bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of an STR panel for a non-native population of an endangered species
Authors: Hill Erin , Linacre Adrian , Toop Simon , Murphy Nicholas , Strugnell Jan
Published: May 2021

The establishment of non-native populations of threatened and legally protected species can have many implications for the areas where these species have been introduced. Non-native populations of threatened species have the potential to be exploited and therefore the subject of legal protection, while conversely, if they have become invasive in their introduced range, there is the likelihood that population control will be carried out to reduce abundance and negative impacts associated with introduced species. From both a legal and invasive species monitoring standpoint, it is important to know how many individuals are present. Short tandem repeats (STRs) were developed for the hog deer, an endangered species that was introduced following European settlement to Victoria, Australia using Illumina MiSeq sequencing technology. These markers were combined with previous STRs characterised for hog deer to create a 29-plex identification system. A total of 224 samples were genotyped across the population in Victoria, and further analyses of null allele frequencies, deviation from Hardy-Weinberg equilibrium, and the removal of monomorphic or low amplifying markers resulted in a final marker panel of 17 loci. Despite low values for number of alleles at each locus (2-4), probability of identity showed sufficient discrimination power, with an average probability of identity at 9.46 × 10−7, and a probability of sibling identity of 7.67 × 10−4 across all sites. These findings show that it is feasible to create an informative DNA profiling system that can distinguish between individuals for applications in both wildlife forensic and population control research.
Transcriptomic profiling of human orbital fat and differentiating orbital fibroblasts
Authors: Kim Dong Won , Taneja Kamil , Hoang Thanh , Santiago Clayton P. , McCulley Timothy J. , Merbs Shannath L. , Mahoney Nicholas R. , Blackshaw Seth , Rajaii Fatemeh
Published: May 2021

Structured Abstract

Purpose

Orbital fat hyperplasia has a central role in the manifestations of thyroid-associated orbitopathy (TAO). To better understand the pathways involved in adipogenesis in TAO, we have used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating orbital fibroblasts (OFs).

Methods

We performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat to compare gene expression in control and TAO patients. We treated cultured OFs derived from TAO patients with media containing dexamethasone, insulin, rosiglitazone, and isobutylmethylxanthine (IBMX) to induce adipogenesis. We used single nuclear RNA-Seq (snRNA-Seq) profiling of treated OFs to compare gene expression over time in order to identify pathways that are involved in orbital adipogenesis in vitro and compared the dynamic patterns of gene expression identify differences in gene expression in control and TAO orbital fat.

Results

Orbital fat from TAO and control patients segregate with principal component analysis (PCA). Numerous signaling pathways are enriched in orbital fat isolated from TAO patients. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of adipocyte-specific genes over the developmental time course. Furthermore, genes that are enriched in TAO orbital fat are also upregulated in orbital adipocytes that differentiate in vitro, while genes that are enriched in control orbital fat are enriched in orbital fibroblasts prior to differentiation.

Conclusions

Differentiating orbital fibroblasts serve as a model to study orbital fat hyperplasia seen in TAO. We demonstrate that the insulin-like growth factor-1 receptor (IGF-1R) and Wnt signaling pathways are differentially expressed early in orbital adipogenesis.

Précis

To understand the pathways involved in adipogenesis in TAO, we used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating OFs. We demonstrate that the IGF-1R and Wnt signaling pathways are differentially expressed during orbital adipogenesis.
New cellular models of undifferentiated pleomorphic sarcoma and malignant peripheral nerve sheath tumor
Authors: Bhalla Angela D. , Landers Sharon M. , Singh Anand K. , Yeagley Michelle G. , Meyerson Gabryella S.B. , Mulder Zachary A. , Delgado-Baez Cristian B. , Dunnand Stephanie , Kochat Veena , Tomczak Katarzyna J. , Nguyen Theresa , Ma Xiaoyan , Bolshakov Svetlana , Menegaz Brian A. , Lamhamedi-Cherradi Salah-Eddine , Ludwig Joseph A. , Beird Hannah C. , Mao Xizeng , Song Xingzhi , Ingram Davis R. , Wang Wei-Lien , Lazar Alexander J. , McCutcheon Ian E. , Slopis John M. , Rai Kunal , Zhang Jianhua , Lev Dina C. , Torres Keila E.
Published: May 2021

Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models led us to generate additional cell lines to study these rare cancers further. Patient-derived tumors were used to establish 5 new UPS models, including one radiation-associated UPS—UPS060.1, UPS271.1, UPS511, UPS0103, and RIS620—and 3 new models of MPNST—MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of each cell line was determined by either subcutaneous injection of cells or implantation of tumor tissue into immunocompromised mice. UPS060.1, UPS271.1, and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Mutation analysis of a panel of genes frequently mutated in sarcomas showed that two of the three MPNST cell lines had NF1 mutations. Two of the three MPNST cell lines had mutations in polycomb repressive complex 2 members. These new cellular models provide the scientific community with powerful tools for detailed studies of sarcomagenesis and investigate novel therapies for UPS and MPNST.
Illuminating the plant rhabdovirus landscape through metatranscriptomics data
Authors: Bejerman Nicolás , Dietzgen Ralf G. , Debat Humberto
Published: May 2021

