title: Screening, Isolation and Characterization of dye degrading bacteria from textile dye effluents.

abstract: Textile dye industry waste is one among the foremost serious issues within the atmosphere. The dye wastes are severely harmful to surface water bodies. The dye degradation and decolorisation processes, that embody several physical and chemical strategies having inherent drawbacks, like cost accounting, economically impracticable (require additional energy and chemicals), unable to get rid of a number of the recalcitrant dyes and production of huge quantity of sludge that if not properly treated, successively will cause secondary pollution. So, biological degradation, being eco-friendly and cheap methodology, is taken into account as an efficient methodology for the removal of nephrotoxic radical dyes. Our present study was therefore aimed to isolate dyestuff decolorizing microorganism from dyeing industry effluent associate degreed to check their characteristics so as to use them as an economical bio agent for decolorizing and mineralizing nephrotoxic radical dyes.Various microorganism like Bacillus subtilis, Aeromonas hydrophila and Bacillus Cereus, fungi & actinomycetes are found to possess dye decolorizing activity. For the aim of finding out their characteristics, water sample was subjected to enrichment culture technique and then isolated on sterile nutrient agar plates containing 0.005%, 0.01%, and 1% of Congo red dye. The probable isolated organism from Congo red dye i.e. Pantoea agglomerans was found which can possess the ability to decolorize Congo red at lower concentration. The probable isolates obtained must be additional investigated relating to varied factors like dye degradation capability, media composition affecting dye degradation & mechanism of dye degrading activity.


title: Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets

abstract:
Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts.

We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway. There are currently no global reports of half-lives in both wild type and NMD defective yeast cells; instead steady-state RNA level changes are used as a proxy. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3{Delta} mutants. We find 230 novel targets, and observe a strong agreement with previous reports of NMD targets, 60 % of our candidates being identified in previous studies.

This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.

category: molecular biology


title: TSC22D4 interacts with Akt1 in response metabolic and stress signals

abstract: Transforming Growth Factor {beta} 1 Stimulated Clone 22 D4 (TSC22D4) is an intrinsically disordered protein that regulates cellular and physiological processes such as cell proliferation, cellular senescence as well as hepatic glucose and lipid metabolism. The molecular mechanism of TSC22D4 action in these cellular and metabolic functions, however, remains largely elusive. Here, we identified TSC22D4 as a novel protein kinase B/Akt1 interacting protein, a critical mediator of insulin/PI3K signaling pathway implicated in diverse set of diseases including type 2 diabetes, obesity and cancer. TSC22D4 interacts with Akt1 not constitutively but rather in a regulatory manner. While glucose and insulin stimulation of cells or refeeding of mice impair the hepatic TSC22D4-Akt1 interaction, inhibition of mitochondria and oxidative stress, promote it; indicating that extra- and intra-cellular cues play a key role in controlling TSC22D4-Akt1 interaction. Our results also demonstrate that together with its dimerization domain, i.e. the TSC box, TSC22D4 requires its intrinsically disordered region (D2 domain) to interact with Akt1. To understand regulation of TSC22D4 function further, we employed tandem mass spectrometry and identified 15 novel phosphorylation sites on TSC22D4. Similar to TSC22D4-Akt1 interaction, TSC22D4 phosphorylation also responds to environmental signals such as starvation, mitochondrial inhibition and oxidative stress. Interestingly, 6 out of the 15 novel phosphorylation sites lie within the TSC22D4 D2 domain, which is required for TSC22D4-Akt1 interaction. Characterization of the regulation and function of these novel phosphorylation sites, in the future, will shed light on our understanding of the role of TSC22D4-Akt1 interaction in both cell biological and physiological functions. Overall, our findings postulate a model whereby TSC22D4 acts as an environmental sensor and interacts with Akt1 to regulate cell proliferation, cellular senescence as well as maintain metabolic homeostasis.


title: TSC22D4 interacts with Akt1 in response to metabolic and stress signals


title: Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

abstract:
BackgroundHow immune-tolerance is lost to pancreatic {beta}-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin ER chaperone glucose-regulated protein (GRP) 94 leads to mishandling of proinsulin, ER stress and activation of the inducible proteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered {beta}-cell MHC-I bound peptidome and inflammasome activation, sensitizing {beta}-cells to immune attack.

MethodsWe used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20{micro}M), or exposed to proinflammatory cytokines interleukin (IL)-1{beta} or IFN{gamma} (15 pg/ml and 10 ng/ml, respectively) for 24 hours. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse phase high performance liquid chromatography (RP-HPLC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NALP1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (I{kappa}B), and (pro) IL-1{beta} expression and secretion were investigated by Western blotting.

ResultsGRP94 KO increased RT1.A expression in {beta}-cells as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptides diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NALP1 expression was upregulated in GRP94 deficient cells along with decreased I{kappa}B content while proIL-1{beta} cellular levels declined, coupled with an increased secretion of mature IL-1{beta}. Our results suggest that limiting {beta}-cell proinsulin chaperoning enhances RT1.A expression, alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress impeding proinsulin handling may sensitize {beta}-cells to immune-attack.


title: Nucleolar targeting in an early-branching eukaryote suggests a general physicochemical mechanism for ribosome protein sorting

abstract:
The eukaryotic cell targets proteins to the organelles in which they function, both membrane-bound (like the nucleus) and non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals, and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in an early-branching eukaryote, Trypanosoma brucei, to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and protein physical properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins in comparison to mitochondrial ribosome proteins followed the same pattern - pointing to adaptation of physicochemical properties to assist segregation.

Summary StatementWe show protein targeting to the nucleolus is mediated by positive charge, likely across eukaryotes, and contributes to sorting of mitochondrial from cytoplasmic ribosome proteins.


title: Neonatal enthesis healing involves non-inflammatory formation of acellular scar through ECM secretion by resident cells

abstract:
Wound healing is a well-orchestrated process that typically recruits the immune and vascular systems to restore the structure and function of the injured tissue. Injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, thereby severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis.

Here, we established an injury model to the Achilles entheses of neonatal mice to study the possibility that at an early age, the enthesis can heal more effectively. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, neonatal enthesis healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including ER stress, autophagy and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and TUNEL assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg.

Collectively, these findings reveal a novel healing mechanism in neonatal entheses, whereby local ECM secretion by resident cells forms an acellular ECM deposit in the absence of inflammation markers, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.


title: Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance

abstract: Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated tEpe1 which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non- cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell-integrity MAP kinase stress signalling pathway, mutations in which alter resistance. Thus, environmental changes provoke a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered.

category: cell biology


title: Single-molecule imaging reveals distinct effects of ligands on CCR5 dynamics depending on its dimerization status

abstract: G protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses (HIV-1). We used TIRF microscopy and an original statistical method to track and classify the motion of different receptors subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on {beta}-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.


title: Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

abstract: Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.


title: Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster

abstract:
Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5 mRNA cap. Drosophila has multiple paralogs of eIF4E, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Other than eIF4E-3, none of these has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 mutants exhibit defects during post-meiotic stages, including a fully penetrant defect in individualization, resulting in failure to produce mature sperm. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, where it regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. eIF4E-5 mutants also have mild defects in spermatid cyst polarization, similar to mutants affecting the cytoplasmic polyadenylation-element binding protein Orb2 and atypical protein kinase C (aPKC). Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Summary StatementThe testis-enriched translation initiation factor eIF4E-5 is needed for spermatid cyst polarization, individualization of mature sperm and male fertility in Drosophila.

