bluuebunny/crossref_metadata_2025 huggingface dataset:
SALTS – SURFR (sncRNA) And LAGOOn (lncRNA) Transcriptomics Suite
Authors: Kasukurthi Mohan V , Houserova Dominika , Huang Yulong , Barchie Addison A. , Roberts Justin T. , Li Dongqi , Wu Bin , Huang Jingshan , Borchert Glen M
Published: February 2021

The widespread utilization of high-throughput sequencing technologies has unequivocally demonstrated that eukaryotic transcriptomes consist primarily (>98%) of non-coding RNA (ncRNA) transcripts significantly more diverse than their protein-coding counterparts.

ncRNAs are typically divided into two categories based on their length. (1) ncRNAs less than 200 nucleotides (nt) long are referred as small non-coding RNAs (sncRNAs) and include microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transfer ribonucleic RNAs (tRNAs), etc., and the majority of these are thought to function primarily in controlling gene expression. That said, the full repertoire of sncRNAs remains fairly poorly defined as evidenced by two entirely new classes of sncRNAs only recently being reported, i.e., snoRNA-derived RNAs (sdRNAs) and tRNA-derived fragments (tRFs). (2) ncRNAs longer than 200 nt long are known as long ncRNAs (lncRNAs). lncRNAs represent the 2ndlargest transcriptional output of the cell (behind only ribosomal RNAs), and although functional roles for several lncRNAs have been reported, most lncRNAs remain largely uncharacterized due to a lack of predictive tools aimed at guiding functional characterizations.

Importantly, whereas the cost of high-throughput transcriptome sequencing is now feasible for most active research programs, tools necessary for the interpretation of these sequencings typically require significant computational expertise and resources markedly hindering widespread utilization of these datasets. In light of this, we have developed a powerful new ncRNA transcriptomics suite, SALTS, which is highly accurate, markedly efficient, and extremely user-friendly. SALTS stands forSURFR (sncRNA)AndLAGOOn (lncRNA)TranscriptomicsSuite and offers platforms for comprehensive sncRNA and lncRNA profiling and discovery, ncRNA functional prediction, and the identification of significant differential expressions among datasets. Notably, SALTS is accessed through an intuitive Web-based interface, can be used to analyze either user-generated, standard next-generation sequencing (NGS) output file uploads (e.g., FASTQ) or existing NCBI Sequence Read Archive (SRA) data, and requires absolutely no dataset pre-processing or knowledge of library adapters/oligonucleotides.

SALTS constitutes the first publically available, Web-based, comprehensive ncRNA transcriptomic NGS analysis platform designed specifically for users with no computational background, providing a much needed, powerful new resource capable of enabling more widespread ncRNA transcriptomic analyses. The SALTS WebServer is freely available online athttp://salts.soc.southalabama.edu.
Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1)
Authors: Quintana Julia , Bernal María I. , Scholle Marleen , Holländer-Czytko Heike , Nga Nguyen , Piotrowski Markus , Mendoza-Cózatl David G. , Haydon Michael J. , Krämer Ute
Published: February 2021

IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron uptake system and indispensable for the completion of the life cycle ofArabidopsis thalianawithout vigorous iron (Fe) supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot mobilization of Fe. We show that theirt1-2 (pam42) mutant over-accumulates Fe in roots, most prominently in the cortex of the differentiation zone, when compared to the wild type. Shoots ofirt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generatedirt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206and His232, which are required for transport activity in yeast. In the transgenic Arabidopsis lines, short-term root Fe uptake rates and secondary substrate Mn accumulation resemble those ofirt1-2, suggesting that these plants remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206Apartially complements rosette dwarfing and leaf chlorosis, as well as root-to-shoot Fe partitioning and gene expression defects ofirt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identifyMYB DOMAIN PROTEIN10,MYB DOMAIN PROTEIN72andNICOTIANAMINE SYNTHASE4as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve iron nutrition and the nutritional quality of agricultural crops.
The protection of Gαz-null NOD mice from hyperglycemia is sexually dimorphic and only partially β-cell autonomous
Authors: Fenske Rachel J. , Peter Darby C. , Wienkes Haley N. , Schaid Michael D. , Reuter Austin , Carbajal Kathryn A. , Kimple Michelle E.
Published: February 2021