Rhabdoviruses infect a large number of plant species and cause significant crop diseases. They have a negative-sense, single-stranded unsegmented or bisegmented RNA genome. The number of plant-associated rhabdovirid sequences has grown in the last few years in concert with the extensive use of high-throughput sequencing platforms. Here, we report the discovery of 27 novel rhabdovirus genomes associated with 25 different host plant species and one insect, which were hidden in public databases. These viral sequences were identified through homology searches in more than 3000 plant and insect transcriptomes from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) using known plant rhabdovirus sequences as the query. The identification, assembly and curation of raw SRA reads resulted in sixteen viral genome sequences with full-length coding regions and ten partial genomes. Highlights of the obtained sequences include viruses with unique and novel genome organizations among known plant rhabdoviruses. Phylogenetic analysis showed that thirteen of the novel viruses were related to cytorhabdoviruses, one to alphanucleorhabdoviruses, five to betanucleorhabdoviruses, one to dichorhaviruses and seven to varicosaviruses. These findings resulted in the most complete phylogeny of plant rhabdoviruses to date and shed new light on the phylogenetic relationships and evolutionary landscape of this group of plant viruses. Furthermore, this study provided additional evidence for the complexity and diversity of plant rhabdovirus genomes and demonstrated that analyzing SRA public data provides an invaluable tool to accelerate virus discovery, gain evolutionary insights and refine virus taxonomy.
A single short reprogramming early in life improves fitness and increases lifespan in old age
Authors: Alle Quentin , Le Borgne Enora , Bensadoun Paul , Lemey Camille , Béchir Nelly , Gabanou Mélissa , Estermann Fanny , Bertrand-Gaday Christelle , Pessemesse Laurence , Toupet Karine , Vialaret Jérôme , Hirtz Christophe , Noël Danièle , Jorgensen Christian , Casas François , Milhavet Ollivier , Lemaitre Jean-Marc
Published: May 2021

Forced and maintained expression of four transcription factors OCT4, SOX2, KLF4 and c-MYC (OSKM), can reprogram somatic cells into induced Pluripotent Stem Cells (iPSCs) and a limited OSKM induction is able to rejuvenate the cell physiology without changing the cell identity. We therefore sought to determine if a burst of OSKM might improve tissue fitness and delay age-related pathologies in a whole animal. For this, we used a sensitive model of heterozygous premature aging mice carrying just one mutated Lamin A allele producing progerin. We briefly treated two months-young heterozygotes mice with OSKM and monitored their natural age-related deterioration by various health parameters. Surprisingly, a single two and a half weeks reprogramming was sufficient to improve body composition and functional capacities, over the entire lifespan. Mice treated early in life had improved tissue structures in bone, lung, spleen, kidney and skin, with an increased lifespan of 15%, associated to a differential DNA methylation signature. Altogether, our results indicate that a single short reprogramming early in life might initiate and propagate an epigenetically related rejuvenated cell physiology, to promote a healthy lifespan.

One Sentence summary

A single short reprogramming early in life rejuvenates cell physiology, improves body composition, tissue fitness and increases lifespan in elderly.
Do soil features condition seed germination of gypsum plants? An analysis of the effect of different natural soils
Authors: Heiden Nathaniel , Cera Andreu , Palacio Sara
Published: May 2021

Gypseous soils are widespread across arid and semiarid environments worldwide. They present remarkable challenges to plants and host a unique flora. We aimed to assess up to what point the specificity and distribution of species on gypsum might be driven by species-specific germination responses to soil gypsum availability. We analyzed the germination of six gypsum specialists and four closely related generalist plant species from the Iberian Peninsula and the Chihuahuan Desert in four different field soils with contrasting concentrations of gypsum, pH and soil texture. Plant restriction to gypsum was unrelated to the germinating ability of seeds on different substrates. Irrespective of their affinity for gypsum, most species germinated better on mixed gypsum-calcareous soil and worse in the acidic soil treatment. Our data suggest soil pH was the main soil feature driving seed germination, while the effect of gypsum content was generally not significant. The main exception was the Iberian gypsum specialist Helianthemum squamatum (L.) Dum. Cours., which showed increased germination on gypseous soils and higher germination in response to increased soil gypsum content. Except for this species, our findings indicate alkaline soils favor the germination of most of the species analyzed, irrespectively of their gypsum content.