category: developmental biology


title: A Meta-Analysis of the 16S-rRNA Gut Microbiome Data in Honeybees (Apis Mellifera)

abstract:
1.Honeybees (Apis Mellifera) perform an essential role in the ecosystem and economy through pollination of insect-pollinated plants, but their population is declining. Many causes of honeybees decline are likely to be influenced by the microbiome which is thought to play an important role in bees and is particularly susceptible to infection and pesticides. However, there has been no systematic review or meta-analysis on honeybee microbiome data. Therefore, we conducted the first systematic meta-analysis of 16S-rRNA data to address this gap in the literature. Four studies were in a usable format - accounting for 336 honeybees worth of data - the largest such dataset to the best of our knowledge. We analysed these datasets in QIIME2 and visualised the results in R-studio. For the first time, we conducted a multi-study evaluation of the core and rare bee microbiome and confirmed previous compositional microbiome data. We established that Snodgrassella, Lactobacillus, Bifidobacterium, Fructobacillus and Saccaribacter form part of the core microbiome and identify 251 rare bacterial genera. Additional components of the core microbiome were likely obscured by incomplete classification. Future studies should refine and add to our existing dataset to produce a more conclusive and high-resolution portrait of the honeybee microbiome. Furthermore, we emphasise the need for an actively curated dataset and enforcement of data sharing standards.

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category: ecology


title: The impact of Piscirickettsia Salmonis infection on genome-wide DNA methylation profile in Atlantic Salmon

abstract: Salmon rickettsial septicaemia (SRS), caused by the intracellular bacteria Piscirickettsia Salmonis, generates significant mortalities to farmed Atlantic salmon, particularly in Chile. Due to its economic importance, a wealth of research has focussed on the biological mechanisms underlying pathogenicity of P. salmonis, the host response, and genetic variation in host resistance. DNA methylation is a fundamental epigenetic mechanism that influences almost every biological process via the regulation of gene expression and plays a key role in the response of an organism to stimuli. In the current study, the role of head kidney and liver DNA methylation in the response to P. salmonis infection was investigated in a commercial Atlantic salmon population. A total of 66 salmon were profiled using reduced representation bisulphite sequencing (RRBS), with head kidney and liver methylomes compared between infected animals (3 and 9 days post infection) and uninfected controls. These included groups of salmon with divergent (high or low) breeding values for resistance to P. salmonis infection, to examine the influence of genetic resistance. Head kidney and liver showed organ-specific global methylation patterns, but with similar distribution of methylation across gene features. Integration of methylation with RNA-Seq data revealed that methylation levels predominantly showed a negative correlation with gene expression, although positive correlations were also observed. Methylation within the first exon showed the strongest negative correlation with gene expression. A total of 911 and 813 differentially methylated CpG sites were identified between infected and control samples in the head kidney at 3 and 9 days respectively, whereas only 30 and 44 sites were differentially methylated in the liver. Differential methylation in the head kidney was associated with immunological processes such as actin cytoskeleton regulation, phagocytosis, endocytosis and pathogen associated pattern receptor signaling. We also identified 113 and 48 differentially methylated sites between resistant and susceptible fish in the head kidney and liver respectively. Our results contribute to the growing understanding of the role of methylation in regulation of gene expression and response to infectious diseases, and in particular reveal key immunological functions regulated by methylation in Atlantic salmon in response to P. salmonis.

category: genomics


title: Molecular Dynamics Simulations of HLA-CW4-B2M-KIR2DL1 Protein and Homology Modeling of a Complex Associated with Psoriasis Disease (HLA-CW6-B 2M-KIR2DS1)

abstract: Molecular dynamics (MD) simulation was used to study the interactions of two immune proteins of HLA-Cw4-{beta}2m-KIR2DL1 complex with small peptide QYDDAVYKL (nine amino acids) in an aqueous solution. This study aims to gain a detailed information about the conformational changes and the dynamics of the complex. The right parameters and force field for performing the MD simulations that was needed to calibrate the complex structure were determined. The non-bonded interactions (Electrostatic and van der Waals contributions), H-bond formation, and salt bridges between the ligand HLA-Cw4 and the receptor KIR2DL1 were estimated using the obtained MD trajectories. The buried surface area due to binding was calculated to get insight into the causes of specificity of receptor to ligand and explains mutations experiment. The study concluded that {beta}2-microglobulin, one part of the complex, is not directly interacting with the peptide at the groove; therefore, it could be neglected from simulation. Our results showed that {beta}2-microglobulin does not have any significant effect on the dynamics of the 3D-structure of the complex. This project will help in understanding to optimize candidate drug design, a small peptide that disrupts the interaction, for the optimal biological effect.

category: biochemistry


title: Genetic advantage and equality of opportunity in education: Two definitions and an empirical application

abstract: The literature of Equality of Opportunity (EOp) has long acknowledged the existence of  talents,  innate ability or  genetic ability. Nonetheless, attempts to explicitly incorporate a measure of innate ability in the quantification of EOp have been rare. On the other hand, the literature of social-science genetics has found credible genetic-based components of EOp, without an explicit quantification of overall EOp. In addition, there exists prevalent disagreement within both kinds of literature on whether innate ability should be perceived as a fair or unfair source of advantage. This paper proposes to quantify EOp while explicitly including a genetic-based measure of innate ability. It proposes two formal definitions of EOp that draw on both stances regarding the compensation of innate ability. Novel testable implications are derived. The educational attainment polygenic index is used as a measure of innate ability while correcting for genetic nurture and accounting for the correlation between genes and other circumstances. An empirical application in the US Health and Retirement Study finds that the share of inequality of opportunity is 26% under the view that genetic differences are unfair sources of advantage and 21% otherwise. A comparative analysis over cohorts reveals that the trend in EOp depends on the definition adopted; if genetic advantage is a fair source of inequality then EOp has improved; the opposite holds if one considers genetic advantage an unfair source of inequality. These results highlight the importance of accounting for genetic differences in the EOp framework.


title: The influence factors of mean particle size and positive quality control of bacterial filtration efficiency system

abstract:
BackgroundThe Coronavirus Disease 2019 (COVID-19) has swept the whole world with high mortality. Since aerosol transmission is the main route of transmission, wearing a mask serves as a crucial preventive measure. An important parameter to evaluate the performance of a mask is the bacteria filtration efficiency (BFE). Aerosol mean particle size (MPS) and positive quality control value are two key indexes of BFE system.

AimTo study the major influence factors of the mean particle size of bacterial aerosols and positive quality control value of BFE system.

Method and ResultsIn this study, we investigated the influence of Anderson sampler, spray flow, medium thickness, and peristaltic pump flow on the MPS of bacterial aerosols and positive quality control value of BFE system, respectively. The results show that the machining accuracy of Anderson sampler has great influence on aerosol MPS and positive quality control value. With the increase of aerosol spray flow rate, the positive quality control value will increase gradually, but the effect on aerosol MPS is not a simple linear relationship. As the agar medium thickness increased, the positive quality control value and aerosol MPS increased gradually. With the increase of peristaltic pump flow, the positive quality control value increased gradually, while the aerosol MPS was basically in a downward trend. When the peristatic pump flow rate was 0.1mL/min, the spray flow rate was 7.2L/min, the agar plate thickness was 27mL, and the Anderson sampler of Beijing Mingjie was used for the experiment, the aerosol MPS and positive quality control value were both within the acceptable range and were the optimal parameters.