The mechanisms that underlie the β-cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our group has defined the unique heterotrimeric G protein alpha-subunit, Gαz, as a key negative regulator of β-cell signal transduction pathways. Non-obese diabetic (NOD) mice lacking Gαzthroughout the body are protected from developing T1D-like hyperglycemia. To determine whether this phenotype is β-cell autonomous, we generated and validated a β-cell-specific Gαzknockout (βKO) on the NOD background and characterized the phenotype of female and male cohorts. Long-term hyperglycemia incidence was lower in GαzβKO mice as compared to wild-type (WT) controls, but, unlike global Gαzknockout mice, this protection was incomplete. While young male and female GαzβKO NOD mice had improved glucose tolerance, WT NOD males were significantly less glucose tolerant than females, and only female GαzβKO mice retained improved glucose tolerance at 28-29 weeks of age. Conversely, β-cell-specific Gαzloss only influenced insulitis in 28-29-week old male NOD mice, a phenotype correlating directly with body burden of glucose during oral glucose challenge. Using surrogates for β-cell function and apoptosis, the partial penetrance of euglycemia in GαzβKO NOD was best explained by an early failure to up-regulate β-cell proliferation. We conclude β-cell Gαzis an important regulator of the sexually-dimorphic T1D-like phenotype of NOD mice. Yet, other factors must be important in imparting full protection from the disease.
Chloroplast redox state changes indicate cell-to-cell signalling during the hypersensitive response
Authors: Lukan Tjaša , Županič Anže , Povalej Tjaša Mahkovec , Brunkard Jacob O. , Juteršek Mojca , Baebler Špela , Gruden Kristina
Published: February 2021

Hypersensitive response (HR)-conferred resistance is associated with an induction of programmed cell death (PCD) and pathogen spread restriction in its proximity. While the pivotal role of salicylic acid (SA) in the restriction of pathogen spread during HR has been confirmed, the exact role of chloroplastic reactive oxygen species and the link between SA signalling and chloroplast redox state during HR remain unexplained. To unravel these relationships, we performed detailed spatiotemporal analysis of chloroplast redox response to potato virus Y (PVY) infection in resistantNy-1-gene-bearing potato and its transgenic counterpart with impaired SA accumulation and compromised resistance. We found that the chloroplasts are highly oxidized in the cells adjacent to the cell death zone at different stages after virus inoculation in both genotypes. Moreover, we detected individual cells with moderately oxidized chloroplasts, which we call “signalling cells”, in close proximity as well as farther from the cell death zone. These are relatively rare in SA-deficient plants, suggesting their role in signalling for HR-conferred resistance. This hypothesis is further supported by highly induced formation of stroma filled tubules that extend from chloroplasts (stromules) in the cells adjacent to signalling cells. Unexpectedly, the cells with the highest occurrence of stromules have chloroplasts in more reduced state than the adjacent ones. In addition, we show that stromules are induced also at the edge of PVY multiplication zone. We conclude that chloroplast redox state and stromule formation are tightly spatiotemporally regulated by SA-signalling which leads to effective HR response.
The putative causal effect of type 2 diabetes in risk of cataract: a Mendelian randomization study in East Asian
Authors: Zhang Haoyang , Xiu Xuehao , Xue Angli , Yang Yuedong , Yang Yuanhao , Zhao Huiying
Published: February 2021

Background

The epidemiological association between type 2 diabetes and cataract has been well-established. However, it remains unclear whether the two diseases share a genetic basis, and if so, whether this reflects a causal relationship.

Methods

We utilized East Asian population-based genome-wide association studies (GWAS) summary statistics of type 2 diabetes (Ncase=36,614,Ncontrol=155,150) and cataract (Ncase=24,622,Ncontrol=187,831) to comprehensively investigate the shared genetics between the two diseases. We performed 1. linkage disequilibrium score regression (LDSC) and heritability estimation from summary statistics (ρ-HESS) to estimate the genetic correlation and local genetic correlation between type 2 diabetes and cataract; 2. multiple Mendelian randomization (MR) analyses to infer the putative causality between type 2 diabetes and cataract; and 3. Summary-data-based Mendelian randomization (SMR) to identify candidate risk genes underling the causality.

Results

We observed a strong genetic correlation (rg=0.58; p-value=5.60×10−6) between type 2 diabetes and cataract. Both ρ-HESS and multiple MR methods consistently showed a putative causal effect of type 2 diabetes on cataract, with estimated liability-scale MR odds ratios (ORs) at around 1.10 (95% confidence interval [CI] ranging from 1.06 to 1.17). In contrast, no evidence supports a causal effect of cataract on type 2 diabetes. SMR analysis identified two novel genesMIR4453HG(βSMR=−0.34, p-value=6.41×10−8) andKCNK17(βSMR=−0.07, p-value=2.49×10−10), whose expression levels were likely involved in the putative causality of type 2 diabetes on cataract.

Conclusions

Our results provided robust evidence supporting a causal effect of type 2 diabetes on the risk of cataract in East Asians, and posed new paths on guiding prevention and early-stage diagnosis of cataract in type 2 diabetes patients.