Highlights

Gypsum soils are one of the most widespread special substrates of the world

We evaluated the effects of natural soils on the germination of gypsum plants

Soil pH was the main soil feature driving seed germination

The effect of gypsum content was not significant

We suggest a potential origin of gypsophiles from plants adapted to alkaline soils
Stomatal CO2-responses at sub- and above-ambient CO2levels employ different pathways in Arabidopsis
Authors: Koolmeister Kaspar , Merilo Ebe , Hõrak Hanna , Kollist Hannes
Published: May 2021

Stomatal pores that control plant CO2uptake and water loss affect global carbon and water cycles. In the era of increasing atmospheric CO2levels and vapor pressure deficit (VPD), it is essential to understand how these stimuli affect stomatal behavior. It is unknown whether stomatal responses to sub-ambient and above-ambient CO2levels are governed by the same regulators and whether these responses depend on VPD. We studied stomatal conductance responses in Arabidopsis stomatal signaling mutants under conditions where CO2levels were either increased from sub-ambient to ambient (400 ppm) or from ambient to above-ambient levels under normal or elevated VPD. We found that guard cell signaling components involved in CO2-induced stomatal closure have different roles in the sub-ambient and above-ambient CO2levels. The CO2-specific regulators prominently affected sub-ambient CO2responses, whereas the lack of guard cell slow-type anion channel SLAC1 more strongly affected the speed of above-ambient CO2-induced stomatal closure. Elevated VPD caused lower stomatal conductance in all and faster CO2-responsiveness in some studied genotypes and CO2-transitions. Our results highlight the importance of experimental set-ups in interpreting stomatal CO2- responsiveness, as stomatal movements under different CO2concentration ranges are controlled by distinct mechanisms. Sometimes elevated CO2and VPD responses also interact. Hence, multi-factor treatments are needed to understand plant behavior under future climate conditions.
Partial response electromyography as a marker of action stopping
Authors: Raud Liisa , Thunberg Christina , Huster René J.
Published: May 2021

Response inhibition is among the core constructs of cognitive control. It is notoriously difficult to quantify from overt behavior, since the outcome of successful inhibition is the lack of a behavioral response. Currently, the most common measure of action stopping, and by proxy response inhibition, is the model-based stop signal reaction time (SSRT) derived from the stop signal task. Recently, partial response electromyography (prEMG) has been introduced as a complementary physiological measure to capture individual stopping latencies. PrEMG refers to muscle activity initiated by the go signal that plummets after the stop signal before its accumulation to a full response. Whereas neither the SSRT nor the prEMG is an unambiguous marker for neural processes underlying response inhibition, our analysis indicates that the prEMG peak latency is better suited to investigate brain mechanisms of action stopping. This study is a methodological resource with a comprehensive overview of the psychometric properties of the prEMG in a stop signal task, and further provides practical tips for data collection and analysis.
Lack of food intake during shift work alters the heart transcriptome and leads to cardiac fibrosis and inflammation in rats
Authors: Trott Alexandra J. , Greenwell Ben J , Karhadkar Tejas R. , Guerrero-Vargas Natali N. , Escobar Carolina , Buijs Ruud M , Menet Jerome S
Published: May 2021

Many epidemiological studies revealed that shift work is associated with increased risk of cardiovascular diseases. However, the underlying mechanisms remain poorly understood. An experimental model of shift work in rats has been shown to recapitulate the metabolic disorders observed in human shift workers, and used to demonstrate that restricting food consumption outside working hours prevents shift work-associated obesity and metabolic disturbance. Here we used this model to characterize the effects of shift work in the heart. We show that experimental shift work reprograms the heart cycling transcriptome independently of food consumption. While phases of rhythmic gene expression are distributed across the 24-hour day in control rats, they are clustered towards discrete times in shift workers. Additionally, preventing food intake during shift work affects the expression level of hundreds of genes in the heart. Many of them are found in transcriptional signatures associated with pressure overload and cardiac hypertrophy, and encode for components of the extracellular matrix and inflammatory markers. Consistent with this, the heart of shift worker rats not eating during work exhibits fibrosis and is colonized by immune cells. While maintaining food access during shift work has less effects on gene expression, genes found in transcriptional signatures of cardiac hypertrophy remain affected, and the heart of shift worker rats exhibits fibrosis without inflammation. Together, our findings provide insights into how shift work affects cardiac function, and suggest that some interventions aiming at mitigating metabolic disorders in shift workers may have adverse effects on cardiovascular diseases.
Scalable mapping of myelin and neuron density in the human brain with micrometer resolution
Authors: Chang Shuaibin , Varadarajan Divya , Yang Jiarui , Chen Ichun Anderson , Kura Sreekanth , Magnain Caroline , Augustinack Jean C. , Fischl Bruce , Greve Douglas N. , Boas David A. , Wang Hui
Published: May 2021