ConclusionsThis study provides guidance for the manufacturers of the BFE system and improves the protective performance of masks, which is important for the human health, especially during the occurrence of viral pandemics such as "COVID-19".

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category: immunology


title: Influence of Pink Pigmented Facultative methylotrophic bacteria (PPFM) as a foliar spraying on the growth and productivity of strawberry(Fragaria xananassa Duch.).

abstract: Pink pigmented facultative methylotrophic bacterium (PPFM) has a favorable impact on plant development and production, it is known as a biostimulator, biofertilizer and biocontroller. Here we investigate the effect of foliar spraying of PPFM, 10% methanol,30% methanol, and their combinations on the growth, fruit quality, and yield of two strawberry cultivars. PPFM was isolated from cotton leaves using imprinting technique. 16S rRNA sequence analysis identified it to be Methylobacterium radiotolerance. Its 16S rRNA sequence were deposited in the Gene Bank under accession number MT644122.1. Two field experiments were conducted during the 2017/2018 and 2018/2019 seasons to investigate the effect of foliar spraying of PPFM and methanol (10 and 30%) on the growth, fruit quality, and yield of two strawberry cultivars. The obtained results showed that, there were no significant differences in the most characteristics between the two cultivars except foliage fresh weight and early yield were higher in cv. Florid, however, Festival cv. recorded higher total yield /plant, anthocyanins and ascorbic acid content in both seasons. Spraying PPFM exhibited the highest values of chlorophyll, fresh weight, total yield and quality. Furthermore, PPFM combined with methanol 10% gave the highest values of leaf area, dry matter %, early yield and some fruit quality. Spraying cv. Florida with PPFM resulted in the best interactions for early yield. However, the best interaction for total yield and most fruit quality features was observed with Festival c.v. and spraying PPFM. It is reasonable to conclude that PPFM is the most effective treatment, increasing strawberry total yield/fed by 28.1 % in the 1st season and 27.91 % in the 2nd season compared to the control.

category: microbiology


title: OligoTRAFTACs: A Generalizable Method for Transcription Factor Degradation

abstract: Dysregulated transcription factors (TFs) that rewire gene expression circuitry are frequently identified as key players in disease. Although several TFs have been drugged with small molecules, the majority of oncogenic TFs are not currently pharmaceutically tractable due to their paucity of ligandable pockets. The first generation of transcription factor targeting chimeras (TRAFTACs) was developed to target TFs for proteasomal degradation by exploiting their DNA binding ability. In the current study, we have developed the second generation TRAFTACs ("oligoTRAFTACs") comprised of a TF- binding oligonucleotide and an E3 ligase-recruiting ligand. Herein, we demonstrate the development of oligoTRAFTACs to induce the degradation of two oncogenic TFs, c-Myc and brachyury. In addition, we show that brachyury can be successfully degraded by oligoTRAFTACs in chordoma cell lines. Furthermore, zebrafish experiments demonstrate in vivo oligoTRAFTAC activity. Overall, our data demonstrate oligoTRAFTACs as a generalizable platform towards difficult-to-drug TFs and their degradability via the proteasomal pathway.

category: cancer biology


title: APOBEC-mediated Editing of SARS-CoV-2 Genomic RNA Impacts Viral Replication and Fitness

abstract:
During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.

One-sentence summaryEfficient Editing of SARS-CoV-2 genomic RNA by Host APOBEC deaminases and Its Potential Impacts on the Viral Replication and Emergence of New Strains in COVID-19 Pandemic


title: The Roles of APOBEC-mediated RNA Editing in SARS-CoV-2 Mutations, Replication, and Fitness


title: A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

abstract: Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 {micro}M range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.


title: SARS-CoV-2 Omicron spike mediated immune escape, infectivity and cell-cell fusion

abstract: The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.


title: Human genetic variants associated with COVID-19 severity are enriched in immune and epithelium regulatory networks

abstract: Human genetic variants can influence the severity of symptoms being infected with SARS-COV-2. Several genome-wide association studies have identified human genomic risk SNPs associated with COVID-19 severity. However, the causal tissues or cell types of COVID-19 severity are uncertain and candidate genes associated with these human risk SNPs were investigated in genomic proximity instead of their functional cellular contexts. Here, we compiled regulatory networks of 77 human contexts and revealed those risk SNPs enriched cellular contexts and associated transcript factors, regulatory elements, and target genes. Twenty-one human contexts were identified and grouped into two categories: immune cells and epithelium cells. We further aggregated the regulatory networks of immune cells, epithelium cells, and immune-epithelium crosstalk and investigated their association with risk SNPs regulation. Two genomic clusters, chemokine receptors cluster and OAS cluster, showed the strongest association with COVID-19 severity and different regulations in immune and epithelium contexts. Our findings were supported by analysis on both microarray and whole genome sequencing based GWAS summary statistics.

category: genetics


title: SARS-CoV-2 Omicron Variant: ACE2 Binding, Cryo-EM Structure of Spike Protein-ACE2 Complex and Antibody Evasion

abstract: The newly reported Omicron variant is poised to replace Delta as the most rapidly spread SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, explaining our finding of similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion, with greater evasion observed in sera obtained from unvaccinated convalescent patients as compared to doubly vaccinated individuals (8-vs 3-fold). The retention of strong interactions at the ACE2 interface and the increase in antibody evasion are molecular factors that likely contribute to the increased transmissibility of the Omicron variant.


title: The P3 O-Tert-Butyl-Threonine is Key to High Cellular and Antiviral Potency for Aldehyde-Based SARS-CoV-2 Main Protease Inhibitors

abstract: As an essential enzyme to SARS-CoV-2, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 sites and the N-terminal protection group, we synthesized a series of MPro inhibitors that contain {beta}-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 site. These inhibitors have a large variation of determined IC50 values that range from 4.8 to 650 nM. The determined IC50 values reveal that relatively small side chains at both P2 and P3 sites are favorable for achieving high in vitro MPro inhibition potency, the P3 site is tolerable toward unnatural amino acids with two alkyl substituents on the -carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. All structures show similar binding patterns of inhibitors at the MPro active site. A covalent interaction between the active site cysteine and a bound inhibitor was observed in all structures. In MPro, large structural variations were observed on residues N142 and Q189. All inhibitors were also characterized on their inhibition of MPro in 293T cells, which revealed their in cellulo potency that is drastically different from their in vitro enzyme inhibition potency. Inhibitors that showed high in cellulo potency all contain O-tert-butyl-threonine at the P3 site. Based on the current and a previous study, we conclude that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for peptidyl aldehyde inhibitors of MPro. This finding will be critical to the development of novel antivirals to address the current global emergency of concerning the COVID-19 pandemic.


title: Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

abstract: Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

category: bioengineering


title: SARS-CoV-2 spike conformation determines plasma neutralizing activity

abstract: Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV-2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.