Key Messages

We utilized genome-wide association studies of type 2 diabetes and cataract in a large Japanese population-based cohort and find a strong genetic overlap underlying the two diseases.

We performed multiple Mendelian randomization models and consistently disclosed a putative causal effect of type 2 diabetes on the development of cataract.

We revealed two candidate genesMIR4453HGandKCNK17whose expression levelss are likely relevant to the causality between type 2 diabetes and cataract.

Our study provided theoretical fundament at the genetic level for improving early diagnosis, prevention and treatment of cataract in type 2 diabetes patients in clinical practice
Patient-specific cell communication networks associate with disease progression in cancer
Authors: Gibbs David L , Aguilar Boris , Thorsson Vésteinn , Ratushny Alexander V , Shmulevich Ilya
Published: February 2021

The maintenance and function of tissues in health and disease depends on cell-cell communication. This work shows how high-level features, representing cell-cell communication, can be defined and used to associate certain signaling ‘axes’ with clinical outcomes. Using cell-sorted gene expression data, we generated a scaffold of cell-cell interactions and define a probabilistic method for creating per-patient weighted graphs based on gene expression and cell deconvolution results. With this method, we generated over 9,000 graphs for TCGA patient samples, each representing likely channels of intercellular communication in the tumor microenvironment. It was shown that particular edges were strongly associated with disease severity and progression, in terms of survival time and tumor stage. Within individual tumor types, there are predominant cell types and the collection of associated edges were found to be predictive of clinical phenotypes. Additionally, genes associated with differentially weighted edges were enriched in Gene Ontology terms associated with tissue structure and immune response. Code, data, and notebooks are provided to enable the application of this method to any expression dataset (https://github.com/IlyaLab/Pan-Cancer-Cell-Cell-Comm-Net).
Harmful Algal Bloom-Forming Organism Responds to Nutrient Stress Distinctly From Model Phytoplankton
Authors: McLean Craig , Haley Sheean T. , Swarr Gretchen J. , Soule Melissa C. Kido , Dyhrman Sonya T. , Kujawinski Elizabeth B.
Published: February 2021

Resources such as nitrogen (N) and phosphorus (P) play an important role in primary production and constraining phytoplankton bloom dynamics. Models to predict bloom dynamics require mechanistic knowledge of algal metabolic shifts in response to resource limitation. For well-studied model phytoplankton like diatoms, this information is plentiful. However, for less-studied groups such as the raphidophytes, there remain significant gaps in understanding metabolic changes associated with nutrient limitation.

Using a novel combination of metabolomics and transcriptomics, we examined how the harmful algal bloom-forming raphidophyteHeterosigma akashiwoshifts its metabolism under N- and P-stress. We choseH. akashiwobecause of its ubiquity within estuarine environments worldwide, where bloom dynamics are influenced by N and P availability.

Our results show that each stress phenotype is distinct in both the allocation of carbon and the recycling of macromolecules. Further, we identified biomarkers of N- and P-stress that may be appliedin situto help modelers and stakeholders manage, predict, and prevent future blooms.

These findings provide a mechanistic foundation to model the metabolic traits and trade-offs associated with N- and P-stress inH. akashiwo, and evaluate the extent to which these metabolic responses can be inferred in other phytoplankton groups.
Single-cell transitional dynamics unravel stimulus- and cell type-dependent signaling outputs of distinct p53 regulatory feedback motifs
Authors: Xie Jun , Zhang Lichun , Zhu Yanting , Liang Xiao , Shi Jue
Published: February 2021

Despite myriad of systemic analysis, our quantitative understanding of signaling networks and their differential outputs is still limited, especially at the dynamic level. Here we employed a network motif approach to dissect key regulatory units of p53 pathway and determined how their collective activities generated context-specific dynamic responses. By combining single-cell imaging and mathematical modeling of dose-dependent p53 dynamics induced by three chemotherapeutics of distinct mechanism-of-action, including Etoposide, Nutlin-3a and 5-Fluouracil, and in five cancer cell types, we identified novel p53 dynamic modes and their mechanistic origins with new insight on previously unknown drug mediators and phenotypic heterogeneity of cancer cells. Our results established the functional roles of unique feedback motifs of p53 pathway in generating the stimulus- and cell type-specific signaling responses and demonstrated that transitional dynamics activated at intermediate stimulus levels can be exploited as novel quantitative readouts to uncover and elucidate the key building blocks of bio-networks.
The Population Structure and Selection Signal Analysis of Shenxian Pigs based on Genome Resequencing Technology
Authors: Diao Liu , Chunlian Lu , Shang Li , Mengyu Jia , Sai Li , Liqin Ren , Yutao Miao , Hongzhan Cao
Published: February 2021

