively normal to undergo functional magnetic resonance imaging (fMRI), positron emission tomography (PET) imaging, and cognitive testing. We conducted analyses to identify regions of significant activation during a well-established face-name pair memory encoding task, and to identify regions of significant asymmetry. We then computed hemispheric asymmetry (negative/positive values indicate left/right asymmetry, respectively) and absolute asymmetry (greater values indicate greater asymmetry in either hemisphere) and investigated their associations with age, sex, education, global cerebral amyloid, global cerebral metabolism, memory encoding task performance, white matter hyperintensities, and multiple domains of cognitive function.

Results

We identified expected regions of significant activation, including the hippocampus, and identified four regions with significant left-hemisphere asymmetry: superior medial frontal gyrus, middle frontal gyrus, supplemental motor area, and medial orbitofrontal gyrus, and two regions with significant right hemisphere asymmetry: putamen and ventral posterolateral nucleus of the thalamus. We found that greater left-hemisphere asymmetry in the middle frontal gyrus was correlated with greater global cerebral glucose metabolism. We also found that better performance in memory, learning, and executive attention was associated with greater absolute symmetry in the thalamus, while better visuospatial performance was associated with greater putamen absolute symmetry.

Discussion

Functional asymmetry is correlated with functional markers (e.g., glucose metabolism) in older cognitively normal adults and may reflect metabolic and cognitive changes. Longitudinal studies may help us better understand these associations and the causal impact of neural activation asymmetry.
Affinity tag coating enables reliable detection of antigen-specific B cells in ImmunoSpot assays
Authors: Köppert Sebastian , Wolf Carla , Becza Noémi , Sautto Giuseppe A. , Franke Fridolin , Kürten Stefanie , Ross Ted M. , Lehmann Paul V. , Kirchenbaum Greg A.
Published: June 2021

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibody in the serum. Rarely does immune monitoring entail assessment of the memory B cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their precursory frequency, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally-suited for antigen-specific B cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen coating approach streamlines characterization of the memory B cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune monitoring efforts of large donor cohorts in general.
EPIDEMIOLOGY OF MAXILLOFACIAL INJURIES IN “HERATSI” NO 1 UNIVERSITY HOSPITAL IN YEREVAN, ARMENIA: A RETROSPECTIVE STUDY
Authors: Aleksanyan Lusine V. , Poghosyan Anna Yu. , Misakyan Martin S. , Minasyan Armen M. , Bablumyan Aren Yu. , Tadevosyan Artashes E. , Muradyan Armen A.
Published: June 2021

Objective

The aim of this study was to perform a retrospective analysis of the prevalence, etiologies, and types of maxillofacial injuries (MFI) and sites of maxillofacial fractures (MFF) and their management in the Department of ENT and Maxillofacial Surgery of <> No. 1 University Hospital in Yerevan, Armenia.

Material and methods

A retrospective cross-sectional study was conducted. Data including age, sex, date of referral, mode of injury, etiology, radiology records and treatment methods were extracted. Study outcomes were measured using percentages, means, standard deviations and tests of proportions. P <.05 was considered significant.

Results

A total of 204 patients had a mean age of 36.26 ±1.08 years (156 males and 48 females), and a total of 259 MFIs were recorded between 2017 and 2020. Interpersonal violence was found to be the most common etiology of MFFs in this study (42.1%), followed by road traffic accidents (RTAs) (27.9%) and falls (18.6%). The nasal bone was the most common injury site (47.5%), followed by the mandible (31.4%) and zygomatic complex (11.7%). The most common fracture site was the mandibular angle (37.9%), followed by the symphysis/parasymphysis (28.1%) and body (12.6%). The majority of MFFs were treated by open reduction and internal fixation.

Conclusion

Interpersonal violence, followed by RTAs and falls, was the most common cause of MFIs. The nasal bone was the most common injury site, followed by the mandible and zygomatic complex. Social education with the objective of reducing aggression and interpersonal conflict should be improved, and appropriate RTA prevention strategies should be strengthened and implemented.