title: Short-term Ketogenic Diet Induces a Molecular Response that is Distinct from Dietary Protein Restriction

abstract: There is increasing interest in utilizing short-term dietary interventions in the contexts of cancer, surgical stress and metabolic disease. These short-term diets may be more feasible than extended interventions and may be designed to complement existing therapies. In particular, the high-fat, low-carbohydrate ketogenic diet (KD), traditionally used to treat epilepsy, has gained popularity as a potential strategy for weight loss and improved metabolic health. In mice, long-term KD improves insulin sensitivity and extends lifespan and healthspan. Dietary protein restriction (PR) causes increased energy expenditure, weight loss and improved glucose homeostasis. Since KD is inherently a low-protein diet (10% of calories from protein vs. 20% in control diet), here we evaluated the potential for mechanistic overlap between PR and KD via activation of a PR response. Mice were fed control, protein-free, or one of four ketogenic diets with varying protein content for 8 days. PF and KD diets both decreased body weight, fat mass, and liver weights, and reduced fasting glucose and insulin levels, compared to mice fed the control diet. However, PF and KD differed with respect to insulin tolerance and hepatic insulin sensitivity, which were increased in PF-fed mice and impaired in KD-fed mice relative to controls. Furthermore, contrary to the PF-fed mice, mice fed ketogenic diets containing at least 5% protein did not increase hepatic Fgf21 or brown adipose Ucp1 expression. Interestingly, mice fed KD lacking protein demonstrated greater elevations in hepatic Fgf21 than mice fed a low-fat PF diet. To further elucidate potential mechanistic differences between PF and KD diets and the interplay between dietary protein and carbohydrate restriction, we conducted RNA-seq analysis on livers from mice fed each of the six diets and identified distinct gene sets which respond to dietary protein content, dietary fat content, and ketogenesis. We conclude that KD with 10% of energy from protein does not induce a protein restriction response, and that the overlapping metabolic benefits of KD and PF diets occur via distinct underlying mechanisms.

category: physiology


title: Lower Aβ-PET signal in white matter lesions relates to higher extracellular free water in mixed small vessel disease and Alzheimer's pathology

abstract:
Background and ObjectivesPositron emission tomography (PET) with amyloid-ligands recently gained interest for evaluating white matter (WM) pathology. Here, we investigated the neurobiological underpinnings of the amyloid-PET signal in the WM using free water (FW) diffusion MRI in a mixed cohort of small vessel disease (SVD) and Alzheimers disease (AD) pathology.

MethodsWe included data from two large cohort imaging studies, including MITNEC-C6 with moderate-to-severe white matter hyperintensity (WMH) burden and ADNI-2 with mild-to-moderate WMH burden, covering the spectrum of cognitively normal to dementia. Subjects underwent diffusion MRI, 18F-AV45 amyloid-PET, and cognitive testing. We calculated WM diffusion metrics including fractional anisotropy (FA) and mean diffusivity (MD). In addition, we applied a bi-tensor diffusion MRI model that differentiates between extracellular (FW fraction) and tissue-specific (FW-adjusted FA) compartments of the WM. We tested associations of all diffusion metrics with 18F-AV45 SUVR in regions of WMH vs. normal-appearing WM (NAWM), and with cognition. We further performed partial-least-square analysis to investigate how the diffusion metrics as well as age, sex, education, WMH volume, and cortical 18F-AV45 SUVR covary with WM 18F-AV45 SUVR.

Results18F-AV45 SUVR was significantly lower in regions of WMH compared to NAWM (t=25.08, P<0.0001, n=115 subjects). Within WMH, lower 18F-AV45 SUVR was associated with higher FW ({beta}=-0.36{+/-}0.13, P=0.005) and lower FA ({beta}=+0.24{+/-}0.12, P=0.046), but not with the tissue-specific metric FW-adjusted FA. Partial-least-square analysis further confirmed that FW had the most influence on 18F-AV45 SUVR in regions of WMH. In contrast, FW-adjusted FA had more influence on 18F-AV45 SUVR in NAWM. Last, correlation with cognitive impairment was higher for FW than FW-adjusted FA, both in regions of WMH (MMSE: {beta}FW=-0.40{+/-}0.13, P=0.003; {beta}FW-adjusted FA=0.14{+/-}0.09, P=0.11) and NAWM (MMSE: {beta}FW=-0.30{+/-}0.11, P=0.01; {beta}FW-adjusted FA=0.21{+/-}0.09, P=0.02).

ConclusionIn a cohort representative of the more common AD population, reduced amyloid-PET uptake in WM lesions may largely reflect the appearance of extracellular FW, while changes in NAWM may associate with tissue-specific damage. Ultimately, our findings may support FW and amyloid-PET as markers of WM abnormalities in SVD and AD.

category: neuroscience


title: Amino acid availability determines plant immune homeostasis in the rhizosphere microbiome

abstract: Microbes possess conserved microbe-associated molecular patterns (MAMPs) such as flagellin that are recognized by plant receptors to induce immunity. Despite containing the same MAMPs as pathogens, commensals thrive in the plant rhizosphere microbiome indicating they must suppress or evade host immunity. The beneficial bacteria Pseudomonas capeferrum WCS358 can suppress Arabidopsis root immunity via acidification by secreting gluconic acid. While gluconic acid is sufficient to suppress immunity, we found that it is not necessary in a second beneficial strain Pseudomonas simiae WCS417, which produces more gluconic acid than WCS358. To uncover mechanisms that contribute to the suppression of Arabidopsis immunity, we performed a forward genetic screen in EMS-mutagenized P. simiae WCS417 using a flagellin-inducible CYP71A12pro:GUS reporter as an Arabidopsis immune readout. We identified a mutant that cannot suppress flagellin-elicited CYP71A12pro:GUS expression or acidify the rhizosphere. Next generation sequencing revealed a mutation in the catabolic site of an ornithine carbamoyltransferase argF, which is required for arginine biosynthesis. The mutant could be complemented by expression of argF from a plasmid, and a {Delta}argF mutant could not suppress immunity. Fungal pathogens can use alkalization through production of ammonia and glutamate, the arginine biosynthetic precursors, to promote their own growth and virulence. Therefore, we hypothesized that the biosynthesis of specific amino acids may be necessary to reduce levels of ammonia and glutamate to prevent rhizosphere alkalization and bacterial overgrowth. Genetically blocking arginine, glutamine, or proline biosynthesis, or by adding corresponding exogenous amino acids, resulted in rhizosphere alkalization. Interestingly, exogenous amino acids caused bacterial overgrowth in a gluconic acid-deficient mutants. Our findings show that bacterial amino acid biosynthesis contributes to acidification by preventing accumulation of glutamate precursors and the resulting alkalization. Collectively this work shows that by regulating nutrient availability, plants have the potential to regulate their immune homeostasis in the rhizosphere microbiome.


title: Transcriptional and post-transcriptional regulation of young genes in plants

abstract: New genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression. Together, these data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.

category: evolutionary biology


title: Characterization Of Interspecific Gene Flows at The Genome-wide Level in a Natural Ecosystem The Massane Forest Reveals New Insights Into Horizontal Transfer In plants.

abstract: Horizontal transfer (HT) refers to the exchange of genetic material between divergent species by mechanisms other than reproduction. In recent years, several studies have demonstrated HTs in eukaryotes, particularly in the context of parasitic relationships and in model species. However, very little is known about HT in natural ecosystems, especially those involving non-parasitic wild species, and the nature of the ecological relationships that promote these HTs. In this work, we conducted a pilot study investigating HTs by sequencing the genomes of 17 wild non-model species from a natural ecosystem, the Massane forest, located in southern France. To this end, we developed a new computational pipeline called INTERCHANGE that is able to characterize HTs at the whole genome level without prior annotation and directly in the raw sequencing reads. Using this pipeline, we identified 12 HT events, half of which occurred between lianas and trees. We found that only LTRs-retrotransposons and predominantly those from the Copia superfamily were transferred between these wild species. This study revealed a possible new route for HTs between non-parasitic plants and provides new insights into the genomic characteristics of horizontally transferred DNA in plant genomes.