Shenxian pigs are the only local black pig of Hebei Province, and were listed in the Genetics of Livestock and Poultry Resources of China in 2016. This breed of pig is considered to be a valuable local pig germplasm genetic resource in China. In the present study, in order to understand the genetic variations of Shenxian pigs, identify selected regions related to superior traits, and accelerate the breeding processes of Shenxian pigs, the whole genome of the Shenxian pigs was resequenced and compared with that of large white pigs. The goal was to explore the germplasm characteristics of Shenxian pigs.The results obtained in this research investigation revealed that the genetic relationships of the Shenxian pig breed were complex, and that sub-populations could be identified within the general population. A total of 23M SNP sites were obtained by whole genome resequencing, and 1,509 selected sites were obtained via bioinformatics analyses. It was determined after annotation that a total of 19 genes were enriched in three items of bioengineering, molecular function, and cell composition.During this research investigation, the aforementioned 19 genes were subjected to GO and KEGG analyses. Subsequently, the candidate genes related to cell proliferation were obtained (DMTF1 and WDR5), which were considered to possibly be related to the slow growth and development of Shenxian pigs. In addition, the candidate genes related to lactation were obtained (CSN2 and CSN3).
Deep learning for robust and flexible tracking in behavioral studies forC. elegans
Authors: Bates Kathleen , Le Kim , Lu Hang
Published: February 2021

Robust and accurate behavioral tracking is essential for ethological studies. Common methods for tracking and extracting behavior rely on user adjusted heuristics that can significantly vary across different individuals, environments, and experimental conditions. As a result, they are difficult to implement in large-scale behavioral studies with complex, heterogenous environmental conditions. Recently developed deep-learning methods for object recognition such as Faster R-CNN have advantages in their speed, accuracy, and robustness. Here, we show that Faster R-CNN can be employed for identification and detection ofCaenorhabditis elegansin a variety of life stages in complex environments. We applied the algorithm to track animal speeds during development, fecundity rates and spatial distribution in reproductive adults, and behavioral decline in aging populations. By doing so, we demonstrate the flexibility, speed, and scalability of Faster R-CNN across a variety of experimental conditions, illustrating its generalized use for future large-scale behavioral studies.
A screen for sleep and starvation resistance identifies a wake-promoting role for the auxiliary channel unc79
Authors: Murakami Kazuma , Palermo Justin , Stanhope Bethany A. , Keene Alex C.
Published: February 2021

The regulation of sleep and metabolism are highly interconnected, and dysregulation of sleep is linked to metabolic diseases that include obesity, diabetes, and heart disease. Further, both acute and long-term changes in diet potently impact sleep duration and quality. To identify novel factors that modulate interactions between sleep and metabolic state, we performed a genetic screen for their roles in regulating sleep duration, starvation resistance, and starvation-dependent modulation of sleep. This screen identified a number of genes with potential roles in regulating sleep, metabolism or both processes. One such gene encodes the auxiliary ion channel UNC79, which was implicated in both the regulation of sleep and starvation resistance. Genetic knockdown or mutation ofunc79results in flies with increased sleep duration, as well as increased starvation resistance. Previous findings have shown thatunc79is required in pacemaker for 24-hour circadian rhythms. Here, we find thatunc79functions in the mushroom body, but not pacemaker neurons, to regulate sleep duration and starvation resistance. Together, these findings reveal spatially localized separable functions ofunc79in the regulation of circadian behavior, sleep, and metabolic function.
A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation
Authors: Xu Zeling , Li Yanran , Cao Huiluo , Si Meiru , Zhang Guangming , Woo Patrick CY , Yan Aixin
Published: February 2021

The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered by difficulties of introducing the class-specific, multi-component effectors in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a complete and highly active I-F Cascade in the notoriously recalcitrant and diverseP. aeruginosagenomes by conjugation. The transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than the Cas9 system in diverse genetic backgrounds, including removal of the large (21-kb) integrated cassette with efficiency and simplicity. An advanced λred-I-F system enabled editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an ‘all-in-one’ I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the mini-CRISPR array expressing desired crRNA in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in non-model isolates of pathogens.
DNA damage-induced phosphorylation of histone H2A at serine 15 is linked to DNA end resection
Authors: Ahmad Salar , Côté Valérie , Côté Jacques
Published: February 2021

