bluuebunny/crossref_metadata_2025 huggingface dataset:
CDK1 controls CHMP7-dependent nuclear envelope reformation
Authors: Gatta Alberto T , Olmos Yolanda , Stoten Caroline L , Chen Qu , Rosenthal Peter B , Carlton Jeremy G
Published: April 2020

Through membrane sealing and disassembly of spindle microtubules, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope (NE) during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III assembly at the NE occurs transiently during mitotic exit and is initiated when CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, interacts with the Inner Nuclear Membrane (INM) protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during mitotic exit. Firstly, we show that CHMP7 plays an important role in the dissolution of LEM2 clusters that form at the NE during M-exit. Secondly, we show that CDK1 phosphorylates CHMP7 upon mitotic entry at Ser3 and Ser441 and that this phosphorylation suppresses CHMP7’s interaction with LEM2, limiting its assembly during M-phase. We show that spatiotemporal differences in the dephosphorylation of CHMP7 license its assembly at the NE during telophase, but restrict its assembly on the ER at this time. Without CDK1 phosphorylation, CHMP7 undergoes inappropriate assembly in the peripheral ER during M-exit, capturing LEM2 and downstream ESCRT-III components. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration.
Escherichia colismall heat shock protein IbpA is an aggregation-sensor that self-regulates its own expression at a translational level
Authors: Miwa Tsukumi , Chadani Yuhei , Taguchi Hideki
Published: April 2020

Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps inEscherichia coli, is regulated by the σ32heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or theibpAgene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsp expression. Oligomeric IbpA self-represses theibpA/ibpBexpression at the translational level, but the self-repression is relieved by the sequestration of IbpA into protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation.

Author summary

All organisms have protein quality control systems against stresses disturbing cellular protein homeostasis (proteostasis). The systems have multiple stages: folding, degradation, and sequestration. Sequestration of denatured proteins is the first step to support other maintenance strategies. Small heat shock proteins (sHsps), which are well-conserved chaperones, are representative “sequestrases” that co-aggregate with denatured proteins. We found that IbpA, anEscherichia colisHsp, is a direct mediator for negative feedback regulation at the translational level. Recruitment of IbpA into the protein aggregates relieves theibpAexpression suppression. This novel mechanism of IbpA as an aggregation-sensor tightly regulates the IbpA level, enabling the sHsp to function as a sequestrase upon aggregation stress.
Conditional antagonism in co-cultures ofPseudomonas aeruginosaandCandida albicans: an intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics
Authors: Doing Georgia , Koeppen Katja , Occipinti Patricia , Hogan Deborah A.
Published: April 2020

Pseudomonas aeruginosaandCandida albicansare opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here we describe the focal role of ethanol in mixed-species co-cultures by dual RNA-seq analyses.P. aeruginosaandC. albicanstranscriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producingC. albicansor aC. albicansmutant lacking the primary ethanol dehydrogenase, Adh1. Analyses using KEGG-pathways and the previously published eADAGE method revealed severalP. aeruginosaresponses toC. albicans-produced ethanol including the induction of a non-canonical low phosphate response mediated by PhoB.C. albicanswild-type, but notC. albicans adh1Δ/Δ, inducesP. aeruginosaproduction of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells. We first demonstrate that PhoB is required for this interaction and that PhoB hyperactivity, via deletion ofpstB,leads to increased production of 5-MPCA even when phosphate concentrations are high, but only in the presence of ethanol. Second, we show that ethanol is only sufficient to promote 5-MPCA production at permissive phosphate concentrations. The intersection of ethanol and phosphate in co-culture is mirrored inC. albicans; theadh1Δ/Δ mutant had increased expression of genes regulated by Pho4, theC. albicanstranscription factor that responds to low phosphate which we confirmed by showing theadh1Δ/Δ strain had elevated Pho4-dependent phosphatase activity. The dual-dependence on ethanol and phosphate concentrations for anti-fungal production highlights how environmental factors modulate microbial interactions and dictate antagonisms such as those betweenP. aeruginosaandC. albicans.