title: A phosphorylation code regulates the multi-functional protein RETINOBLASTOMA-RELATED1 in Arabidopsis thaliana

abstract: The RETINOBLASTOMA-RELATED (RBR) proteins play a central role coordinating cell division, cell differentiation and cell survival within an environmental and developmental context. These roles reflect RBR ability to engage in multiple protein-protein interactions (PPIs), which are regulated by multi-site phosphorylation. However the functional outcomes of RBR phosphorylation in multicellular organisms remain largely unexplored. Here we test the hypothesis that phosphorylation allows diversification of RBR functions in multicellular context. Using a representative collection of transgenic loss- and gain of function point mutations in RBR phosphosites, we analysed their complementation capacity in Arabidopsis thaliana root meristems. While the number of mutated residues often correlated to the phenotypic strength of RBR phosphovariants, phospho-sites contributed differentially to distinct phenotypes. For example, the pocket-domain has a greater influence on meristematic cell proliferation, whereas the C-terminal region associates to stem cell maintenance. We found combinatorial effects between the T406 phopspho-site with others in different protein domains. Moreover, a phospho-mimetic and a phospho-defective variant, both promoting cell death, indicate that RBR controls similar cell fate choices by distinct mechanisms. Thus, additivity and specificity of RBR phospho-sites fine tune RBR activity across its multiple roles. Interestingly, a mutation disrupting RBR interactions with the LXCXE motif suppresses dominant phospho-defective RBR phenotypes. By probing protein-protein interactions of RBR variants, we found that LXCXE-containing members of the DREAM complex constitute an important component of phosphorylation-regulated RBR function, but also that RBR participates in stress or environmental responses independently of its phosphorylation state. We conclude that developmental-related, but not stress- or environmental-related functions of RBR are defined and separable by a combinatorial phosphorylation code.

category: plant biology


title: IDENTIFYING PLANT GENES SHAPING MICROBIOTA COMPOSITION IN THE BARLEY RHIZOSPHERE

abstract: A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here we used metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identified a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS locus, emerged as a major determinant of microbiota composition. We then subjected soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allowed us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide novel insights into the footprint of crop improvement on the plants capacity of shaping rhizosphere microbes.


title: A lineage-specific Exo70 is required for receptor kinase-mediated immunity in barley

abstract: In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity, but lack causal association. Here, we show that Rps8-mediated resistance in barley to the fungal pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: LRR-RK and Exo70FX12, which are together necessary and sufficient. The Rps8 LRR-RK is the ortholog of rice extracellular immune receptor Xa21 and Exo70FX12 is a member of the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae, and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Rps8 LRR-RK immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.


title: Molecular evidence for segmental duplication across chromosomes of soybean using transcription factor gene family

abstract: Duplication of genome is an important genetic innovation. Large genome size (1.1 Gb) along with ancient and recent duplication events make the soybean genome more complex. Analyzing the distribution and duplication event in soybean transcription family genes, the segmental duplication within chromosomes was revealed. Our study provides a strong evidence that the large segmental duplication event in genome architecture and evolution of soybean genome using simple method of sequence and order analysis of TF genes. Finally, a scheme for interrelationship of different chromosomes has been proposed.


title: EARBOX: An open tool for high-throughput measurements of the spatial organization of maize ears and inference of novel traits

abstract:
BackgroundCharacterizing plant genetic resources and their response to the environment through accurate measurement of relevant traits is crucial to genetics and breeding. The spatial organization of the maize ear provides insights into the response of grain yield to environmental conditions. Current automated methods for phenotyping the maize ear do not capture these spatial features.

ResultsWe developed EARBOX, a low-cost, open-source system for automated phenotyping of maize ears. EARBOX integrates open-source technologies for both software and hardware that facilitate its deployment and improvement for specific research questions. The imaging platform consists of a customized box in which ears are repeatedly imaged as they rotate via motorized rollers. With deep learning based on convolutional neural networks, the image analysis algorithm uses a two-step procedure: ear-specific grain masks are first created and subsequently used to extract a range of trait data per ear, including ear shape and dimensions, the number of grains and their spatial organisation, and the distribution of grain dimensions along the ear. The reliability of each trait was validated against ground-truth data from manual measurements. Moreover, EARBOX derives novel traits, inaccessible through conventional methods, especially the distribution of grain dimensions along grain cohorts, relevant for ear morphogenesis, and the distribution of abortion frequency along the ear, relevant for plant response to stress, especially soil water deficit.

ConclusionsThe proposed system provides robust and accurate measurements of maize ear traits including spatial features. Future developments include grain type and colour categorization. This method opens avenues for high-throughput genetic or functional studies in the context of plant adaptation to a changing environment.


title: Disentangling the genetic basis of rhizosphere microbiome assembly in tomato

abstract: Microbiomes play a pivotal role in plant growth and health, but the genetic factors involved in microbiome assembly remain largely elusive. Here, 16S amplicon and metagenomic features of the rhizosphere microbiome were mapped as quantitative traits of a recombinant inbred line population of a cross between wild and domesticated tomato. Gene content analysis of prioritized tomato QTLs suggested a genetic basis for differential recruitment of various rhizobacterial lineages, including a Streptomyces-associated 6.31-Mbp region harboring tomato domestication sweeps and encoding, among others, the iron regulator FIT and the aquaporin SlTIP2.3. Within metagenome-assembled genomes of the rhizobacterial lineages Streptomyces and Cellvibrio, we identified microbial genes involved in metabolism of plant polysaccharides, iron, sulfur, trehalose, and vitamins, whose genetic variation associated with either modern or wild tomato QTLs. Integrating  microbiomics and quantitative plant genetics pinpointed putative plant and reciprocal microbial traits underlying microbiome assembly, thereby providing the first step towards plant-microbiome breeding programs.


title: Passage of SARS-CoV-2 in cells expressing human and mouse ACE2 selects for mouse-adapted and ACE2-independent viruses

abstract: Human ACE2 (hACE2) is the key cell attachment and entry receptor for SARS-CoV-2, with the original SARS-CoV-2 isolates unable to use mouse ACE2 (mACE2). Herein we describe a new system for generating mouse-adapted SARS-CoV-2 in vitro by serial passaging virus in co-cultures of cell lines expressing hACE2 and mACE2. Mouse-adapted viruses emerged with up to five amino acid changes in the spike protein, all of which have been seen in human isolates. Mouse-adapted viruses replicated to high titers in C57BL/6J mouse lungs and nasal turbinates, and caused severe lung histopathology. One mouse-adapted virus was also able to replicate efficiently in ACE2-negative cell lines, with ACE2-independent entry by SARS-CoV-2 representing a new biology for SARS-CoV-2 that has potential widespread implications for disease and intervention development.