Optical Coherence Tomography (OCT) is an emerging 3D imaging technique that allows quantification of intrinsic optical properties such as scattering coefficient and back-scattering coefficient, and has proved useful in distinguishing delicate microstructures in the human brain. The origins of scattering in brain tissues are contributed by the myelin content, neuron size and density primarily; however, no quantitative relationships between them have been reported, which hampers the use of OCT in fundamental studies of architectonic areas in the human brain and the pathological evaluations of diseases. To date, histology remains the golden standard, which is prone to errors and can only work on a small number of subjects. Here, we demonstrate a novel method that uses serial sectioning OCT to quantitatively measure myelin content and neuron density in the human brain. We found that the scattering coefficient possesses a strong linear relationship with the myelin content across different regions of the human brain, while the neuron density serves as a secondary contribution that only slightly modulates the overall tissue scattering.
Scale-dependent tipping points of bacterial colonization resistance
Authors: Karita Yuya , Limmer David T. , Hallatschek Oskar
Published: May 2021

Bacteria are efficient colonizers of a wide range of secluded micro-habitats, such as soil pores, skin follicles, or intestinal crypts. How the structural diversity of these habitats modulates microbial self-organization remains poorly understood, in part because of the challenge to specifically manipulate the physical structure of microbial environments. Using a microfluidic device to grow bacteria in crypt-like incubation chambers of systematically varied lengths, we show that small variations in the physical structure of the micro-habitat can drastically alter bacterial colonization success and resistance against invaders. Small crypts are un-colonizable, intermediately sized crypts can stably support dilute populations, while beyond a second critical lengthscale, populations phase-separate into a dilute and a jammed region. The jammed state is characterized by extreme colonization resistance, even if the resident strain is suppressed by an antibiotic. Combined with a flexible biophysical model, we demonstrate that colonization resistance and associated priority effects can be explained by a crowding-induced phase transition, which results from a competition between proliferation and density-dependent cell leakage. The emerging sensitivity to scale underscores the need to control for scale in microbial ecology experiments. Systematic flow-adjustable lengthscale variations may serve as a promising strategy to elucidate further scale-sensitive tipping points and to rationally modulate the stability and resilience of microbial colonizers.
Mesothelial cells are not a source of adipocytes in mice
Authors: Westcott Gregory P. , Emont Margo P. , Li Jin , Jacobs Christopher , Tsai Linus , Rosen Evan D.
Published: May 2021

Visceral adipose tissue (VAT) depots are associated with the adverse metabolic consequences of obesity, such as insulin resistance. The developmental origin of VAT depots and the identity and regulation of adipocyte progenitor cells have been active areas of investigation. In recent years, a paradigm of mesothelial cells as a source of VAT adipocyte progenitor cells has emerged based on lineage-tracing studies using the Wilms’ tumor gene, Wt1, as a marker for cells of mesothelial origin. Here we show that Wt1 expression in adipose tissue is not limited to the mesothelium, but is also expressed by a distinct preadipocyte population in both mice and humans. We identify keratin 19 (Krt19) as a highly-specific marker for the adult mouse mesothelium, and demonstrate that Krt19-expressing mesothelial cells do not differentiate into visceral adipocytes. These results contradict the assertion that the VAT mesothelium can serve as a source of adipocytes.
DeGlyPHER: an ultrasensitive method for analysis of viral spike N-glycoforms
Authors: Baboo Sabyasachi , Diedrich Jolene K , Martínez-Bartolomé Salvador , Wang Xiaoning , Schiffner Torben , Groschel Bettina , Schief William R , Paulson James C , Yates John R
Published: May 2021

Viruses can evade the host immune system by displaying numerous glycans on their surface “spike-proteins” that cover immune epitopes. We have developed an ultrasensitive “single pot” method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.
The EphB2-MYC Axis is a Major Determinant of Barrett’s Pathobiology and a Therapeutic Vulnerability in Esophageal Cancer
Authors: Venkitachalam Srividya , Babu Deepak , Ravillah Durgadevi , Katabathula Ramachandra M. , Joseph Peronne , Singh Salendra , Udhayakumar Bhavatharini , Miao Yanling , Martinez-Uribe Omar , Hogue Joyce A. , Kresak Adam M. , Dawson Dawn , LaFramboise Thomas , Willis Joseph E. , Chak Amitabh , Garman Katherine S. , Blum Andrew E. , Varadan Vinay , Guda Kishore
Published: May 2021

Esophageal adenocarcinoma (EAC), a highly aggressive cancer with limited therapeutic options, often arises in the backdrop of a molecularly-complex esophageal metaplasia disorder, Barrett’s Esophagus (BE). Using transcriptomics and systems biology analyses of treatment-naïve malignant/pre-malignant biopsy tissues, we found Eph receptor B2 (EphB2) tyrosine kinase signaling to be frequently hyperactivated during early stages of EAC development, and across the BE-EAC continuum. Functional studies revealed EphB2 to be an upstream post-translational regulator of c-MYC activity and as a key molecular dependency in BE/EAC. Single-cell transcriptomics in a porcine esophageal 3D spheroid model showed enhanced EphB2 and MYC activity to be significantly associated with BE-like cell fate. shRNA-based knockdown of EphB2 or small molecule inhibitors of MEK, that modulate MYC protein stability, proved effective in suppressing EAC tumor growth in vivo. These findings point to EphB2-MYC axis as an early promoter of EAC and a novel therapeutic vulnerability in this increasingly-prevalent esophageal malignancy.