KEY MESSAGES

What is already known on this subject

Traumatic injuries continue to be important causes of morbidity and mortality in both developed and developing countries.

The incidence rates, etiologies, types, and injuries associated with MFFs vary among different countries.

RTAs are the most frequent cause of MFIs in developing countries, and in contrast, the most frequent cause of MFFs in developed countries is IV or assault

What this study adds

Interpersonal violence was found to be the most common etiology of MFF in this study, followed by RTAs and falls.

Patients aged 21-30 years were likely to have sustained nasal bone fractures and mandible fractures in equal proportions.

The nasal bones were found to be the most common injury site, followed by the mandible and zygomatic complex.
Whole blood-based measurement of SARS-CoV-2-specific T cell responses reveals asymptomatic infection and vaccine efficacy in healthy subjects and patients with solid organ cancers
Authors: Scurr Martin J. , Zelek Wioleta M. , Lippiatt George , Somerville Michelle , Burnell Stephanie E. A. , Capitani Lorenzo , Davies Kate , Lawton Helen , Tozer Thomas , Rees Tara , Roberts Kerry , Evans Mererid , Jackson Amanda , Young Charlotte , Fairclough Lucy , Wills Mark , Westwell Andrew D. , Morgan B. Paul , Gallimore Awen , Godkin Andrew
Published: June 2021

Accurate assessment of SARS-CoV-2 immunity in the population is critical to evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS-CoV-2-specific T cell responses are a critical feature of the immune response that will likely form a key correlate of protection against COVID-19. Here, we developed and optimised a high-throughput whole blood-based assay to determine the T cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 156 healthy donors and 67 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were harvested and analysed for TH1-type effector cytokines (IFN-γ and IL-2). Amongst healthy donors, highly significant differential IFN-γ+/IL-2+ SARS-CoV-2-specific T cell responses were seen amongst vaccinated or previously infected COVID-19-positive individuals in comparison to unknown/naïve individuals (P < 0.0001). IL-2 production from T cells in response to SARS-CoV-2 derived antigens was a highly predictive diagnostic assay (P < 0.0001; 96.0% sensitivity, 93.9% specificity); measurement of IFN-γ+ SARS-CoV-2 specific T cell responses was equally effective at identifying asymptomatic (antibody and T cell positive) participants. A single dose of COVID-19 vaccine induced IFN-γ and/or IL-2 SARS-CoV-2-specific T cell responses in 28/29 (96.6%) of healthy donors, reducing significantly to 27/56 (48.2%) when measured in cancer patients (P = 0.0003). Overall, this cost-effective standardisable test ensures accurate and comparable assessments of SARS-CoV-2-specific T cell responses amenable to widespread population immunity testing.
Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment
Authors: Maes Margaret E , Wögenstein Gabriele M , Colombo Gloria , Casado-Polanco Raquel , Siegert Sandra
Published: June 2021

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette.Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.
The REF6-dependent H3K27 demethylation establishes transcriptional competence to promote germination in Arabidopsis
Authors: Pan Jie , Zhang Huairen , Zhan Zhenping , Zhao Ting , Jiang Danhua
Published: June 2021

Seed germination is a critical developmental switch from a dormant state to active growth, which involves extensive changes in metabolism, gene expression and cellular identity. However, our understanding of epigenetic and transcriptional reprogramming during this process is limited. The histone H3 lysine 27 trimethylation (H3K27me3) plays a key role in regulating gene repression and cell fate specification. Here, we profile H3K27me3 dynamics and dissect the function of H3K27 demethylation during germination. Our temporal genome-wide profiling of H3K27me3 and transcription reveal delayed H3K27me3 reprogramming compared with transcriptomic changes during germination, with H3K27me3 changes mainly occurring when the embryo is entering into vegetative development. REF6-mediated H3K27 demethylation promotes germination but does not significantly contribute to H3K27me3 dynamics during germination, but rather stably establishes an H3K27me3-depleted state permissive to transcription. By analyzing REF6 genomic binding, we show that it is absent from mature embryo chromatin and gradually establishes occupancy during the course of germination to counteract increased PRC2 activity. Our study provides key insights into the dynamics of gene expression and H3K27me3 during seed germination and suggests the function of H3K27me3 in facilitating cell fate switch. Furthermore, we reveal the importance of H3K27 demethylation-established transcriptional competence in germination and likely other developmental processes.
The Hidden Side of Diversity: Effects of Imperfect Detection on Multiple Dimensions of Biodiversity
Authors: Richter Aline , Nakamura Gabriel , Iserhard Cristiano Agra , da Silva Duarte Leandro
Published: June 2021