Author Summary

Pseudomonas aeruginosaandCandida albicansare opportunistic pathogens that are frequently isolated from co-infections. Using a Dual-Seq approach in combination with genetics approaches, we found that ethanol produced byC. albicansstimulates the PhoB regulon inP. aeruginosaasynchronously with activation of the Pho4 regulon inC. albicans.In doing so, we demonstrate that eADAGE-based analysis can improve the understanding of theP. aeruginosaresponse to ethanol-producingC. albicansas measured by transcriptomics: we identify a subset of PhoB-regulated genes as differentially expressed in response to ethanol. We validate our result by showing that PhoB is necessary for multiple roles in co-culture including the competition for phosphate and the production of 5-methyl-phenazine-1-carboxylic acid, and that theP. aeruginosaresponse toC. albicans-produced ethanol depends on phosphate availability. The conditional stimulation of virulence production in response to sub-inhibitory concentrations of ethanol only under phosphate limitation highlights the importance of considering nutrient concentrations in the analysis of co-culture interactions.
Genotypic and phenotypic characterization ofStreptococcus mutansisolated from dental caries
Authors: Hossain Md. Shahadat , Alam Sadab , Nibir Yead Morshed , Tusty Tahrima Arman , Bulbul Sayyeed Mahmud , Islam Shahidul , Hossain Mohammad Shahnoor
Published: April 2020

Streptococcus mutans, considered as principal causative agent of dental caries, maintains a biofilm lifestyle in the dental plaque. The oral cavity harbors numerousS. mutansstrains, which displayed remarkable genotypic and phenotypic diversity. This study evaluated the genotypic and phenotypic diversity of 209S. mutansstrains isolated from 336 patients with dental caries and compared with the universal reference strain UA159. Our study has revealed a high degree of genotypic and phenotypic variability among the clinical strains. We observed significant differences in colony morphology, generation time, biofilm formation, bacteriocin and acid production while growing in culture medium. All the clinical isolates were able to lower pH while growing in THY broth. In consistent with phenotypic variations, we also observed tremendous level of genotypic variation by AP-PCR and gene specific PCR. AP-PCR analysis suggested that most of the patients with dental caries have distinct type ofS. mutansstrains. Genes related to various two component systems were highly conserved among the strains, however, bacteriocin encoding genes such asnlmAB, nlmCwere absent in half of the clinical isolates. In sum, our study highlights the genotypic and phenotypic diversity ofS. mutansclinical isolates and indicates the presence of diverse mechanism to initiate and establish the biofilm lifestyle which leads to tooth decay.
Efficacy of transoral laser surgery versus linear accelerator radiotherapy for the treatment of T1 glottic carcinoma: a meta-analysis of oncologic outcomes
Authors: Zhu Jiawei , Shen Jing , Zheng Ziye , Lian Xin , Miao Zheng , Hu Ke , Zhang Fuquan
Published: April 2020

Objective

A meta-analysis was conducted to compare oncologic outcomes for patients of T1 glottic carcinoma who were treated with transoral laser surgery (TLS) or linear accelerator radiotherapy (linac RT).

Methods

All related studies published up to September 2019 were acquired by searching Pubmed, EMBASE, and Cochrane, with the index words: glottic, vocal, laryngeal, radiation, radiotherapy, irradiation, laser, surgery, cordectomy, carcinoma and cancer. Relative studies which compared oncologic outcomes between linac RT and TLS were included. Sensitivity analysis were performed to evaluate heterogeneity.

Results

A total of twelve eligible studies were included for the analysis, which contained three prospective studies and nine retrospective studies. Patients who underwent TLS had increased overall survival (OR = 1.40, 95% CI=1.02-1.94,P=0.04) and laryngeal preservation (OR = 5.37, 95% CI = 3.05-9.44,P< 0.00001) versus who underwent linac RT. No statistical difference was observed between TLS group and linac RT group in terms of local control (OR=0.88, 95% CI = 0.62-1.24,P= 0.47), disease-specific survival (OR = 0.61, 95% CI = 0.26-1.43,P= 0.26), and disease-free survival (OR = 1.63, 95% CI = 0.70-3.81,P= 0.14).

Conclusions

The results of this meta-analysis indicate that there were clinical benefits for patients with glottic carcinoma after TLS compared with linac RT with respect to overall survival and laryngeal preservation. However, more multi-center randomized controlled trials would be urgently needed to prove these differences.
Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish genepsen2
Authors: Jiang Haowei , Pederson Stephen Martin , Newman Morgan , Dong Yang , Lardelli Michael
Published: April 2020

PRESENILIN 2(PSEN2) is one of the genes mutated in early onset familial Alzheimer’s disease (EOfAD).PSEN2shares significant amino acid sequence identity with another EOfAD-related genePRESENILIN 1(PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions ofPSEN1andPSEN2is not yet understood. In this study, we performed targeted mutagenesis of the zebrafishpsen2gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation,psen2S4Ter, are viable and fertile, and adults do not show any gross pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive topsen2but notpsen1activity during embryogenesis did not reveal decreasedpsen2function. Transcripts containing theS4Termutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions ofpsen2S4Ter5’ mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that thepsen2S4Termutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins that obey the “reading frame preservation rule” ofPRESENILINEOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygouspsen2S4Terfemale siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.
Anatomy of epithemal hydathodes in four monocotyledon plants of economic and academic relevance
Authors: Jauneau Alain , Cerutti Aude , Auriac Marie-Christine , Noël Laurent D.
Published: April 2020