STATEMENT OF SIGNIFICANCE

We identify EphB2 signaling as a potential master regulator and early promoter of esophageal adenocarcinoma, and the proto-oncogene MYC as a key downstream effector of EphB2 function. Targeting the EphB2-MYC axis could be a promising therapeutic strategy for these often refractory and lethal EAC tumors.
Molecular diversity and lineage commitment of human interneuron progenitors
Authors: Velmeshev Dmitry , Chavali Manideep , Nowakowski Tomasz J. , Bhade Mohini , Mayer Simone , Goyal Nitasha , Alvarado Beatriz , Mancia Walter , Wang Shaohui , Speir Matthew , Haeussler Maximilian , Rowitch David , Alvarez-Buylla Arturo , Huang Eric J. , Paredes Mercedes , Kriegstein Arnold
Published: May 2021

Cortical interneurons are indispensable for proper function of neocortical circuits. Changes in interneuron development and function are implicated in human disorders, such as autism spectrum disorder and epilepsy. In order to understand human-specific features of cortical development as well as the origins of neurodevelopmental disorders it is crucial to identify the molecular programs underlying human interneuron development and subtype specification. Recent studies have explored gene expression programs underlying mouse interneuron specification and maturation. We applied single-cell RNA sequencing to samples of second trimester human ganglionic eminence and developing cortex to identify molecularly defined subtypes of human interneuron progenitors and immature interneurons. In addition, we integrated this data from the developing human ganglionic eminences and neocortex with single-nucleus RNA-seq of adult cortical interneurons in order to elucidate dynamic molecular changes associated with commitment of progenitors and immature interneurons to mature interneuron subtypes. By comparing our data with published mouse single-cell genomic data, we discover a number of divergent gene expression programs that distinguish human interneuron progenitors from mouse. Moreover, we find that a number of transcription factors expressed during prenatal development become restricted to adult interneuron subtypes in the human but not the mouse, and these adult interneurons express species- and lineage-specific cell adhesion and synaptic genes. Therefore, our study highlights that despite the similarity of main principles of cortical interneuron development and lineage commitment between mouse and human, human interneuron genesis and subtype specification is guided by species-specific gene programs, contributing to human-specific features of cortical inhibitory interneurons.
The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions
Authors: Gopalakrishnan Rajaraman , Winston Fred
Published: May 2021

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5’ ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.
Influenza A virus defective viral genomes are inefficiently packaged into virions relative to wild-type genomic RNAs
Authors: Alnaji Fadi G. , Reiser William K. , Velthuis Aartjan te , Brooke Christopher B.
Published: May 2021

Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hotspots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild type genomic RNAs. Surprisingly, packaged DelVGs exhibited no signs of enrichment for specific deletion characteristics suggesting that all DelVGs are equally limited in packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the IAV replication machinery and shed light on the mechanisms that underly DelVG formation.

Importance

Defective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes, hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain elusive. Here, we used a specialized sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This dataset provides an unbiased profile of the deletion-forming preferences of the influenza virus replicase. Additionally, by comparing the early intracellular DelVGs with those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.
The role of accelerated growth plate fusion in the absence of SOCS2 on osteoarthritis vulnerability
Authors: Samvelyan Hasmik J. , Huesa Carmen , Lin Lucy Cui , Farquharson Colin , Staines Katherine A.
Published: May 2021

Osteoarthritis is the most prevalent systemic musculoskeletal disorder characterised by articular cartilage degeneration and subchondral bone (SCB) sclerosis. Here we sought to examine the contribution of accelerated growth to osteoarthritis development using a murine model of excessive longitudinal growth. Suppressor of cytokine signalling 2 (SOCS2) is a negative regulator of growth hormone (GH) signalling, thus mice deficient in SOCS2 (Socs2-/-) display accelerated bone growth. We examined vulnerability ofSocs2-/-mice to osteoarthritis following surgical induction of disease (destabilisation of the medial meniscus (DMM)), and with ageing, by histology and micro-CT. We observed significant increase in number (WT DMM: 532±56; WT sham: 495±45; KO DMM: 169±49; KO sham: 187±56; P<0.01) and density (WT DMM: 2.2±0.9; WT sham: 1.2±0.5; KO DMM: 13.0±0.5; KO sham: 14.4±0.7) of growth plate bridges inSocs2-/-in comparison to wild-type (WT). Histological examination of WT andSocs2-/-knees revealed articular cartilage damage with DMM in comparison to sham (WT DMM: 3.4±0.4; WT sham: 0.3±0.05 (P<0.05); KO DMM: 3.2±0.8; KO sham: 0.8±0.3). Articular cartilage lesion severity scores (mean and maximum) were similar in WT andSocs2-/-mice with either DMM, or with ageing. Micro-CT analysis revealed significant decreases in SCB thickness, epiphyseal trabecular number and thickness in the medial compartment ofSocs2-/-, in comparison to WT (P<0.001). DMM had no effect on the SCB thickness in comparison to sham in either genotype. Together these data suggest that enhanced GH signalling through SOCS2 deletion accelerates growth plate fusion, however this has no effect on osteoarthritis vulnerability in this model.