The repair of DNA double-strand breaks (DSBs) occurs in chromatin and several histone post-translational modifications have been implicated in the process. Modifications of histone H2A N-terminal tail has also been linked to DNA damage response, through acetylation or ubiquitination of lysine residues that regulate repair pathway choice. Here, we characterize a new DNA damage-induced phosphorylation on chromatin, at serine 15 of H2A in yeast. We show that this SQ motif functions independently of the classical S129 C-terminal site (γH2A) and mutant mimicking constitutive phosphorylation increases cell sensitivity to DNA damage. H2AS129ph is induced by Tel1ATMand Mec1ATR, and loss of Lcd1ATRIPor Mec1 signaling decreases γH2A spreading distal to the DSB. In contrast, H2AS15ph is completely dependent on Lcd1ATRIP, indicating that this modification only happens when end resection is engaged. This is supported by an increase of RPA and a decrease in DNA signal near the DSB in theH2AS-15Ephosphomimic mutant, indicating higher resection. This serine is replaced by a lysine in mammals (H2AK15), which undergoes an acetyl-monoubiquityl switch to regulate binding of 53BP1 and resection. This regulation seems functionally conserved with budding yeast H2AS15 and 53BP1-homolog Rad9, using different post-translational modifications between organisms but achieving the same function.
“Susceptibility and risk of suffering SARS-COV-2 infection by demographic characteristics and pre-existing medical conditions among middle-aged and older adults in Tarragona, Spain: results from the COVID19 TARRACO Cohort Study, March-June 2020”
Authors: Satué-Gracia EM. , Vila-Córcoles A. , de Diego-Cabanes C. , Vila-Rovira A. , Torrente-Fraga C. , Gómez-Bertomeu F. , Hospital-Guardiola I. , Ochoa-Gondar O. , Martín-Luján F.
Published: February 2021

Objective

To analyse susceptibility/risk of suffering COVID19 among adults with distinct underlying medical conditions.

Methods

Cohort study (population-based) including 79,083 people ≥50 years-old in Tarragona (Southern Catalonia, Spain). At study start (01/03/2020) baseline cohort characteristics (demographic, previous comorbidities, chronic medications and vaccinations’ history) were recorded. Primary outcome was laboratory-confirmed COVID19 incurred in cohort members throughout 01/03/2020-30/06/2020. Risk of suffering COVID19 was evaluated by Cox regression, estimating multivariable hazard ratios (HRs) adjusted for age/sex and previous comorbidities.

Results

Across study period, 536 laboratory-confirmed COVID19 cases were observed (mean incidence: 39.5 cases per 100,000 persons-week). In multivariable-analysis, age/years (HR: 1.01; 95% CI: 1.00-1.02; p=0.050), nursing-home (HR: 20.19; 95% CI: 15.98-25.51; p<0.001), neurological disease (HR: 1.35; 95% CI: 1.03-1.77; p=0.029), taking diuretics (HR: 1.39; 95% CI: 1.10-1.75; p=0.006), antiplatelet (HR: 1.36; 95% CI: 1.05-1.76; p=0.021) and benzodiazepines (HR: 1.24; 95% CI:1.00-1.53; p=0.047) significantly increased risk; while smoking (HR: 0.57; 95%CI: 0.41-0.80; p=0.001), angiotensin converting enzyme inhibitors (HR: 0.78; 95% CI: 0.61-1.00; p=0.048), angiotensin II receptor blockers (HR: 0.70; 95%CI: 0.51-0.96; p=0.027) and statins (HR: 0.75; 95% CI: 0.58-0.96; p=0.025) were associated with reduced risk. Among non-institutionalised persons, cancer, renal and cardiac disease appeared also related to increased risk, whereas influenza vaccination was associated with reduced risk.

Conclusion

In a setting with relatively low incidence of COVID19 across the first wave of pandemic period, age, nursing-home residence and multiple comorbidities appear predisposing for COVID19 among middle-aged/older adults. Conversely, statins, angiotensin-receptor blockers/inhibitors and influenza vaccination were related with decreased risk.
In vivooptogenetic stimulation of the primate retina activates the visual cortex after long-term transduction
Authors: Chaffiol Antoine , Provansal Matthieu , Joffrois Corentin , Blaize Kévin , Labernede Guillaume , Goulet Ruben , Burban Emma , Brazhnikova Elena , Duebel Jens , Pouget Pierre , Alain Sahel José , Picaud Serge , Gauvain Gregory , Arcizet Fabrice
Published: February 2021

Over the last 15 years, optogenetics has changed fundamental research in neuroscience, and is now reaching toward therapeutic applications. Vision restoration strategies using optogenetics are now at the forefront of these new clinical opportunities. But applications to human patients suffering from retinal diseases leading to blindness rise important concerns on the long-term functional expression of optogenes and the efficient signal transmission to higher visual centers. Here we demonstrate in non-human primates, continued expression and functionality at the retina level ∼20 months after delivery of our construct. We also performedin-vivorecordings of visually evoked potentials in the primary visual cortex of anaesthetized animals. Using synaptic blockers, we isolated thein-vivocortical activation resulting from the direct optogenetic stimulation of primate retina.