Studies on ecological communities often address patterns of species distribution and abundance, but few consider uncertainty in counts of both species and individuals when computing diversity measures.

We evaluated the extent to which imperfect detection may influence patterns of taxonomic, functional, and phylogenetic diversity in ecological communities.

We estimated the true abundance of fruit-feeding butterflies sampled in canopy and understory strata in a subtropical forest. We compared the diversity values calculated by observed and estimated abundance data through the hidden diversity framework. This framework evaluates the deviation of observed diversity when compared with diversities derived from estimated true abundances, and if such deviation represents a bias or a noise in the observed diversity pattern.

The hidden diversity values differed between strata for all diversity measures, except for functional richness. The taxonomic measure was the only one where we observed an inversion of the most diverse stratum when imperfect detection was included. Regarding phylogenetic and functional measures, the strata showed distinct responses to imperfect detection, despite the tendency to overestimate observed diversity. While the understory showed noise for the phylogenetic measure, since the observed pattern was maintained, the canopy had biased diversity for the functional metric. This bias occurred since no significant differences were found between strata for observed diversity, but rather for estimated diversity, with the canopy being more clustered.

We demonstrate that ignore imperfect detection may lead to unrealistic estimates of diversity and hence, to erroneous interpretations of patterns and processes that structure biological communities. For fruit-feeding butterflies, according to their phylogenetic position or functional traits, the undetected individuals triggered different responses in the relationship of the diversity measures to the environmental factor. This highlights the importance to evaluate and include the uncertainty in species detectability before calculating biodiversity measures to describe communities.
Resource and seasonality drive interspecific variability in a Dynamic Energy Budget model
Authors: Mariño Joany , Dufour Suzanne C. , Hurford Amy , Récapet Charlotte
Published: June 2021

Animals show a vast array of phenotypic traits in time and space. These variation patterns have traditionally been described as ecogeographical rules; for example, the tendency of size and clutch size to increase with latitude (Bergman’s and Lack’s rules, respectively). Despite considerable research into these patterns, the processes behind trait variation remain controversial. Here, we show how food variability, which determines individual energy input and allocation trade-offs, can drive interspecific trait variation. Using a dynamic energy budget (DEB) model, we simulated different food environments as well as interspecific variability in the parameters for energy assimilation, mobilization, and allocation to soma. We found that interspecific variability is greater when the resource is non-limiting in both constant and seasonal environments. Our findings further show that individuals can reach larger biomass and greater reproductive output in a seasonal environment than in a constant environment of equal average resource due to the peaks of food surplus. Our results agree with the classical patterns of interspecific trait variation and provide a mechanistic understanding that supports recent hypotheses which explain them: the resource and the eNPP (net primary production during the growing season) rules. Due to the current alterations to ecosystems and communities, disentangling trait variation is increasingly important to understand and predict biodiversity dynamics under environmental change.
Transposable element accumulation drives size differences among polymorphic Y chromosomes in Drosophila
Authors: Nguyen Alison H. , Bachtrog Doris
Published: June 2021