Hydathode is a plant organ responsible for guttation in vascular plants, i.e. the release of droplets at leaf margin or surface. Because this organ connects the plant vasculature to the external environment, it is also a known entry site for some vascular pathogens. In this study, we present a detailed microscopic examination of monocot hydathodes for three crops (maize, rice and sugarcane) and the model plantBrachypodium distachyon. Our study highlights both similarities and specificities of those epithemal hydathodes. These observations will serve as a foundation for future studies on the physiology and the immunity of hydathodes in monocots.
An image J plugin for the high throughput image analysis ofin vitroscratch wound healing assays
Authors: Suarez-Arnedo Alejandra , Figueroa Felipe Torres , Clavijo Camila , Arbeláez Pablo , Cruz Juan C. , Muñoz-Camargo Carolina
Published: April 2020

In vitroscratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. There are open-source programs such as imageJ to analyze images ofin vitroscratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. For that reason, we developed an optimized plugin for imageJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Our plugin is easy to install and can be used with different operating systems. It can be adapted to analyze both individual images and stacks. Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of differentin vitrowound parameters with high accuracy compared with other tools and manual identification.
An immunohistochemical prostate cell identification key indicates that aging shifts procollagen 1A1 production from myofibroblasts to fibroblasts in dogs prone to prostate-related urinary dysfunction
Authors: Ruetten Hannah , Cole Clara , Wehber Marlyse , Wegner Kyle A. , Girardi Nicholas M. , Peterson Nelson T. , Scharpf Brandon R. , Romero Michael F. , Wood Michael W. , Colopy Sara A. , Bjorling Dale E. , Vezina Chad M.
Published: April 2020

Background

The identity and spatial distribution of prostatic cell types has been determined in humans but not in dogs, even though aging- and prostate-related voiding disorders are common in both species and mechanistic factors, such as prostatic collagen accumulation, appear to be shared between species. In this publication we characterize the regional distribution of prostatic cell types in the young intact dog to enable comparisons with human and mice and we examine how the cellular source of procollagen 1A1 changes with age in intact male dogs.

Methods

A multichotomous decision tree involving sequential immunohistochemical stains was validated for use in dog and used to identify specific prostatic cell types and determine their distribution in the capsule, peripheral, periurethral and urethral regions of the young intact canine prostate. Prostatic cells identified using this technique include perivascular smooth muscle cells, pericytes, endothelial cells, luminal, intermediate, and basal epithelial cells, neuroendocrine cells, myofibroblasts, fibroblasts, fibrocytes, and other hematolymphoid cells. Corresponding images were made freely accessible through the GUDMAP database athttps://doi.org/10.25548/16-WMM4.

Results

The prostatic peripheral region harbors the largest proportion of epithelial cells. Aging does not change the density of hematolymphoid cells, fibroblasts, and myofibroblasts in the peripheral region or in the fibromuscular capsule, regions where we previously observed aging- and androgen-mediated increases in prostatic collagen abundance Instead, we observed aging-related changes the procollagen 1A1 positive prostatic cell identity from a myofibroblast to a fibroblast.

Conclusions

Hematolymphoid cells and myofibroblasts are often identified as sources of collagen in tissues prone to aging-related fibrosis. We show that these are not the likely sources of pathological collagen synthesis in older intact male dogs. Instead, we identify an aging-related shift in the prostatic cell type producing procollagen 1A1 that will help direct development of cell type and prostate appropriate therapeutics for collagen accumulation.
Motor and non-motor sequence prediction is equally affected in children with Developmental Coordination Disorder
Authors: Opitz Bertram , Brady Daniel , Leonard Hayley C.
Published: April 2020

Children with Developmental Coordination Disorder (DCD) are diagnosed based on motor difficulties. However, they also exhibit difficulties in several other cognitive domains, including visuospatial processing, executive functioning and attention. One account of the difficulties seen in DCD proposes an impairment in internal forward modelling, i.e., the ability to (i) detect regularities of a repetitive perceptual or motor pattern, (ii) predict future outcomes of motor actions, and (iii) adapt behaviour accordingly. Using electroencephalographic recordings, the present study aimed to delineate these different aspects of internal forward modelling across several domains. To this end, 24 children with DCD and 23 typically-developing children (aged 7-10 years) completed a serial prediction task in the visual, temporal, spatial and motor domains. This task required them to learn short sequences and to indicate whether a sequence was disrupted towards its end. Analyses revealed that, across all domains, children with DCD showed poorer discrimination between intact and disrupted sequences, accompanied by a delayed late parietal positivity elicited by disrupted sequences. These results indicate an impairment in explicit sequence discrimination in DCD across motor and cognitive domains. However, there is no evidence for an impairment in implicit performance on the motor task in DCD. These results suggest an impairment of the updating of an internal forward model in DCD resulting in a blurred representation of that model and consequently in a reduced ability to detect regularities in the environment (e.g., sequences). Such a detailed understanding of internal forward modelling in DCD could help to explain the wide range of co-occurring difficulties experienced by those with a diagnosis of DCD.
Abortion and various associated risk factors in dairy cow and sheep in Ili, China
Authors: Deng Xiaoyu , Zhang Huan , Shao Zhiran , Zhao Xiaoli , Yang Qin , Song Shengnan , Wang Zhen , Wang Yong , Wang Yuanzhi , Sheng Jinliang , Chen Chuangfu
Published: April 2020