In conclusion, our work indicates long-term transgene expression and transmission of the signal generated in the macaque retina to the visual cortex, two important features for future clinical applications.
Plasmodium falciparumhydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway
Authors: Scott Alan F. , Deery Evelyne , Lawrence Andrew D. , Warren Martin J.
Published: February 2021

The production of uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified inPlasmodium falciparumbut it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). In this present study it is demonstrated thatP. falciparumHmbS does not have uroporphyrinogen III synthase activity. This was demonstrated by the failure of a codon optimisedP. falciparum hemCgene, encoding HmbS, to compliment a definedE. coli hemD-mutant (SASZ31) deficient in uroporphyrinogen III synthase activity. Furthermore, HPLC analysis of the oxidsed reaction product from recombinant, purified HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, thus showing thatP. falciparumHmbS does not have UroS activity and can only catalyse the formation of hydroxymethylbilane from porphobilinogen.
Reversible blood-brain barrier opening utilizing the membrane active peptide melittinin vitroandin vivo
Authors: Linville Raleigh M. , Komin Alexander , Lan Xiaoyan , DeStefano Jackson G. , Chu Chengyan , Liu Guanshu , Walczak Piotr , Hristova Kalina , Searson Peter C.
Published: February 2021

The blood-brain barrier (BBB) tightly controls entry of molecules and cells into the brain, restricting the delivery of therapeutics. Blood-brain barrier opening (BBBO) utilizes reversible disruption of cell-cell junctions between brain microvascular endothelial cells to enable transient entry into the brain. Development of BBBO techniques has been hindered by a lack of physiological models forin vitrostudy. Here, we utilize anin vitrotissue-engineered microvessel model to demonstrate that melittin, a membrane active peptide present in bee venom, supports BBBO. From endothelial and neuronal viability studies, we identify the accessible concentration range for BBBO. We then use a tissue-engineered model of the human BBB to optimize dosing and elucidate the mechanism of opening. Melittin and other membrane active variants transiently increase paracellular permeability via disruption of cell-cell junctions. In mice, we demonstrate a minimum clinically effective intra-arterial dose of 3 μM·min melittin, which is reversible within one day and neurologically safe. Melittin-induced BBBO represents a novel platform for delivery of therapeutics into the brain.
A comparative study of genomic adaptations to low nitrogen availability in Genlisea aurea
Authors: Goldsborough Thibaut
Published: February 2021

Genlisea aureais a carnivorous plant that grows on nitrogen-poor waterlogged sandstone plateaus and is thought to have evolved carnivory as an adaptation to very low nitrogen levels in its habitat. The carnivorous plant is also unusual for having one of the smallest genomes among flowering plants. Genomic DNA is known to have a high nitrogen content and yet, to the author’s knowledge, no published study has linked nitrogen starvation ofG. aureawith genome size reduction. This comparative study of the carnivorous plantG. aurea, the model organismArabidopsis thaliana(Brassicaceae) and the nitrogen fixingTrifolium pratense(Fabaceae) attempts to investigate whether the genome, transcriptome and proteome ofG. aureashowed evidence of adaptations to low nitrogen availability. It was found that althoughG. aurea’s genome, CDS and non-coding DNA were much lower in nitrogen than the genome ofT. pratenseandA. thalianathis was solely due to the length of the genome, CDS and non-coding sequences rather than the composition of these sequences.
A Human 3D neural assembloid model for SARS-CoV-2 infection
Authors: Wang Lu , Sievert David , Clark Alex E. , Federman Hannah , Gastfriend Benjamin D. , Shusta Eric , Palecek Sean P. , Carlin Aaron F. , Gleeson Joseph
Published: February 2021

Clinical evidence suggests the central nervous system (CNS) is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although mechanisms remain unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points1. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Prior to infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral ‘replication hubs’, with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new ‘assembloid’ model2that supports SARS-CoV-2 entry and replication in neural tissue, and PCCOs serve as an experimental model for neural infection.
Synaptotagmin 7 is enriched at the plasma membrane through γ-secretase
processing to promote vesicle docking and control synaptic plasticity in mouse
hippocampal neurons
Authors: Vevea Jason D. , Kusick Grant F. , Chen Erin , Courtney Kevin C. , Watanabe Shigeki , Chapman Edwin R.
Published: February 2021

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function,
but its localization and precise function in the synaptic vesicle cycle remain
unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate
release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal
neurons. We analyzed asynchronous release, paired pulse facilitation, and
synaptic vesicle replenishment, and found that SYT7 contributes to each of these
processes to different degrees. ‘Zap-and-freeze’ electron microscopy revealed
that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and
the recovery of depleted synaptic vesicles after a stimulus train. To execute
these functions, SYT7 must be targeted to the plasma membrane via
γ-secretase-mediated cleavage of the amino terminus, followed by palmitoylation.
The complex sorting itinerary of SYT7 endows this
Ca2+-sensor with the ability to control crucial forms of
synaptic function and plasticity.