Y chromosomes of many species are gene poor and show low levels of nucleotide variation, yet often display high amounts of structural diversity. Dobzhansky cataloged several morphologically distinct Y chromosomes inDrosophila pseudoobscurathat differ in size and shape, but the molecular causes of their dramatic size differences are unclear. Here we use cytogenetics and long-read sequencing to study the sequence content of polymorphic Y chromosomes inD. pseudoobscura. We show that Y chromosomes differ by almost 2-fold in size, ranging from 30 to 60 Mb. Most of this size difference is caused by a handful of active transposable elements (TEs) that have recently expanded on the largest Y chromosome, with different elements being responsible for Y expansion on differently sizedD. pseudoobscuraY’s. We show that Y chromosomes differ in their heterochromatin enrichment, expression of Y-enriched TEs, and also influence expression of dozens of autosomal and X-linked genes. Intriguingly, the same helitron element that showed the most drastic amplification on the largest Y inD. pseudoobscuraindependently amplified on a polymorphic large Y chromosome inD. affinis, suggesting that some TEs are inherently more prone to become deregulated on Y chromosomes.
Gene regulatory networks underlying human microglia maturation
Authors: Han Claudia Z. , Li Rick Z. , Hansen Emily , Bennett Hunter R. , Poirion Olivier , Buchanan Justin , Challacombe Jean F. , Fixsen Bethany R. , Trescott Samantha , Schlachetzki Johannes C.M. , Preissl Sebastian , Wang Allen , O’Connor Carolyn , Warden Anna S. , Shriram Shreya , Kim Roy , Nguyen Celina T. , Schafer Danielle M. , Ramirez Gabriela , Anavim Samuel A. , Johnson Avalon , Sajti Eniko , Gupta Mihir , Levy Michael L. , Ben-Haim Sharona , Gonda David D. , Laurent Louise , Glass Christopher K. , Coufal Nicole G.
Published: June 2021

The fetal period is a critical time for brain development, characterized by neurogenesis, neural migration, and synaptogenesis1-3. Microglia, the tissue resident macrophages of the brain, are observed as early as the fourth week of gestation4 and are thought to engage in a variety of processes essential for brain development and homeostasis5-11. Conversely, microglia phenotypes are highly regulated by the brain environment12-14. Mechanisms by which human brain development influences the maturation of microglia and microglia potential contribution to neurodevelopmental disorders remain poorly understood. Here, we performed transcriptomic analysis of human fetal and postnatal microglia and corresponding cortical tissue to define age-specific brain environmental factors that may drive microglia phenotypes. Comparative analysis of open chromatin profiles using bulk and single-cell methods in conjunction with a new computational approach that integrates epigenomic and single-cell RNA-seq data allowed decoding of cellular heterogeneity with inference of subtype- and development stage-specific transcriptional regulators. Interrogation of in vivo and in vitro iPSC-derived microglia models provides evidence for roles of putative instructive signals and downstream gene regulatory networks which establish human-specific fetal and postnatal microglia gene expression programs and potentially contribute to neurodevelopmental disorders.
Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids
Authors: Criss Zachary K. , Bhasin Nobel , Di Rienzi Sara C. , Rajan Anubama , Deans-Fielder Kali , Swaminathan Ganesh , Kamyabi Nabiollah , Zeng Xi-Lei , Chakravarti Deepavali , Estrella Clarissa , Yu Xiaomin , Patil Ketki , Fleet James C. , Verzi Michael P. , Christakos Sylvia , Helmrath Michael A. , Arimura Sumimasa , DePinho Ronald A. , Britton Robert , Maresso Anthony , Grande-Allen Jane , Blutt Sarah E. , Crawford Sue E. , Estes Mary K. , Ramani Sasirekha , Shroyer Noah F.
Published: June 2021

Background & Aims

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity.

Methods

RNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways.

Results

PERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures.

Conclusions

Surprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.
Single-Cell Transcriptional Profiling of the Adult Corticospinal Tract Reveals Forelimb and Hindlimb Molecular Specialization
Authors: Golan Noa , Kauer Sierra , Ehrlich Daniel B , Ravindra Neal , van Dijk David , Cafferty William BJ
Published: June 2021