We studied livestock abortion and various associated risk factors in the Ili region of northwest China. Livestock abortion prevalence was estimated and correlated with infections (Brucellosis, Salmonellosis,MycoplasmaandChlamydiaseropositivity) and management (farming type and contact with other herds/flocks) risk factors. The prevalence of cow and sheep abortion induced byBrucellawas 76.8% (P<0.0001) and 84.1% (P<0.0001), andMycoplasmacaused an estimated 15.5% (P=0.025) and 17.6% (P<0.001) abortions, respectively. Abortion-related risk factors included mixed farming (cowP=0.001, sheepP<0.001), contact with other flocks (cowP=0.007, sheepP=0.003), brucellosis positivity (cowP<0.001, sheepP<0.001) andMycoplasmapositivity (cowP=0.031, sheepP<0.001). A total of 2996 serum samples (1402 cow, 1594 sheep) were identified by RBPT (Rose Bengal Plate Test), and they showed the seroprevalence of brucellosis in X county was cow 7.1%, sheep 9.1%; in H county was cow 11.7%, sheep 10.7%; and in Q county was cow 4.2%, sheep 9.1%. The seroprevalence ofMycoplasmain X county was cow 3.4%, sheep 7.9%; in H county was cow 5.3%, sheep 9.9%; and in Q county was cow 2.1%, sheep 4.3%. A total of 54 samples, including aborted cow (22), sheep (30) fetuses and milk samples (2), were identified asBrucella melitensis(B. melitensis) positive. A total of 38Brucellawere isolated from 16 aborted cow, 20 sheep fetuses and 2 milk samples. All of these isolates were identified, and confirmed, asB. melitensis. A phylogenetic tree showed that theBrucellaisolates closely matched theB. melitensisbiovar 3 isolated in Inner Mongolia, China, andB. melitensisisolated from Norway and India. These results suggest thatB. melitensisbiovar 3 is the main pathogen responsible for cow and sheep abortion and also pose a human health risk. Additionally, livestock reproduction can also be influenced byMycoplasmainfection and managerial factors (farming type and contact with other herds/flocks), especially in remote areas.
Ligand Sensing Enhances Bacterial Flagellar Motor Output via Stator Recruitment
Authors: Naaz Farha , Agrawal Megha , Chakraborty Soumyadeep , Tirumkudulu Mahesh S. , Venkatesh K.V.
Published: April 2020

The phenomenon of chemotaxis in bacteria, where the cells migrate towards or away from chemicals, has been extensively studied in the past. For flagellated bacteria such asEscherichia coli, a change in chemical concentration in its environment is sensed by a chemoreceptor and communicated via a well-characterised signalling pathway to the flagellar motor. It has been widely accepted that the signals change the rotation bias of the motor without influencing the motor speed. Here, we present results to the contrary and show that the bacteria is also capable of modulating motor speed on merely sensing a ligand. Step changes in concentration of non-metabolisable ligand cause temporary recruitment of stators leading to a momentary increase in motor speeds. For metabolisable ligand, the combined effect of sensing and metabolism leads to higher motor speeds for longer durations. Swimming speeds measured at the population level corroborate the observations. Experiments performed with mutant strains delineate the role of metabolism and sensing in the modulation of motor speed and show how speed changes along with changes in bias can significantly enhance bacteria’s response to changes in its environment.
Antibiotic-resistantSalmonella speciesandEscherichia coliin broiler chickens from farms, abattoirs and open markets in selected districts of Zambia
Authors: Phiri Nelson , Mainda Geoffrey , Mukuma Mercy , Sinyangwe Ntazana N. , Banda Luke John , Kwenda Geoffrey , Muligisa-Muonga Elizabeth , Flavien Bumbangi Nsoni , Mwansa Mwaba , Yamba Kaunda , Munyeme Musso , Muma John Bwalya
Published: April 2020