Summary statement

Deletion of SOCS2 results in accelerated growth plate fusion, however this has no effect on osteoarthritis vulnerability.
Overdominant mutations restrict adaptive loss of heterozygosity at linked loci
Authors: Fisher Kaitlin J. , Vignogna Ryan C. , Lang Gregory I.
Published: May 2021

Loss of heterozygosity is a common mode of adaptation in asexual diploid populations. Because mitotic recombination frequently extends the full length of a chromosome arm, the selective benefit of loss of heterozygosity may be constrained by linked heterozygous mutations. In a previous laboratory evolution experiment with diploid yeast, we frequently observed homozygous mutations in the WHI2 gene on the right arm of Chromosome XV. However, when heterozygous mutations arose in the STE4 gene, another common target on Chromosome XV, loss of heterozygosity at WHI2 was not observed. Here we show that mutations at WHI2 are partially dominant and that mutations at STE4 are overdominant. We test whether beneficial heterozygous mutations at these two loci interfere with one another by measuring loss of heterozygosity at WHI2 over 1,000 generations for ∼300 populations that differed initially only at STE4 and WHI2. We show that the presence of an overdominant mutation in STE4 reduces, but does not eliminate, loss of heterozygosity at WHI2. By sequencing 40 evolved clones, we show that populations with linked overdominant and partially dominant mutations show less parallelism at the gene level, more varied evolutionary outcomes, and increased rates of aneuploidy. Our results show that the degree of dominance and the phasing of heterozygous beneficial mutations can constrain loss of heterozygosity along a chromosome arm, and that conflicts between partially dominant and overdominant mutations can affect evolutionary outcomes.

SIGNIFICANCE STATEMENT

In diploid populations, it is beneficial for partially dominant beneficial mutations to lose heterozygosity, but it is deleterious for overdominant beneficial mutations to do so. Because loss-of-heterozygosity tracts often encompass entire chromosome arms, a conflict will arise when a partially dominant beneficial mutation and an overdominant beneficial mutation exist in close proximity. We demonstrate that this conflict occurs, and that it restricts loss of heterozygosity, resulting in more variable evolutionary outcomes.
iSubGen: Integrative Subtype Generation by Pairwise Similarity Assessment
Authors: Fox Natalie S. , Haider Syed , Li Constance H. , Boutros Paul C.
Published: May 2021

There are myriad types of biomedical data– genetics, transcriptomics, clinical, imaging, wearable devices and many more. When a group of patients with the same underlying disease exhibit similarities across multiple types of data, this is called a subtype. Disease subtypes can reflect etiology and sometimes predict clinical behaviour. Existing subtyping approaches struggle to simultaneously handle multiple diverse data types, particularly when there is missing information, as is common in most real-world clinical datasets. To improve subtype discovery, we exploited changes in the correlation-structure between different data types to create iSubGen, an algorithm forintegrativesubtypegeneration. iSubGen can combine arbitrary data types for subtype discovery, such as merging molecular, mutational signature, pathway and micro-environmental data. iSubGen recapitulates known subtypes across multiple diseases, even in the face of substantial missing data. It identifies groups of patients with divergent clinical outcomes, and can combine arbitrary data types for subtype discovery, such as merging molecular, mutational signature, pathway and micro-environmental data. iSubGen can accommodate any feature that can be compared with a similarity-metric, and provides a versatile approach for creating subtypes. It is available athttps://CRAN.R-project.org/package=iSubGen.
A platform for deep sequence-activity mapping and engineering antimicrobial peptides
Authors: DeJong Matthew P. , Ritter Seth C. , Fransen Katharina A. , Tresnak Daniel T. , Golinski Alexander W. , Hackel Benjamin J.
Published: May 2021