title: ACE2-independent SARS-CoV-2 infection and mouse adaption emerge after passage in cells expressing human and mouse ACE2


title: PlantRNA 2.0 : an updated database dedicated to tRNAs of photosynthetic eukaryotes

abstract: PlantRNA (http://plantrna.ibmp.cnrs.fr/) is a comprehensive database of transfer RNA (tRNA) gene sequences retrieved from fully annotated nuclear, plastidial and mitochondrial genomes of photosynthetic organisms. In the first release (PlantRNA 1.0), tRNA genes from 11 organisms were annotated. In this second version, the annotation was implemented to 48 photosynthetic species covering the whole phylogenetic tree of photosynthetic organisms, from the most basal group of Archeplastida, the glaucophyte Cyanophora paradoxa, to various land plants. Transfer RNA genes from lower photosynthetic organisms such as streptophyte algae or lycophytes as well as extremophile photosynthetic species such as Eutrema parvulum were incorporated in the database. As a whole, circa 35 000 tRNA genes were accurately annotated. In the frame of the tRNA genes annotation from the genome of the Rhodophyte Chondrus crispus, putative unconventional splicing sites in the D- or T-regions of tRNA molecules were experimentally determined to strengthen the quality of the database. As for PlantRNA 1.0, comprehensive biological information including 5- and 3-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences and tRNA mitochondrial import are included.


title: Mixture Density Regression reveals frequent recent adaptation in the human genome

abstract: How much genome differences between species reflect neutral or adaptive evolution is a central question in evolutionary genomics. In humans and other mammals, the prevalence of adaptive versus neutral genomic evolution has proven particularly difficult to quantify. The difficulty notably stems from the highly heterogenous organization of mammalian genomes at multiple levels (functional sequence density, recombination, etc.) that complicates the interpretation and distinction of adaptive vs. neutral evolution signals. Here, we introduce Mixture Density Regressions (MDRs) for the study of the determinants of recent adaptation in the human genome. MDRs provide a flexible regression model based on multiple Gaussian distributions. We use MDRs to model the association between recent selection signals and multiple genomic factors likely to affect positive selection, if the latter was common enough in the first place to generate these associations. We find that a MDR model with two Gaussian distributions provides an excellent fit to the genome-wide distribution of a common sweep summary statistic (iHS), with one of the two distributions likely capturing the positively selected component of the genome. We further find several factors associated with recent adaptation, including the recombination rate, the density of regulatory elements in immune cells and testis, GC-content, gene expression in immune cells, the density of mammal-wide conserved elements, and the distance to the nearest virus-interacting gene. These results support that strong positive selection was relatively common in recent human evolution and highlight MDRs as a powerful tool to make sense of signals of recent genomic adaptation.


title: A Modified TurboID Approach Identifies Tissue-Specific Centriolar Components In C. elegans

abstract: Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and Bld10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.


title: Spatial and temporal requirement of Mlp60A isoforms during muscle development and function in Drosophila melanogaster

abstract: Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. In this paper, we show that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to late pupal lethality, with the surviving flies being majorly flight defective. Mlp60A null flies show developmental lethality, and muscle defects in the individuals surviving till the third instar larval stage. This lethality could be rescued partially by muscle-specific overexpression of the short isoform. Almost 90% of the long isoform-specific P-element insertion mutant flies show a compromised flight ability and have reduced sarcomere length. Hence, our data shows that the two Mlp60A isoforms are functionally specialized, to ensuring normal embryonic muscle development and adult flight muscle function.


title: Single-cell protein analysis reveals metastable states during the transition to a sensory organ fate

abstract: Cell fate decisions can be envisioned as bifurcating dynamical systems, and the decision that Drosophila cells make to undergo sensory organ differentiation has been sucessfully described as such. We have extended these studies by focusing on the Senseless protein, which orchestrates the sensory fate transition. Wing cells contain intermediate Senseless numbers prior to their fate transition, after which they express much greater numbers of Senseless molecules as they differentiate. However, the dynamics are not consistent with it being a simple bistable system. Cells with intermediate Senseless are best modeled as residing in four discrete states, each with a distinct protein number and occupying a specific region of the tissue. Although the four states are stable over time, the number of molecules in each state vary with time. Remarkably, the fold-change in molecule number between adjacent states is invariant and robust to absolute protein number variation. Thus, cells transitioning to sensory fates exhibit metastability with relativistic properties.


title: Bacterial Outer Membrane Vesicles Induce a Transcriptional Shift in Arabidopsis Towards Immune System Activation Leading to Suppression of Pathogen Growth in Planta

abstract: Gram negative bacteria form spherical blebs on their cell periphery, which later dissociate and released into the surrounding environment. Previous studies have shown that these nano scale structures, derived primarily from the bacterial outer membrane and are termed outer membrane vesicles (OMVs), induce typical immune outputs in both mammals and plants. On the other hand, these same structures have been shown to promote infection and disease. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated samples at 2, 6 and 24 h post challenge. We found that the most pronounced transcriptional shift occurred in the first two time points, as was reflected by both the number of differentially expressed genes (DEGs) and the average fold change. Gene ontology enrichment analysis revealed that OMVs induce a major transcriptional shift in Arabidopsis towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors and transcriptional factors. Comparing Arabidopsis response to OMVs and to single purified elicitors, revealed that while OMVs induce a similar suite of genes and pathways as single elicitors, some differential pathways activated by OMVs were detected including response to drug and apoptosis, which may indicate exposure to toxic compounds via OMV. To examine whether the observed transcriptional shift in Arabidopsis leads to an effective immune response, plants were pretreated with OMVs and then inoculated with a bacterial pathogen. OMV-mediated priming led to a significant reduction in bacterial titer in inoculated leaves two days following inoculation. Mutations in the elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) receptor, did not significantly affect OMV-priming. All together these results show that OMV induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change is reflected in the ability to better resist bacterial infection.


title: The catscape: spatial manifestation of a pet cat population with outdoor access

abstract: The domestic cat (Felis catus) is the most popular companion animal and the most abundant carnivore globally. It is also a pet with an immense ecological footprint, because even non-feral and food-subsidized cats are prolific predators. Whereas knowledge about the spatial behavior of individual domestic cats is growing, we still know little about how a local population of free-ranging pet cats occupies the landscape. Using a citizen science approach, we GPS-tagged 92 pet cats with outdoor access living in a residential area in southern Norway. The resulting position data allowed us to construct both individual home range kernels and a population-level utilization distribution. Our results reveal a dense predatory blanket that outdoor cats drape over and beyond the urban landscape. It is this population-level intensity surface - the "catscape" - that potential prey have to navigate. There were almost no gaps in the catscape within our residential study area and therefore few terrestrial refuges from potential cat predation. However, cats spent on average 79% of their outdoor time within 50 meters to their owners home, which suggests that the primary impact is local and most acute for wildlife in the vicinity to homes with cats. We discuss the catscape as a conceptual and quantitative tool for better understanding and mitigating the environmental impact of domestic cats.


title: Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2

abstract: Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). To identify additional ways of reducing ataxin-2 levels, we performed a genome-wide screen in human cells for regulators of ataxin-2 and identified RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a top hit. RTN4R knockdown, or treatment with a peptide inhibitor, was sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. Remarkably, we observed that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.


title: Insilico and Invitro optimization of Naringin and rutin molecules targeting DNA damage in breast cancer cells

abstract:
Discovering the molecular mechanisms of DNA damage response pathways has led to new therapeutic approaches in oncology. Our study optimized DNA damage-targeting molecules naringin and rutin in breast cancer cells.

Our study involved MTT assays for detection of its toxicity and proliferative activity in breast cancer cells and normal cancer cells. Our studies determined the molecules antioxidant properties using the DPPH assay. The role in reducing free radicals has been evaluated using a variety of free radical scavenging activity assays.

Further evaluation of the molecules was carried out by high alkaline comet assay (pH >13) to test for genotoxicity. Human Dermal Fibroblast cells (2DD) (1x105 cells/ml) and breast cancer cells (MDA-MB-231) were pre-incubated with Naringin and Rutin (10 {micro}M) for one hour.