Salmonellaspecies andEscherichia coliare major bacterial enteropathogens of global public health importance that cause foodborne diseases, thereby contributing to increased human morbidity and mortality. Both pathogens have also been found to contribute towards the spread of antimicrobial resistance through the food chain, especially in poultry. The aim of this study was to determine the occurrence of antibiotic-resistantSalmonella sp. andE. coliin broiler chickens at farm level, abattoirs and open markets in selected districts of Zambia. A cross-sectional study was undertaken in seven districts of Zambia to determine the resistance profiles ofSalmonella sp. andE. coliobtained from broiler chickens at farms, abattoirs and open markets. A total of 470 samples were collected, including litter, cloacal swabs and carcass swabs. Samples were inoculated into buffered peptone water, sub-cultured onto MacConkey and Xylose Lysine Deoxycholate agar plates. Identification ofSalmonella sp. and E. coliwas done using the API-20E kit and confirmation by 16S rDNA sequencing. Confirmed isolates were tested against a panel of 10 antibiotics using the Kirby-Bauer disc-diffusion method and interpreted according to the Clinical Laboratory Standards Institute guidelines. Analysis of the antibiotic susceptibility test results was done using WHONET 2018 software. Overall, 4Salmonella spp. and 280E. coliwere isolated. One of theSalmonella sp. was resistant to ampicillin (25%), amoxicillin/clavulanic acid (25%) and cefotaxime (25%).E. coliantibiotic resistance was highest to tetracycline (81.4%) and lowest to imipenem (0.7%). The antibiotic susceptibility profile revealed 55% (154/280) multidrug resistantE. coli, with the highest multidrug resistance profile (20.7%) in the ampicillin-tetracycline-trimethoprim/sulfamethoxazole drug combination. Furthermore, 4.3% (12/280) of the isolates showed Extensive Drug resistance. The levels of antimicrobial resistance toE. coliandSalmonellaobserved in market-ready chickens is of public health concern.
Agonist-induced Functional Analysis and Cell Sorting, a novel tool to select and analyze neurons: Fragile X as a proof of concept
Authors: Castagnola Sara , Cazareth Julie , Lebrigand Kevin , Jarjat Marielle , Magnone Virginie , Delhaye Sebastien , Brau Frederic , Bardoni Barbara , Maurin Thomas
Published: April 2020

To get a better insight into the dynamic interaction between cells and their environment, we developed the agonist-induced Functional Analysis and Cell Sorting (aiFACS) technique, which allows the simultaneous recording and sorting of cells in real-time according to their immediate and individual response to a stimulus. By modulating the aiFACS selection parameters, testing different developmental times, using various stimuli and multiplying the analysis of readouts, it is possible to analyze cell populations of any tissue, including tumors. The association of aiFACS to single-cell transcriptomic allows to build a tissue cartography based on specific functional response/s of cells.

As proof of concept, we used aiFACS on the dissociated mouse brain, a highly heterogenous tissue, enriching it in interneurons upon stimulation with an agonist of the glutamate receptors and upon sorting based on calcium levels. Further single-cell RNA-seq of these aiFACS-selected interneurons resulted in a nine-cluster classification. Furthermore, we used aiFACS on interneurons derived from the brain of theFmr1-KO mouse, a rodent model of Fragile X syndrome. We show here that these interneurons manifest a generalized defective pharmacological response compared to wild type, affecting all the analyzed cell clusters at one specific post-natal developmental time.
Mechanical and signaling mechanisms that guide pre-implantation embryo movement
Authors: Flores Diana , Madhavan Manoj , Wright Savannah , Arora Ripla
Published: April 2020

How a mammalian embryo determines and arrives at its site of attachment is a mystery that has puzzled researchers for decades. Additionally, in multiparous species, embryos face a unique challenge of achieving adequate spacing to avoid competition for maternal resources. Using our enhanced confocal imaging and 3D image reconstruction technology, we evaluate murine embryo location in the uterus along the longitudinal oviductal-cervical axis. Our analysis reveals three distinct pre-implantation stages: a) Embryo entry; b) Unidirectional movement of embryo clusters; and c) Bidirectional scattering and spacing of embryos. We show that unidirectional movement of embryo clusters is facilitated by a mechanical stimulus of the embryo as a physical object and is regulated by adrenergic uterine smooth muscle contractions. Embryo scattering, on the other hand, relies on embryo-uterine communication reliant on the LPAR3 signaling pathway and is independent of adrenergic muscle contractions. We propose that the presence of embryo clusters in the uterine horn provides an opportunity for the uterus to sense and count the embryos, followed by scattering and spacing these embryos along the given length of the horn. Thus, uterine implantation sites in mice are neither random nor predetermined but are guided by the number of embryos entering the uterine lumen. These studies have implications for understanding how embryo-uterine communication is key to determining an optimal implantation site, which is necessary for the success of a pregnancy.