SYT7 mediated asynchronous release, paired pulse facilitation,
and synaptic vesicle replenishment was observed optically at
individual hippocampal synapses

Localization, trafficking, and stability of SYT7 is dependent on
processing by γ-secretase

Short term plasticity defects arise in SYT7KOs due to decreased
docking of synaptic vesicles after stimulation

SYT7 promotes paired-pulse facilitation and asynchronous release
via distinct mechanisms
Streptococcus pyogenesforms serotype and local environment-dependent inter-species protein complexes
Authors: Chowdhury Sounak , Khakzad Hamed , Ertürk Bergdahl Gizem , Lood Rolf , Ekstrom Simon , Linke Dirk , Malmström Lars , Happonen Lotta , Malmström Johan
Published: February 2021

Streptococcus pyogenesis known to cause both mucosal and systemic infections in humans. In this study, we used a combination of quantitative and structural mass spectrometry techniques to determine the composition and structure of the interaction network formed between human plasma proteins and the surface of differentS. pyogenesserotypes. Quantitative network analysis revealed thatS. pyogenesform serotype-specific interaction networks that are highly dependent on the domain arrangement of the surface-attached M protein. Subsequent structural mass spectrometry analysis and computational modelling on one of the M proteins, M28 revealed that the network structure changes across different host microenvironments. We report that M28 binds secretory IgA via two separate binding sites with high affinity in saliva. During vascular leakage mimicked by increasing plasma concentrations in saliva, the binding of secretory IgA was replaced by binding of monomeric IgA and C4BP. This indicates that an upsurge of C4BP in the local microenvironment due to damage of the mucosal membrane drives binding of C4BP and monomeric IgA to M28. The results suggest thatS. pyogeneshas evolved to form microenvironment-dependent host-pathogen protein complexes to combat the human immune surveillance during both mucosal and systemic infections.
New exon ignites accelerated evolution of placental gene Nrk in the ancestral lineage of eutherians
Authors: Liu Guopeng , Zhang Chunxiao , Wang Yuting , Dai Guangyi , Liu Shu-Qun , Wang Wenshuai , Pan Yi-Hsuan , Ding Jianping , Li Haipeng
Published: February 2021

Accelerated evolution is often driven by the interaction between environmental factors and genes. However, it remains unclear whether accelerated evolution can be ignited. Here, we focused on adaptive events during the emergence of chorioallantoic placenta. We scanned the chromosome X and identified eight accelerated regions in the ancestral lineage of eutherian mammals. Five of these regions (P = 5.61 × 10−11 ~ 9.03 × 10−8) are located in the five exons of Nik-related kinase (Nrk), which is essential in placenta development and fetoplacental induction of labor. Moreover, a eutherian-specific exogenous exon lack of splice variant was found to be conserved. Structure modelling of NRK suggests that the accelerated exons and the eutherian-specific exon could change the enzymatic activity of eutherian NRK. Since the eutherian-specific exon was surrounded by accelerated exons, it indicates that the accelerated evolution of Nrk may be ignited by the emergence of the new exon in the ancestral lineage of eutherian mammals. The new exon might shift the function of Nrk and provide a new fitness landscape for eutherian species to explore. Although multiple exons were accelerated in both of the Nrk catalytic and regulatory domains, positive selection can only be revealed on the regulatory domain if the branch specific nonsynonymous and synonymous rate test was performed by PAML. Thus, it may be important to detect accelerated evolution when studying positive selection on coding regions. Overall, this work suggests that the fundamental process of placental development and fetoplacental induction of labor has been targeted by positive Darwinian selection. Identifying positively selected placental genes provides insights into how eutherian mammals gain benefits from the invasive chorioallantoic placenta to form one of the most successful groups among terrestrial vertebrates.
Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection
Authors: Taschner Michael , Basquin Jérôme , Steigenberger Barbara , Schaefer Ingmar , Soh Young-Min , Basquin Claire , Lorentzen Esben , Räschle Markus , Scheltema Richard A. , Gruber Stephan
Published: February 2021

Eukaryotic cells employ three SMC complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.
Impact of scaffolding protein TNRC6 paralogs on gene expression and splicing
Authors: Johnson Samantha T. , Chu Yongjun , Liu Jing , Corey David R.
Published: February 2021