In normal cells, rutin and naringin molecules do not cause genotoxicity, but they cause DNA damage in breast cancer cells when they are diluted to 10{micro}M. The results from our study indicate that both molecules cause 60-70% DNA damage in breast cancer cells.


title: Platr4 is an ESC-specific lncRNA that exhibits its function downstream on meso/endoderm lineage commitment

abstract: The mammalian genome encodes thousands of long non-coding RNAs (lncRNAs) that are developmentally regulated and differentially expressed across tissues, suggesting possible roles in cellular differentiation. Despite this expression pattern, little is known about how lncRNAs influence lineage commitment at the molecular level. Here, we reveal that perturbation of an embryonic stem cell (ESC)-specific lncRNA, Pluripotency associated transcript 4 (Platr4), in ESCs directly influences the downstream meso/endoderm differentiation program without affecting pluripotency. We further show that Platr4 interacts with the TEA domain transcription factor 4 (Tead4) to regulate the expression of a downstream target gene crucial in the cardiac lineage program known as connective tissue growth factor (Ctgf). Importantly, Platr4 knockout mice exhibit myocardial atrophy, valve mucinous degenration associated with reduced cardiac output and sudden heart failure. Together, our findings provide evidence that Platr4 expression in undifferentiated ESCs is critical for downstream lineage differentiation, highlighting its importance in disease modeling and regenerative medicine.


title: Human embryos arrest in a quiescent-like state characterized by metabolic and zygotic genome activation problems

abstract: Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3-8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a quiescent-like state, marked by cell cycle arrest, the downregulation of ribosomes and histones and downregulation of MYC and p53 activity. Mechanistically, the arrested embryos can be divided into three types. Type I embryos fail to complete the maternal-zygotic transition, and type II/III embryos have erroneously low levels of glycolysis and variable levels of oxidative phosphorylation. Treatment with resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype. The mechanism of reactivation involves the upregulation of SIRT1, and activation of glycolysis and fatty acid oxidation which forces the embryos out of a quiescent state. Overall, our data reveal how human embryo arrest can be overcome by modulating metabolic pathways.


title: The N-Terminal Carbamate is Key to High Cellular and Antiviral Potency for Boceprevir-Based SARS-CoV-2 Main Protease Inhibitors

abstract: Boceprevir is an HCV NSP3 inhibitor that has been explored as a repurposed drug for COVID-19. It inhibits the SARS-CoV-2 main protease (MPro) and contains an -ketoamide warhead, a P1 {beta}-cyclobutylalanyl moiety, a P2 dimethylcyclopropylproline, a P3 tert-butyl-glycine, and a P4 N-terminal tert-butylcarbamide. By introducing modifications at all four positions, we synthesized 20 boceprevir-based MPro inhibitors including PF-07321332 and characterized their MPro inhibition potency in test tubes (in vitro) and human host cells (in cellulo). Crystal structures of MPro bound with 10 inhibitors and antiviral potency of 4 inhibitors were characterized as well. Replacing the P1 site with a {beta}-(S-2-oxopyrrolidin-3-yl)-alanyl (opal) residue and the warhead with an aldehyde leads to high in vitro potency. The original moieties at P2, P3 and the P4 N-terminal cap positions in boceprevir are better than other tested chemical moieties for high in vitro potency. In crystal structures, all inhibitors form a covalent adduct with the MPro active site cysteine. The P1 opal residue, P2 dimethylcyclopropylproline and P4 N-terminal tert-butylcarbamide make strong hydrophobic interactions with MPro, explaining high in vitro potency of inhibitors that contain these moieties. A unique observation was made with an inhibitor that contains an P4 N-terminal isovaleramide. In its MPro complex structure, the P4 N-terminal isovaleramide is tucked deep in a small pocket of MPro that originally recognizes a P4 alanine side chain in a substrate. Although all inhibitors show high in vitro potency, they have drastically different in cellulo potency in inhibiting ectopically expressed MPro in human 293T cells. All inhibitors including PF-07321332 with a P4 N-terminal carbamide or amide have low in cellulo potency. This trend is reversed when the P4 N-terminal cap is changed to a carbamate. The installation of a P3 O-tert-butyl-threonine improves in cellulo potency. Three molecules that contain a P4 N-terminal carbamate were advanced to antiviral tests on three SARS-CoV-2 variants. They all have high potency with EC50 values around 1 M. A control compound with a nitrile warhead and a P4 N-terminal amide has undetectable antiviral potency. Based on all observations, we conclude that a P4 N-terminal carbamate in a boceprevir derivative is key for high antiviral potency against SARS-CoV-2.


title: Structure and mechanism of NALCN-FAM155A-UNC79-UNC80 channel complex

abstract: NALCN channel mediates sodium leak currents and is important for maintaining proper resting membrane potential. NALCN and FAM155A form the core complex of the channel, the activity of which essentially depends on the presence of both UNC79 and UNC80, two auxiliary proteins. NALCN, FAM155A, UNC79, and UNC80 co-assemble into a large hetero-tetrameric channel complex. Genetic mutations of NALCN channel components lead to neurodevelopmental diseases. However, the structure and mechanism of the intact channel complex remain elusive. Here, we present the cryo-EM structure of the mammalian NALCN-FAM155A-UNC79-UNC80 quaternary complex. The structure showed that UNC79-UNC80 form a large piler-shaped heterodimer which was tethered to the intracellular side of the NALCN channel through tripartite interactions with the cytoplasmic loops of NALCN. Two interactions are essential for proper cell surface localization of NALCN. The other interaction relieves the self-inhibition of NALCN by pulling the auto-inhibitory CTD Interacting Helix (CIH) out of its binding site.

category: biophysics


title: Sentimental Tweets Classification of Symptomatic COVID 19

abstract: The approach I described is straightforward, related to COVID-19 SARS based tweets and the symptoms, that people tweet about. Also, social media mining for health application reports was shared in many different tasks of 2021. The motto at the back of this observe is to analyses tweets of COVID-19 based symptoms. By performing BERT model and text classification with XLNET with which uses to classify text and purpose of the texts (i.e.) tweets. So that I can get a deep understanding of the texts. When developing the system, I used two models the XLNet and DistilBERT for the text sorting task, but the outcome was XLNET out-performs the given approach to the best accuracy achieved. Now I discover a whole lot vital for as it should be categorizing tweets as encompassing self-said COVID-19 indications. Whether or not a tweets associated with COVID-19 is a non-public report or an information point out to the virus. Which gives test accuracy to an F1 score of 96%.

category: scientific communication and education


title: Small molecule v-ATPase inhibitor Etidronate lowers levels of ALS protein ataxin-2

abstract: Antisense oligonucleotide therapy targeting ATXN2--a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis--has entered clinical trials in humans. Additional methods to lower ataxin-2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin-2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v-ATPase) as regulators of ataxin-2 levels. We demonstrate that Etidronate--a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v-ATPase--lowers ataxin-2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.