Significance Statement

In mammals that carry multiple offspring in one gestation, embryos seemingly acquire even embryo spacing. Such even distribution would imply a guided interaction between the mother and the fetus very early on in pregnancy to allow favorable pregnancy outcomes. Thus, it is essential to understand quantitatively if and when such a uniform distribution of embryos is established. Further, uncovering the physical and biological mechanisms that allow for such equal distribution of embryos, will improve our understanding of early pregnancy events and provide for novel targets for improving pregnancy success in case of infertility and artificial reproductive technologies as well as to develop non-hormonal therapies for contraception.
Cortical ChAT+neurons co-transmit acetylcholine and GABA in a target- and brain-region specific manner
Authors: Granger Adam J , Wang Wengang , Robertson Keiramarie , El-Rifai Mahmoud , Zanello Andrea , Bistrong Karina , Saunders Arpiar , Chow Brian , Nuñez Vicente , Garcia Miguel Turrero , Harwell Corey , Gu Chenghua , Sabatini Bernardo L
Published: April 2020

The cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the neurotransmitters released by cortical ChAT+neurons and their synaptic connectivity are unknown. We show that the nearly all cortical ChAT+neurons are specialized VIP+interneurons that release GABA strongly onto other inhibitory interneurons and acetylcholine sparsely onto layer 1 interneurons and other VIP+/ChAT+interneurons. This differential transmission of ACh and GABA based on the postsynaptic target neuron is reflected in VIP+/ChAT+interneuron pre-synaptic terminals, as quantitative molecular analysis shows that only a subset of these are specialized to release acetylcholine. In addition, we identify a separate, sparse population of non-VIP ChAT+neurons in the medial prefrontal cortex with a distinct developmental origin that robustly release acetylcholine in layer 1. These results demonstrate both cortex-region heterogeneity in cortical ChAT+interneurons and target-specific co-release of acetylcholine and GABA.
ATR inhibition enhances 5-fluorouracil sensitivity independent of non-homologous end-joining and homologous recombination repair pathway
Authors: Ito Soichiro S. , Nakagawa Yosuke , Matsubayashi Masaya , Sakaguchi Yoshihiko M. , Kobashigawa Shinko , Matsui Takeshi K. , Nanaura Hitoki , Nakanishi Mari , Kitayoshi Fumika , Kikuchi Sotaro , Kajihara Atsuhisa , Tamaki Shigehiro , Sugie Kazuma , Kashino Genro , Takahashi Akihisa , Hasegawa Masatoshi , Mori Eiichiro , Kirita Tadaaki
Published: April 2020

The anticancer agent, 5-fluorouracil (5-FU), is typically applied in the treatment of various types of cancers because of its properties. Thought to be an inhibitor of the enzyme thymidylate synthase which plays a role in nucleotide synthesis, 5-FU has been found to induce single- and double-strand DNA breaks. The activation of ATR occurs as a reaction to UV- and chemotherapeutic drug-induced replication stress. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity. Using western blotting, we found that 5-FU treatment led to the phosphorylation of ATR. Surviving fractions were remarkably decreased in 5-FU with ATR inhibitor (ATRi) compared to 5-FU with other major DNA repair kinases inhibitors. ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU could induce the activation of intra-S checkpoint. Surprisingly,BRCA2-deficient cells were sensitive to 5-FU in the presence of ATRi. In addition, ATR inhibition enhanced the efficacy of 5-FU treatment, independent of non-homologous end-joining and homologous recombination repair pathways. Findings from the present study suggest ATR as a potential therapeutic target for 5-FU chemotherapy.
Greenhouse spatial effects detected in the barley (Hordeum vulgareL.) epigenome underlie stochasticity of DNA methylation
Authors: Konate Moumouni , Wilkinson Michael J. , Taylor Julian , Scott Eileen S. , Berger Bettina , Rodriguez Lopez Carlos Marcelino
Published: April 2020

Environmental cues are known to alter the methylation profile of genomic DNA, and thereby change the expression of some genes. A proportion of such modifications may become adaptive by adjusting expression of stress response genes but others been shown to be highly stochastic, even under controlled conditions. The influence of environmental flux on plants adds an additional layer of complexity that has potential to confound attempts to interpret interactions between environment, methylome and plant form. We therefore adopt a positional and longitudinal approach to study progressive changes to barley DNA methylation patterns in response to salt exposure during development under greenhouse conditions. Methylation-Sensitive Amplified Polymorphism (MSAP) and phenotypic analyses of nine diverse barley varieties were grown in a randomized plot design, under two salt treatments (0 mM and 75 mM NaCl). Combining environmental, phenotypic and epigenetic data analyses, we show that at least part of the epigenetic variability, previously described as stochastic, is linked to environmental micro-variations during plant growth. Additionally, we show that differences in methylation increase with time of exposure to micro-variations in environment. We propose that subsequent epigenetic studies take into account microclimate-induced epigenetic variability.
Chloroplast chaperonin-mediated targeting of a thylakoid membrane protein
Authors: Klasek Laura , Inoue Kentaro , Theg Steven M.
Published: April 2020