Developing potent antimicrobials, and platforms for their study and engineering, is critical as antibiotic resistance grows. A high-throughput method to quantify antimicrobial peptide and protein (AMP) activity across a broad continuum can elucidate sequence-activity landscapes and identify potent mutants. We developed a platform to perform sequence-activity mapping of AMPs via depletion (SAMP-Dep): a bacterial host culture is transformed with an AMP mutant library, induced to express AMPs, grown, and deep sequenced to quantify mutant frequency. The slope of mutant growth rate versus induction level indicates potency. Using SAMP-Dep, we screened 170,000 mutants of oncocin, a proline-rich AMP, for intracellular activity against Escherichia coli. Clonal validation of 36 mutants supported SAMP-Dep sensitivity and accuracy. The efficiency and accuracy of SAMP-Dep enabled mapping the oncocin sequence-activity space with remarkable detail and scale and guided focused, successful synthetic peptide library design, yielding a mutant with two-fold enhancement in both intracellular and extracellular activity.
Differential receptor crosstalk in DRD1-DRD2 heterodimer upon phasic and tonic dopamine signals
Authors: Kim Hyunbin , Nam Min-Ho , Jeong Sohyeon , Lee Hyowon , Oh Soo-Jin , Kim Jeongjin , Choi Nakwon , Seong Jihye
Published: May 2021

In response to phasic and tonic release, dopamine neurotransmission is regulated by its receptor subtypes, mainly dopamine receptor type 1 and 2 (DRD1 and DRD2). These dopamine receptors are known to form a heterodimer, however the receptor crosstalk between DRD1 and DRD2 was only suspected by measuring their downstream signaling products, due to the lack of methodology for selectively detecting individual activity of different dopamine receptors. Here, we develop red DRD1 sensor (R-DRD1) and green DRD2 sensor (G-DRD2) which can specifically monitor the real-time activity of DRD1 and DRD2, and apply these multicolor sensors to directly measure the receptor crosstalk in the DRD1-DRD2 heterodimer. Surprisingly, we discover that DRD1 activation in the heterodimer is inhibited only at micromolar phasic concentration of dopamine, while DRD2 activation is selectively inhibited at nanomolar tonic dopamine level. Differential receptor crosstalk in the DRD1-DRD2 heterodimer further modulates their downstream cAMP level. These data imply a novel function of the DRD1-DRD2 heterodimer at physiological dopamine levels of phasic and tonic release. Our approach utilizing multicolor receptor sensors will be useful to discover novel function of GPCR heterodimers.
Assessing the role of cold-shock protein C: A novel regulator of Acinetobacter baumannii biofilm formation and virulence
Authors: Tomlinson Brooke R. , Denham Grant A. , Brzozowski Robert S. , Allen Jessie L. , Eswara Prahathees J. , Shaw Lindsey N.
Published: May 2021

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings and can spread quickly via healthcare personnel despite preventive measures. This resilience is often attributed to a proclivity for biofilm formation, which grants A. baumannii a higher tolerance towards external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Herein, we performed RNA-seq on biofilm and planktonic populations for the multidrug resistant isolate, AB5075, and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), produced a mucoidal, non-mucoviscous colony phenotype. RNA-sequencing of this mutant revealed the down regulation of pili and fimbriae in the cspC mutant, which would explain the decreased biofilm observed. Using MIC assays, we note that the mutant displayed increased antibiotic tolerance that we hypothesize is mediated by overexpression of multi-drug efflux pumps and altered mRNA stability of their corresponding transcriptional repressor. Finally, we show that CspC is required for survival during oxidative stress and challenge by the human immune system, and plays a pivotal role during systemic infection. Collectively, our work identifies a cadre of new biofilm associated genes within A. baumannii and provides insight into the global regulatory network of this emerging human pathogen.
SAR11 Cells Rely on Enzyme Multifunctionality to Transport and Metabolize a Range of Polyamine Compounds
Authors: Noell Stephen E. , Barrell Gregory E. , Suffridge Christopher , Morré Jeff , Gable Kevin P. , Graff Jason R. , VerWey Brian J. , Hellweger Ferdi L. , Giovannoni Stephen J.
Published: May 2021

In the ocean surface layer and cell culture, the polyamine transport protein PotD of SAR11 bacteria is often one of the most abundant proteins detected. Polyamines are organic cations produced by all living organisms and are thought to be an important component of dissolved organic matter (DOM) produced in planktonic ecosystems. We hypothesized that SAR11 cells transport and metabolize multiple polyamines and use them as sources of carbon and nitrogen. Metabolic footprinting and fingerprinting were used to measure the uptake of five polyamine compounds (putrescine, cadaverine, agmatine, norspermidine, and spermidine) in two SAR11 strains that represent the majority of SAR11 cells in the surface ocean environment, Ca. Pelagibacter st. HTCC7211 and C. P. ubique st. HTCC1062. Both strains transported all five polyamines and concentrated them to micromolar or millimolar intracellular concentrations. Both strains could use most of the polyamines to meet their nitrogen requirements, but we did not find evidence of use as carbon sources. We propose potABCD transports cadaverine, agmatine, and norspermidine, in addition to its usual substrates of spermidine and putrescine, and that spermidine synthase, speE, is reversible, catalyzing the breakdown of spermidine and norspermidine, in addition to its usual biosynthetic role. These findings provide support for the hypothesis that enzyme multifunctionality enables streamlined cells in planktonic ecosystems to increase the range of DOM compounds they oxidize.