The corticospinal tract (CST) is refractory to repair after CNS trauma, resulting in chronic debilitating functional motor deficits after spinal cord injury. While novel pro-axon growth activators have stimulated plasticity and regeneration of corticospinal neurons (CSNs) after injury, robust functional recovery remains elusive. These repair strategies are sub-optimal in part due to underexplored molecular heterogeneity within the developing and adult CST. In this study, we combine retrograde CST tracing with single-cell RNA sequencing to build a comprehensive atlas of CSN subtypes. By comparing CSNs to non-spinally projecting neurons in layer Vb, we identify pan-CSN markers including Wnt7b. By leveraging retrograde tracing, we are able to compare forelimb and hindlimb projecting CSNs, identifying subtype-specific markers, including Cacng7 and Slc16a2 respectively. These markers are expressed in embryonic and neonatal CSNs and can be used to study early postnatal patterning of the CST. Our results provide molecular insight into the differences between anatomically distinct CSN subtypes and provide a resource for future screening and exploitation of these subtypes to repair the damaged CST after injury and disease.
Ex vivo infection of human skin with herpes simplex virus 1 reveals mechanical wounds as insufficient entry portals via the skin surface
Authors: De La Cruz Nydia , Möckel Maureen , Wirtz Lisa , Sunaoglu Katharina , Malter Wolfram , Zinser Max , Knebel-Mörsdorf Dagmar
Published: June 2021

Herpes simplex virus 1 (HSV-1) enters its human host via the skin or mucosa. The open question is how the virus invades this highly protective tissue in vivo to approach its receptors in the epidermis and initiate infection. Here, we performed ex vivo infection studies in human skin to investigate how susceptible the epidermis and dermis are to HSV-1 and whether wounding facilitates viral invasion. Upon ex vivo infection of complete skin, only sample edges demonstrated infected cells. After removal of the dermis, HSV-1 efficiently invaded the basal layer, and from there, gained access to suprabasal layers supporting a high susceptibility of the epidermis. In contrast, only single infected cells were detected in the papillary layer of the separated dermis. Interestingly, after wounding, nearly no infection of the epidermis was observed via the skin surface. However, if the wounding of the skin samples led to breaks through the dermis, HSV-1 infected mainly keratinocytes via the wounded dermis. The application of latex beads revealed only occasional entry via the wounded dermis, however, facilitated penetration via the wounded skin surface. Thus, we suggest that the wounded human skin surface allows particle penetration but still provides barriers that prevent HSV-1 invasion.
A female-specific role for Calcitonin Gene-Related Peptide (CGRP) in rodent pain models
Authors: Paige Candler , Plasencia-Fernandez Isabel , Kume Moeno , Papalampropoulou-Tsiridou Melina , Lorenzo Louis-Etienne , Mejia Galo L. , Driskill Christopher , Ferrini Francesco , Feldhaus Andrew L. , Garcia-Martinez Leon F. , Akopian Armen N. , De Koninck Yves , Dussor Gregory , Price Theodore J.
Published: June 2021

We aimed to investigate a potentially sexually dimorphic role of Calcitonin Gene-Related Peptide (CGRP) in mouse and rat models of pain. Based on findings in migraine where CGRP has a preferential pain-promoting effect in female rodents, we hypothesized that CGRP antagonists and antibodies would attenuate pain sensitization more efficaciously in female than male mice and rats. In hyperalgesic priming induced by activation of interleukin 6 (IL-6) signaling, CGRP receptor antagonists, olcegepant and CGRP8-37, both given intrathecally, blocked and reversed hyperalgesic priming only in females. A monoclonal antibody against CGRP, given systemically, blocked priming specifically in female rodents but failed to reverse it. In the spared nerve injury (SNI) model, there was a transient effect of both CGRP antagonists, given intrathecally, on mechanical hypersensitivity in female mice only. Consistent with these findings, intrathecally applied CGRP caused a long-lasting, dose-dependent mechanical hypersensitivity in female mice but more transient effects in males. This CGRP-induced mechanical hypersensitivity was reversed by the KCC2 activator, CLP257 suggesting a role for anionic plasticity in the dorsal horn in the pain-promoting effects of CGRP in females. In spinal dorsal horn slices, CGRP shifted GABAA reversal potentials to significantly more positive values but, again, only in female mice. Therefore, CGRP may regulate KCC2 expression and/or activity specifically in females. However, KCC2 hypofunction promotes mechanical pain hypersensitivity in both sexes because CLP257 alleviated hyperalgesic priming in male and female mice. We conclude that CGRP promotes pain plasticity in female mice, but has a limited impact in male mice.