Post-translational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins post-translationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in Plsp1’s thylakoid targeting. In isolated thylakoids, the integration of Plsp1 decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon which inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60’s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, while cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment.

One-sentence summary

The chloroplast chaperonin captures and releases Plastidic type I signal peptidase 1 during its targeting to the thylakoid membrane.
Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis
Authors: Choudhary Vineet , Atab Ola El , Mizzon Giulia , Prinz William A. , Schneiter Roger
Published: April 2020

Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected, and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG synthases and additional LD biogeneiss factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.
Identification of a novel efficient transcriptional activation domain from Chinese fir (Cunninghamia lanceolata)
Authors: Zhu Tengfei , Tang Wenyu , Chen Delan , Zheng Renhua , Li Jian , Su Jun
Published: April 2020

Activation domains are used as critical components of artificial gene modification tools for genetic breeding. The high efficiency of the activation domain relies on the host plant. However, no activation domain has been identified that originates from Chinese fir (Cunninghamia lanceolate). In this study, a novel strong activator was identified from the whole Chinese fir cDNA library. This plant conserved activator was named TAC 3 (Transcriptional Activation domain from Chinese fir 3). C-terminal 70 amino acids of TAC (TAC3d) have a stronger ability than the commonly used strong activation domain of the virus protein VP16, or the strong plant activation domain, EDLL, in Chinese fir. Through Dual-luciferase assay, phenomic analysis and FT (Flowering Locus T [FT]) quantification, it was shown that, TAC3d can overcome the transcriptional repression of strong plant repressors (Flowering Locus C [FLC]) when fused to its C-terminal domain, thus inhibit the repression of FT expression. In conclusion, for the first time, an activation domain has been identified from Chinese fir. TAC3, which can be used for precise gene activation in Chinese fir in the future, and its function in the plant is more powerful than the commonly used strong activation domain (such as VP16 and EDLL).

Highlight

TAC3 is the first transcriptional activation domain identified from Chinese fir and its function is more powerful than some commonly used strong transcriptional activators (such as VP16 and EDLL)
Exceptional diversity and selection pressure on SARS-CoV and SARS-CoV-2 host receptor in bats compared to other mammals
Authors: Frank Hannah K. , Enard David , Boyd Scott D.
Published: April 2020

Pandemics originating from pathogen transmission between animals and humans highlight the broader need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection. Here, we investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2. We find that the ACE2 gene is under strong selection pressure in bats, the group in which the progenitors of SARS-CoV and SARS-CoV-2 are hypothesized to have evolved, particularly in residues that contact SARS-CoV and SARS-CoV-2. We detect positive selection in non-bat mammals in ACE2 but in a smaller proportion of branches than in bats, without enrichment of selection in residues that contact SARS-CoV or SARS-CoV-2. Additionally, we evaluate similarity between humans and other species in residues that contact SARS-CoV or SARS-CoV-2, revealing potential susceptible species but also highlighting the difficulties of predicting spillover events. This work increases our understanding of the relationship between mammals, particularly bats, and coronaviruses, and provides data that can be used in functional studies of how host proteins are bound by SARS-CoV and SARS-CoV-2 strains.
Melanocyte differentiation and epidermal pigmentation is regulated by polarity proteins
Authors: Knapp Sina K. , Iden Sandra
Published: April 2020

Pigmentation serves various purposes such as protection, camouflage, or attraction. In the skin epidermis, melanocytes react to certain environmental signals with melanin production and release, thereby ensuring photo-protection. For this, melanocytes acquire a highly polarized and dendritic architecture that facilitates interactions with surrounding keratinocytes and melanin transfer. How the morphology and function of these neural crest-derived cells is regulated remains poorly understood. Here, using mouse genetics and primary cell cultures, we show that conserved proteins of the mammalian Par3-aPKC polarity complex are required for epidermal pigmentation. Melanocyte-specific deletion of Par3 in mice caused skin hypopigmentation, reduced expression of components of the melanin synthesis pathway, and altered dendritic morphology. Mechanistically, Par3 was necessary downstream of α-melanocyte stimulating hormone (α-MSH) to elicit melanin production. Strikingly, pharmacologic activation of MITF using a salt-inducible kinase inhibitor was sufficient to restore melanocyte differentiation and skin pigmentation in the absence of Par3. This data reveals a central role of polarity proteins in transmitting external pigment-inducing signals through the α-MSH/Mc1R/MITF ‘tanning pathway’, exposing unexpected links between polarity signaling and melanogenesis with new insights for pigment cell biology.
Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates
Authors: Ahmed Nabeel , Friedrich Ulrike A. , Sormanni Pietro , Ciryam Prajwal , Altman Naomi S. , Bukau Bernd , Kramer Günter , O’Brien Edward P.
Published: April 2020