title: Neuronal circuits integrating visual motion information in Drosophila

abstract: The detection of visual motion enables sophisticated animal navigation, and studies in flies have provided profound insights into the cellular and circuit basis of this neural computation. The flys directionally selective T4 and T5 neurons respectively encode ON and OFF motion. Their axons terminate in one of four retinotopic layers in the lobula plate, where each layer encodes one of four cardinal directions of motion. While the input circuitry of the directionally selective neurons has been studied in detail, the synaptic connectivity of circuits integrating T4/T5 motion signals is largely unknown. Here we report a 3D electron microscopy reconstruction, wherein we comprehensively identified T4/T5s synaptic partners in the lobula plate, revealing a diverse set of new cell types and attributing new connectivity patterns to known cell types. Our reconstruction explains how the ON and OFF motion pathways converge. T4 and T5 cells that project to the same layer, connect to common synaptic partners symmetrically, that is with similar weights, and also comprise a core motif together with bilayer interneurons, detailing the circuit basis for computing motion opponency. We discovered pathways that likely encode new directions of motion by integrating vertical and horizontal motion signals from upstream T4/T5 neurons. Finally, we identify substantial projections into the lobula, extending the known motion pathways and suggesting that directionally selective signals shape feature detection there. The circuits we describe enrich the anatomical basis for experimental and computations analyses of motion vision and bring us closer to understanding complete sensory-motor pathways.


title: Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen

abstract: Spacing of the BNT162b2 mRNA doses beyond 3 weeks raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously-infected donors. This regimen elicited robust RBD-specific B cell responses whose kinetics differed between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting did not increase magnitude of CD4+ T cell responses further compared to the first dose, unsupervised clustering analyses of single-cell features revealed phenotypic and functional shifts over time and between cohorts. Integrated analysis showed longitudinal immune component-specific associations, with early Thelper responses post-first dose correlating with B cell responses after the second dose, and memory Thelper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.


title: Multiscale 3D Genome Reorganization during Skeletal Muscle Stem Cell Lineage Progression and Muscle Aging

abstract: 3D genome rewiring is known to influence spatiotemporal expression of lineage-specific genes and cell fate transition during stem cell differentiation and aging processes. Yet it is unknown how 3D architecture remodels and orchestrates transcriptional changes during skeletal muscle stem cell (also called satellite cell, SC) activation, proliferation and differentiation course. Here, using in situ Hi-C we comprehensively map the 3D genome topology reorganization at multiscale levels during mouse SC lineage progression and integrate with transcriptional and chromatin signatures to elucidate how 3D genome rewiring dictates gene expression program. Specifically, rewiring at compartment level is most pronounced when SC becomes activated. Striking loss in TAD border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can also form TAD clusters and super-enhancer containing TAD clusters orchestrate stage-specific gene expression during SC early activation. Furthermore, we elucidate 3D chromatin regulation of key transcription factor, PAX7 and identify cis-regulatory elements that are crucial for local chromatin architecture and Pax7 expression. Lastly, 3D genome remodeling is profiled in SCs isolated from naturally aging mice, unveiling that geriatric SCs display a prominent gain in long-range contacts and loss of TAD border insulation. Genome compartmentalization and chromatin looping are evidently altered in aged SC while geriatric SC display a more prominent loss in strength of TAD borders. Together, our results implicate 3D chromatin extensively reorganizes at multiple architectural levels and underpin the transcriptome remodeling during SC lineage development and SC aging.


title: OMA1 mediates local and global stress responses against protein misfolding in CHCHD10 mitochondrial myopathy

abstract:
Mitochondrial stress triggers a response in the cells mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a novel knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, inhibiting mitochondrial fusion, and signaled outside the mitochondria, activating the integrated stress response. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a novel component of this response. Our results demonstrate that OMA1 is essential for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.

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title: Polarized α-synuclein trafficking and transcytosis across Brain Endothelial Cells via Rab7-decorated carriers

abstract:
Parkinsons disease is mainly caused by aggregation of -synuclein (-syn) in the brain. Exchange of -syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of -syn across the blood brain barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of -syn monomers and oligomers across an in vitro BBB model system. Both -syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric -syn in brain endothelial cells, we investigated co-localization of -syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric -syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric -syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric -syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric -syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric -syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinsons disease and related synucleinopathies.

Significance StatementIn the submitted manuscript, we describe the use of a state-of-the-art porcine blood-brain barrier model based on primary cells to get information about this important issue. We identify several hitherto undescribed cellular pathways to mediate polarized transport of alpha-synuclein. One of these paths we find regulated by Rab7 and can be inhibited by targeting several intracellular proteins such as VPS35, Caveolin1 and Rab7. New knowledge describing brain endothelial intracellular transport systems are highly warranted for identifying new target to alleviate Parkinsons disease. We believe that our findings could be the seed to establish new therapeutic strategies against Parkinsons disease and related synucleinopathies.


title: Extracellular Matrix protein gelatin provides higher expansion, reduces size heterogeneity, and maintains cell stiffness in a long-term culture of mesenchymal stem cells.

abstract: Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behaviour of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behaviour. However, the influence of ECM protein coating on long term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 g/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of Gelatin showed less size heterogeneity among the cells when expanded from passages P3 to P9. Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins provides a better platform for mesenchymal stem cell expansion.


title: GLP is critical for oogenesis exhibiting a G9A-independent role in transcriptional repression

abstract: GLP (EHMT1) functions as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with G9A (EHMT2). Here, we investigated the role of GLP in oocyte and embryo development in comparison to G9A using oocyte-specific conditional knockout mouse models (G9a cKO, Glp cKO, G9a-Glp cDKO). Loss of GLP in oogenesis severely impairs oocyte maturation, fertilization and embryo development, resulting in lethality before embryonic day E12.5. In contrast, loss of G9A has a milder effect with a proportion of embryos producing viable offspring. The Glp cKO also showed loss of G9A protein and, hence, was phenotypically very similar to the G9a-Glp cDKO. H3K9me2 was equally depleted in all cKO genotypes, whereas H3K9me1 was decreased only in Glp cKO and G9a-Glp cDKO oocytes. Furthermore, the transcriptome, DNA methylome and proteome were markedly more affected in G9a-Glp cDKO than G9a cKO oocytes, demonstrating that in the absence of GLP there are widespread epigenetic and gene expression changes in the oocyte independent of H3K9me2. Gene dysregulation with coupled changes in DNA methylation suggest localised loss of chromatin repression, resulting in upregulated protein expression. Together, our findings demonstrate that GLP can function independently of G9A in the oocyte and is required for oocyte developmental competence.


title: FUCCI-based live imaging platform reveals cell cycle dynamics and identifies pro-proliferative compounds in human iPSC-derived cardiomyocytes

abstract: One of the major goals in cardiac regeneration research is to replace lost ventricular tissue with new cardiomyocytes. However, cardiomyocyte proliferation drops to low levels in neonatal hearts and is no longer efficient in compensating for the loss of functional myocardium in heart disease. We generated a human induced pluripotent stem cell (iPSC)-derived cardiomyocyte-specific cell cycle indicator system (TNNT2-FUCCI) to characterize regular and aberrant cardiomyocyte cycle dynamics. We visualized cell cycle progression in TNNT2-FUCCI and found G2 cycle arrest in endoreplicating cardiomyocytes. Moreover, we devised a live-cell compound screening platform to identify pro-proliferative drug candidates. We found that the alpha-adrenergic receptor agonist clonidine induced cardiomyocyte proliferation in vitro and increased cardiomyocyte cell cycle entry in neonatal mice. In conclusion, the TNNT2-FUCCI system is a valuable tool to characterize cardiomyocyte cell cycle dynamics and identify pro-proliferative candidates with regenerative potential in the mammalian heart.