TNRC6 is a scaffolding protein that bridges interactions between small RNAs, argonaute (AGO) protein, and effector proteins to control gene expression. There are three paralogs in mammalian cells,TNRC6A, TNRC6B, and TNRC6C. These paralogs have ~40% amino acid sequence identity and the extent of their unique or redundant functions is unclear. Here, we use knockout cell lines, enhanced crosslinking immunoprecipitation (eCLIP), and high-throughput RNA sequencing (RNAseq) to explore the roles of TNRC6 paralogs in RNA-mediated control of gene expression. We find that that the paralogs are largely functionally redundant and changes in levels of gene expression are well-correlated with those observed inAGOknockout cell lines. Splicing changes observed inAGOknockout cell lines are observed inTNRC6knockout cells. These data further define the roles of the TNRC6 isoforms as part of the RNA interference (RNAi) machinery.
Specification of axial identity by Hoxa2 distinguishes between a phenotypic and molecular ground state in mouse cranial neural crest cells
Authors: Pushel Irina , Trainor Paul A , Krumlauf Robb
Published: February 2021

Hoxgenes play a key role in head formation by specifying the axial identity of neural crest cells (NCCs) migrating into embryonic pharyngeal arches. In the absence ofHoxa2, NCC derivatives of the second pharyngeal arch (PA2) undergo a homeotic transformation and duplicate structures formed by first arch (PA1) NCCs. Current models postulate that PA1 represents a NCC ‘ground state’ and loss ofHoxa2causes a reversion of PA2 NCCs to the PA1 ‘ground state’. We use bulk and single-cell RNAseq to investigate the molecular mechanisms driving this phenotypic transformation in the mouse. InHoxa2-/-mutants, PA2 NCCs generally maintain expression of the PA2 transcriptional signature and fail to strongly upregulate a PA1 transcriptional signature. Our analyses identify putative HOXA2 targets and suggest that subsets of NCCs may respond to HOXA2 activity in distinct manners. This separation of phenotypic and molecular states has significant implications for understanding craniofacial development.
Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells
Authors: Bernadskaya Yelena Y. , Yue Haicen , Copos Calina , Christiaen Lionel , Mogilner Alex
Published: February 2021

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that these multicellular arrangements confer biochemical and biomechanical properties that contribute to tissue scale organization. The cardiopharyngeal progenitors of the tunicate Ciona provide the simplest possible model of collective cell migration. They form cohesive bilateral cell pairs, leader-trailer polarized along the migration path as they migrate between the ventral epidermis and trunk endoderm. Here, circumventing difficulties in quantifying cellular mechanics in live embryos, we use the Cellular Potts Model to computationally probe the distributions of forces consistent with the shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we first determine that cardiopharyngeal progenitors display hallmarks of supracellular organization, with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. Combined 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy that contributes to aligning collective polarity with the direction of migration, as observed with three or more cells bothin silicoandin vivo. Our approach reveals emerging properties of the migrating collective. Specifically, cell pairs are more persistent, thus migrating over longer distances, and presumably with higher accuracy. Finally, simulations suggest that polarized cell pairs literally join forces to deform the trunk endoderm, as they migrate through the extracellular space. We thus propose that the polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.
High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples
Authors: Pater Adrian A. , Bosmeny Michael S. , Parasrampuria Mansi , Eddington Seth B. , Ovington Katy N. , White Adam A. , Barkau Christopher L. , Metz Paige E. , Sylvain Rourke J. , Chilamkurthy Ramadevi , Yinusa Abadat O. , Benzinger Scott W. , Hebert Madison M. , Gagnon Keith T.
Published: February 2021

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking movement and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using online visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable ‘how-to’ reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and next-generation nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.
Population density affects the outcome of competition in co-cultures of Gardnerella species isolated from the human vaginal microbiome
Authors: Khan Salahuddin , Hill Janet E.
Published: February 2021

Negative frequency-dependent selection is one possible mechanism for maintenance of rare species in communities, but the selective advantage of rare species may be checked at lower overall population densities where resources are abundant. Gardnerella spp. belonging to cpn60 subgroup D, are detected at low levels in vaginal microbiomes and are nutritional generalists relative to other more abundant Gardnerella spp., making them good candidates for negative frequency-dependent selection. The vaginal microbiome is a dynamic environment and the resulting changes in density of the microbiota may explain why subgroup D never gains dominance. To test this, we co-cultured subgroup D isolates with isolates from the more common and abundant subgroup C. Deep amplicon sequencing of rpoB was used to determine proportional abundance of each isolate at 0 h and 72 h in 152 co-cultures, and to calculate change in proportion. D isolates had a positive change in proportional abundance in most co-cultures regardless of initial proportion. Initial density affected the change in proportion of subgroup D isolates either positively or negatively depending on the particular isolates combined, suggesting that growth rate, population density and other intrinsic features of the isolates influenced the outcome. Our results demonstrate that population density is an important factor influencing the outcome of competition between Gardnerella spp. isolated from the human vaginal microbiome.