Variation in translation-elongation kinetics along a transcript’s coding sequence plays an important role in the maintenance of cellular protein homeostasis by regulating co-translational protein folding, localization, and maturation. Translation-elongation speed is influenced by molecular factors within mRNA and protein sequences. For example, the presence of proline in the ribosome’s P- or A-site slows down translation, but the effect of other pairs of amino acids, in the context of all 400 possible pairs, has not been characterized. Here, we studySaccharomyces cerevisiaeusing a combination of bioinformatics, mutational experiments, and evolutionary analyses, and show that many different pairs of amino acids and their associated tRNA molecules predictably and causally encode translation rate information when these pairs are present in the A- and P-sites of the ribosome independent of other factors known to influence translation speed including mRNA structure, wobble base pairing, tripeptide motifs, positively charged upstream nascent chain residues, and cognate tRNA concentration. The fast-translating pairs of amino acids that we identify are enriched four-fold relative to the slow-translating pairs acrossSaccharomyces cerevisiae’s proteome, while the slow-translating pairs are enriched downstream of domain boundaries. Thus, the chemical identity of amino acid pairs contributes to variability in translation rates, elongation kinetics are causally encoded in the primary structure of proteins, and signatures of evolutionary selection indicate their potential role in co-translational processes.
Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species
Authors: Hendrix Bill , Zheng Wei , Bauer Matthew J. , Havecker Ericka R. , Mai Jennifer T. , Hoffer Paul H. , Sanders Rick , Eads Brian D. , Caruano-Yzermans Amy , Taylor Danielle N. , Hresko Chelly , Oakes Janette , Iandolino Alberto B. , Bennett Michael J. , Deikman Jill
Published: April 2020

22 nt miRNAs or siRNAs have been shown to specifically induce production of transitive (secondary) siRNAs for targeted mRNAs. An abrasion method to deliver dsRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes inNicotiana benthamianaandAmaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves ofN. benthamiana, but systemic silencing ofGFPoccurred only when the guide strand contained 22 nt. Silencing in the treated leaves ofN. benthamianawas demonstrated for 3 endogenous genes:magnesium cheletase subunit I(CHL-I),magnesium cheletase subunit H(CHL-H), andGUN4. However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced forGFPin response to treatment with 22 nt siRNAs, but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also had more transitive siRNAs produced in response to 22 nt siRNAs, but the response varied from weak (CHL-I) to strong (CHL-H). 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs forGFP, CHL-HandGUN4inN. benthamiana. The special activity of 22 nt siRNAs in producing a greater local phenotype and induction of elevated levels of transitive siRNAs was also shown inA. cruentusfor theCHL-Hgene. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.
Antibiotics and the developing intestinal microbiome, metabolome and inflammatory environment: a randomized trial of preterm infants
Authors: Russell Jordan T. , Ruoss J. Lauren , de la Cruz Diomel , Li Nan , Bazacliu Catalina , Patton Laura , McKinley Kelley Lobean , Garrett Timothy J. , Polin Richard A. , Triplett Eric W. , Neu Josef
Published: April 2020

Antibiotic use in neonates can have detrimental effects on the developing gut microbiome, increasing the risk of morbidity. A majority of preterm neonates receive antibiotics after birth without clear evidence to guide this practice. Here microbiome, metabolomic, and immune marker results from the Routine Early Antibiotic use in SymptOmatic preterm Neonates (REASON) study are presented. The REASON study is the first trial to randomize symptomatic preterm neonates to receive or not receive antibiotics in the first 48 hours after birth. Using 16S rRNA sequencing of stool samples collected longitudinally for 91 neonates, the effect of such antibiotic use on microbiome diversity is assessed. The results illustrate that type of nutrition shapes the early infant gut microbiome. By integrating data for the gut microbiome, stool metabolites, stool immune markers, and inferred metabolic pathways, an association was discovered betweenVeillonellaand the neurotransmitter gamma-aminobutyric acid (GABA). These results suggest early antibiotic use may impact the gut-brain axis with the potential for consequences in early life development, a finding that needs to be validated in a larger cohort.