Significance Statement

The majority of patients impacted by chronic pain are women. Mechanistic studies in rodents are creating a clear picture that molecular events promoting chronic pain are different in male and female animals. Far more is known about chronic pain mechanisms in male animals. We sought to build on recent evidence showing that CGRP is a more potent and efficacious promoter of headache pain in female than in male rodents. To test this, we used hyperalgesic priming and the spared nerve injury (SNI) neuropathic pain models in mice. Our findings show a clear sex dimorphism wherein CGRP promotes pain in female but not male mice. Our work suggests that CGRP antagonists could be tested for efficacy in women for a broader variety of pain conditions.
Why Blood Pressure and Body Mass Should be Controlled for in Resting-State Functional Magnetic Resonance Imaging Studies
Authors: Sjuls Guro Stensby , Specht Karsten
Published: June 2021

Replicability has become an increasing focus within the scientific communities with the ongoing “replication crisis”. One area that appears to struggle with unreliable results is resting-state functional magnetic resonance imaging (rs-fMRI). Therefore, the current study aimed to improve the knowledge of endogenous factors that contribute to inter-individual variability. Arterial blood pressure, body mass, hematocrit, and glycated hemoglobin were investigated as potential sources of between-subject variability in rs-fMRI, in healthy individuals. Whether changes in resting state-networks (rs-networks) could be attributed to variability in the BOLD-signal, changes in neuronal activity, or both, was of special interest. Within-subject parameters were estimated utilizing Dynamic Causal Modelling (DCM) as it allows to make inferences on the estimated hemodynamic (BOLD-signal dynamics) and neuronal parameters (effective connectivity) separately. The results of the analyses imply that blood pressure and body mass can cause between-subject and between-group variability in the BOLD-signal and that all the included factors can affect the underlying connectivity. Given the results of the current and previous studies, rs-fMRI results appear to be susceptible to a range of factors, which is likely to contribute to the low degree of replicability of these studies. Interestingly, the highest degree of variability seems to appear within the much-studied Default Mode Network and its connections to other networks.
PyPlr: A versatile, integrated system of hardware and software for researching the human pupillary light reflex
Authors: Martin Joel T. , Pinto Joana , Bulte Daniel , Spitschan Manuel
Published: June 2021

We introduce PyPlr—a versatile, integrated system of hardware and software to support a broad spectrum of research applications concerning the human pupillary light reflex (PLR). PyPlr is a custom Python library for integrating a research-grade video-based eye-tracker system with a light source and streamlining stimulus design, optimisation and delivery, device synchronisation, and extraction, cleaning, and analysis of pupil data. We additionally describe how full-field, homogenous stimulation of the retina can be realised with a low-cost integrating sphere that serves as an alternative to a more complex Maxwellian view setup. Users can integrate their own light source, but we provide full native software support for a high-end, commercial research-grade 10-primary light engine that offers advanced control over the temporal and spectral properties of light stimuli as well as spectral calibration utilities. Here, we describe the hardware and software in detail and demonstrate its capabilities with two example applications: 1) pupillometer-style measurement and parametrisation of the PLR to flashes of white light, and 2) comparing the post-illumination pupil response (PIPR) to flashes of long and short-wavelength light. The system holds promise for researchers who would favour a flexible approach to studying the PLR and the ability to employ a wide range of temporally and spectrally varying stimuli, including simple narrowband stimuli.
EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes
Authors: Koester Anna M. , Laidlaw Kamilla M. , Morris Silke , Cutiongco Marie F.A. , Stirrat Laura , Gadegaard Nikolaj , Boles Eckhard , Black Hannah L. , Bryant Nia J. , Gould Gwyn W.
Published: June 2021