Importance

Genome streamlining in SAR11 bacterioplankton has resulted in a small repertoire of genes, yet paradoxically they consume a substantial fraction of primary production in the oceans. Enzyme multifunctionality is hypothesized to be an adaptation that increases the range of organic compounds oxidized by cells in environments where selection favors genome minimization. We provide experimental support for this hypothesis by demonstrating that SAR11 cells use multiple polyamine compounds and propose that a small set of multifunctional genes catalyze this metabolism. We also report polyamine uptake rates can exceed metabolism, resulting in high intracellular concentrations of these nitrogen-rich compounds and an increase in cell size. Increases in cytoplasmic solute concentrations during transient episodes of high nutrient exposure has previously been observed in SAR11 cells and may be a feature of their strategy for maximizing the share of labile DOM acquired when in competition with other cell types.
Efficacy and Safety of Polyherbal formulation as an add-on to the standard of care in mild to moderate COVID-19: A randomized, double-blind, placebo-controlled trial
Authors: Patankar Suresh B. , Rangnekar Hrishikesh , Joshi Kalpana , Suryawanshi Kishor , Soni Pravin , Gorde Anupama , Shah Tejas , Patankar Sagar , Jha Diwakar , Raje Rajesh
Published: May 2021

Objective

To assess the efficacy and safety of polyherbal formulation (designated as IP) in comparison to placebo as add on to the standard of care (SoC) among patients with mild to moderate novel corona virus disease 2019 (COVID-19)

Methods

Laboratory proved patients of mild to moderate COVID-19 disease were randomized to either placebo or IP as an add-on to SoC. Using quantitative reverse transcription-polymerase chain reaction (qRTPCR), we assessed the effect on viral load (VL). Change in immunological parameters such as blood lymphocyte subset, serum immunoglobulin was determined. The clinical improvement was assessed using a numerical rating scale (NRS) and WHO ordinal scale. Patients were followed for 30 days after randomization.

Results

In total, 72 patients were randomized to either placebo (n=33) and IP (n=39). Fifty-two patients (n=21 in placebo and n=31 in IP arm) had qRT-PCR on day 0 and day 4. There was significant reduction in VL in IP arm (from 662081 copies/mL on day 0 to 48963 copies/mL on day 4; p=0.002)) but not in the placebo arm (from 385670 copies/mL on day 0 to 66845 copies/mL on day 4, p=0.106). Change in the NRS score and WHO ordinal scale score was significant in both treatment arms. However, the difference between the two groups was statistically significant in favour of drug group,. The increase in Th1 response was significant in the IP arm (p=0.023) but not in the placebo arm (p=0.098), thus implying immunomodulatory activity in the drug. No safety concerns were observed in any of the trial participants.

Conclusion

This study finds that polyherbal formulation reduces viral load and contributes to immunomodulation and improvement in clinical conditions when used as add-on to the standard care in patients with mild to moderate COVID-19 without any side effects. These findings need to be further confirmed in a large, prospective, randomized study.
A chemotactic sensor controlsSalmonella-host cell interaction
Authors: Hoffmann Stefanie , Gendera Kathrin , Schmidt Christiane , Kirchweger Peter , Imhof Axel , Bogdan Christian , Muller Yves A. , Hensel Michael , Gerlach Roman G.
Published: May 2021

Intimate cell contact and subsequent type three secretion system-dependent cell invasion are key steps in host colonization ofSalmonella. Adhesion to complex glycostructures at the apical membrane of polarized cells is mediated by the giant adhesin SiiE. This protein is secreted by a type 1 secretion system (T1SS) and needs to be retained at the bacterial surface to exert its adhesive function. Here, we show that SiiE surface expression was linked to the presence of L-aspartate sensed by theSalmonella-specific methyl-accepting chemotaxis protein CheM. Bacteria lacking CheM were attenuated for invasion of polarized cells, whereas increased invasion was seen withSalmonellaexposed to the non-metabolizable aspartate analog α-methyl-D, L-aspartate (MeAsp). While components of the chemotaxis phosphorelay or functional flagella were dispensable for the increased invasion, CheM directly interacted with proteins associated with the SiiE T1SS arguing for a novel non-canonical signaling mechanism. As a result, CheM attractant signaling caused a shift from secreted to surface-retained and adhesion-competent SiiE. Thus, CheM controls the virulence function of SiiE in a precise spatio-temporal fashion depending on the host micro-milieu.