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.
Automated ChIPmentation procedure on limited biological material of the human blood fluke Schistosoma mansoni
Authors: Lasica Chrystelle , de Carvalho Augusto Ronaldo , Mone Helene , Mouahid Gabriel , Chaparro Cristian , Veillard Anne-Clemence , Zelisko-Schmidt Agnieszka , Grunau Christoph
Published: June 2021

Automated ChIPmentation procedure is a convenient alternative to native chromatin immunoprecipitation (N-ChIP). It is now routinely used for ChIP-Seq. Using the human parasite Schistosoma mansoni, whose production requires scarifying animals and should therefore kept to a minimum, we show here that the automated ChIPmentation is suitable for limited biological material. We define as operational limit ≥20,000 cells. We also present a streamlined protocol for the preparation of ChIP input libraries.
Deep-rooted plant species recruit distinct bacterial communities in 3 m deep subsoil
Authors: Bak Frederik , Lyhne-Kjærbye Annemette , Tardif Stacie , Dresbøll Dorte Bodin , Nicolaisen Mette H.
Published: June 2021

Deep-rooted plants can obtain water and nutrients from the subsurface, making them more resilient to climatic changes such as drought. In addition, the deeper root network also allow the plants to recruit bacteria from a larger reservoir in the soil. These bacteria might contribute to nutrient acquisition and provide other plant beneficial traits to the plant. However, the deep rhizosphere communities’ compositions and their assembly dynamics are unknown. Here, we show, using three perennial crops, Kernza, lucerne and rosinweed, grown in 4 m RootTowers, that deep rhizosphere bacterial communities are plant specific, but clearly distinct from the shallow communities. We found that the diversity decreased with depth in the rhizosphere, whereas abundance of 16S rRNA gene copies did not change with depth in lucerne and rosinweed. Furthermore, we identified a subgroup (4-8%) of ASVs in the rhizosphere communities that could not be retrieved in the corresponding bulk soil communities. The abundances of genes determined by qPCR involved in N-cycling: amoA, nifH, nirK, nirS and nosZ differed significantly between plant species, suggesting differences in N content in the root exudates of the plant species. Our results suggest that colonization of the rhizosphere by bulk soil bacteria is not limited by carbon supply, but rather by dispersal. Furthermore, the abundance of N cycling genes indicate that deep rhizosphere bacteria have the potential to provide N through nitrogen fixation.
Phytochemical Investigation of the Crude and Fractionated Extracts of two Nigerian Herbs, Mitragyna Inermis (Wild) and Lawsonia Inermis (Linn)
Authors: Onuh O.A. , Odugbo M. , Oladipo O.O. , Olobayotan I.W.
Published: June 2021

Investigation was done to determine the phytochemicals present in the crude extracts and fractions of Mitragyna inermis (Wild) of the family, Rubiceae and Lawsonia inermis (Linn) of the family, Lythraceae- Loose - Strife using standard protocols to ascertain their bioactive molecules of pharmaceutical potentials. Successive extraction of the plant parts was carried out using hexane, ethyl acetate, methanol and water (aqueous). Eleven fractions were eluted from both plants using column and thin-layer chromatography. The study revealed the presence of alkaloids, flavonoids, saponins, phenols, tannins, steroids and carbohydrates in both plants while anthraquinone was present in only L. inermis. Quantitative study of some of the phytochemicals revealed various percentages ranging from 0.56% (flavonoids) to 21.58% (saponins). Alkaloids and phenols were present in more fractions (M6 – M11) of M. inermis extracts while tannins and anthraquinones were present in more fractions (L6 – L11) of L. inermis extracts than in other fractions. The phytochemicals present in these two plants could be responsible for their various therapeutic uses that have been reported. It is recommended that further research is done to isolate these bioactive molecules in their pure forms and evaluate the dosage required and toxicity levels.
Functional Human Genes Typically Exhibit Epigenetic Conservation
Authors: Rud Daniel , Marjoram Paul , Siegmund Kimberly , Shibata Darryl
Published: June 2021

Recent DepMap CRISPR-Cas9 single gene disruptions have identified genes more essential to proliferation in tissue culture. It would be valuable to translate these finding with measurements more practical for human tissues. Here we show that DepMap essential genes and other literature curated functional genes exhibit cell-specific preferential epigenetic conservation when DNA methylation measurements are compared between replicate cell lines and between intestinal crypts from the same individual. Culture experiments indicate that epigenetic drift accumulates through time with smaller differences in more functional genes. In NCI-60 cell lines, greater targeted gene conservation correlated with greater drug sensitivity. These studies indicate that two measurements separated in time allow normal or neoplastic cells to signal through conservation which human genes are more essential to their survival in vitro or in vivo.
Prediction of pancreatic adenocarcinoma patient risk status using alternative splicing events
Authors: Kumar Rajesh , Lathwal Anjali , Raghava Gajendra P.S.
Published: June 2021

In literature, several mRNA, miRNA, lncRNA based biomarkers are identified by genomic analysis to stratify the patients into high and low risk groups of pancreatic adenocarcinoma (PAAD). The identified biomarkers are of limited use in terms of sensitivity and prediction ability. Thus, we aimed to identify the prognostic alternative splicing events and their related mutations in the PAAD. PAAD splicing data of 174 samples (17874 AS events in 6209 genes) and corresponding clinical information was obtained from the SpliceSeq and The Cancer Genome Atlas (TCGA), respectively. Prognostic-index based modeling was used to obtain the best predictive models for the seven AS types. However, model based on multiple spliced events genes (APP; LATS1; MRPL4; LAS1L; STARD10; PHF21A; NMRAL1) outperformed the single event models with a remarkable HR of 9.13 (p-value = 6.42e-10) as well as other existing models. Results from g:Profiler suggest that transcription factors ZF5, ER81, E2F-1/2/3, ER81, Erg, and PEA3 are most related to the prognostic spliced genes. We also identified 565 mutations across 160 spliced genes that have a strong association with the prognostic AS events. The analysis revealed that around 560 of these mutations were not reported before in context to splice event/region. Overall, we conclude that altered AS events may serve as strong indicators for overall survival in pancreatic cancer patients, and novel linkage of the known mutations to the survival-related AS events may provide a new dimension to the advancement of diagnostic and therapeutic interventions in these patients.
Disinfection performance of a drinking water bottle system with a UVC LED cap against waterborne pathogens and heterotrophic contaminants
Authors: Mariita Richard M. , Blumenstein Sébastien A. , Beckert Christian M. , Gombas Thomas , Randive Rajul V.
Published: June 2021

Background:

The purgaty One systems (cap+bottle) are portable stainless-steel water bottles with ultraviolet subtype C (UVC) disinfection capability. This study examines the bottle design, verifies disinfection performance against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae and heterotrophic contaminants and addresses the public health relevance of heterotrophic bacteria.

Methods:

Bottles were inoculated with bacterial strains and disinfection efficacy examined using colony forming unit (CFU) assay. The heterotrophic plate count (HPC) method was used to determine the disinfection performance against environmental contaminants at day 0 and after 3 days of water stagnation. All UVC irradiation experiments were performed under stagnant conditions to confirm that the preset application cycle of 55 seconds offers the desired disinfection performance under worst case condition. To determine effectiveness of purgaty One systems (cap+bottle) in disinfection, inactivation efficacy or log reduction value (LRV) was determined using bacteria concentration between UVC ON condition and controls (UVC OFF). The study utilized the 16S rRNA gene for isolate characterization by identifying HPC bacteria to confirm if they belong to groups that are of public health concern.

Results:

Purgaty One systems fitted with Klaran UVC LEDs achieved 99.99% inactivation (LRV4) efficacy against E. coli and 99.9% inactivation (LRV3) against P. aeruginosa, V. cholerae and heterotrophic contaminants. Based on the 16S rRNA gene analyses, the study determined that the identified HPC isolates enriched by UVC irradiation are of rare public health concern.

Conclusion:

The bottles satisfactorily inactivated the target pathogenic bacteria and HPC contaminants even after 3 days of water stagnation.