title: Transformation from local image contrast to location-independent numerosity tuning in human extrastriate cortex

abstract: Human early visual cortex response amplitudes monotonically increase with numerosity (object number), regardless of object size and spacing. However, numerosity is typically considered a high-level visual or cognitive feature, while early visual responses follow image contrast in the spatial frequency domain. We found that, at fixed contrast, aggregate Fourier power (at all orientations and spatial frequencies) followed numerosity closely but nonlinearly with little effect of object size, spacing or shape. This would allow straightforward numerosity estimation from spatial frequency domain image representations. Using 7T fMRI, we showed monotonic responses originate in primary visual cortex (V1) at the stimuluss retinotopic location. Responses here and in neural network models followed aggregate Fourier power more closely than numerosity. Truly numerosity tuned responses emerged after lateral occipital cortex and were independent of retinotopic location. We propose numerositys straightforward perception and neural responses may have built on behaviorally beneficial spatial frequency analyses in simpler animals.

category: neuroscience


title: Numerosity tuning in human association cortices and local image contrast representations in early visual cortex


title: Associations Between Task-Related Modulation of Motor-Evoked Potentials and EEG Event-Related Desynchronization in Children with ADHD

abstract: Children with attention-deficit/hyperactivity disorder (ADHD) have previously shown a decreased magnitude of event-related desynchronization (ERD) during a finger-tapping task, with a large between-group effect. Because the neurobiology underlying several TMS measures have been studied in multiple contexts, we compared ERD and three TMS measures (Resting Motor Threshold [RMT], Short-Interval Cortical Inhibition [SICI] and Task-Related Up-Modulation [TRUM]) within 14 participants with ADHD (ages 8-12y) and 17 control children. The TD group showed a correlation between greater RMT and greater magnitude of alpha (10-13Hz, here) ERD, and there was no diagnostic interaction effect, consistent with a rudimentary model of greater needed energy input to stimulate movement. Similarly, inhibition measured by SICI was also greater in the TD group when the magnitude of movement-related ERD was higher; there was a miniscule diagnostic interaction effect. Finally, TRUM during a response-inhibition task showed an unanticipated pattern: in TD children, the greater TMS task modulation (TRUM) was associated with a smaller magnitude of ERD during finger-tapping. The ADHD group showed the opposite direction of association: greater TRUM was associated with larger-magnitude ERD. Prior EEG results have demonstrated specific alterations of task-related modulation of cortical physiology, and the current results provide a fulcrum for multimodal study.


title: Asymmetric structures and conformational plasticity of the uncleaved full-length human immunodeficiency virus (HIV-1) envelope glycoprotein trimer

abstract:
The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. Gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pre-triggered, "closed" (State-1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (States 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and crosslinking decreased the exposure of pNAb epitopes on cell-surface gp160; however, after detergent solubilization, crosslinked and BMS-806-treated gp160 sampled non-State-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume State-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a State-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs.

IMPORTANCEThe envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage to a persistent virus like HIV-1.

category: molecular biology


title: A TBP-independent mechanism for RNA Polymerase II transcription

abstract: Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the Pre-Initiation Complex onto promoter DNA. Decades of in vitro and yeast research have shown that the TATA-box binding protein (TBP) is essential to Pol II initiation by triggering the binding of other general transcription factors, and ensuring proper Pol II loading. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells (mESCs) has no global effect on ongoing Pol II transcription. Surprisingly, Pol II transcriptional induction through the Heat Shock Response or cellular differentiation also occurs normally in the absence of TBP. In contrast, acute TBP depletion severely impairs initiation by RNA Polymerase III. Lastly, we show that a metazoan-specific paralog of TBP is expressed in mESCs and that it binds to promoter regions of active Pol II genes even in the absence of TBP. Taken together, our findings reveal an unexplored TBP-independent process in mESCs that points to a diversity in Pol II transcription initiation mechanisms.


title: Unconventional Translation Initiation Factor EIF2A is required for Drosophila spermatogenesis

abstract: The eukaryotic initiation factor EIF2A is an unconventional translation factor required for initiation of protein synthesis from non-AUG codons from a variety of transcripts, including oncogenes and stress related genes in mammalian cells. Its function in multicellular organisms has not been reported. Here, we identify and characterize mutant alleles of the CG7414 gene, which encodes the Drosophila EIF2A ortholog. We identified that CG7414 undergoes sex-specific splicing that regulates its male-specific expression. We characterized a Mi{Mic} transposon insertion that disrupts the coding regions of all predicted isoforms and is a genetic null allele, and a PBac transposon insertion into an intron, which is a hypomorph. The Mi{Mic} allele is homozygous lethal, while the viable progeny from the hypomorphic PiggyBac allele are male sterile and female fertile. In dEIF2A mutant flies, sperm failed to individualize due to defects in F-actin cones and failure to form and maintain cystic bulges, ultimately leading to sterility. These results demonstrate that EIF2A is essential in a multicellular organism, both for normal development and spermatogenesis, and provide an entree into the elucidation of the role of EIF2A and unconventional translation in vivo.

category: developmental biology


title: Maxent modeling for predicting the potential distribution of global talaromycosis

abstract:
Talaromycosis, an invasive mycosis caused by Talaromyces marneffei (Tm), has rapidly increased in recent years, becoming an emerging pathogenic fungal disease. However, The driving factors and potential distribution of global talaromycosis is still unclear. Here, we developed maxent ecology model using environmental variables, Rhizomys distribution and HIV/AIDS epidemic to forecast ecological niche of talaromycosis worldwhile, as well as Identify the drivering factors. The constructed model had excellent performance with the area under the curve (AUC) of the receiver operating curve (ROC) of 0.997 in training data and 0.991 in testing data. Our model revealed that Rhizomys distribution, mean temperature of warmest quarter, precipitation of wettest month, HIV/AIDS epidemic and mean temperature of driest quarter were the top 5 important variables affecting talaromycosis distribution. In addition to traditional talaromycosis epidemic areas (South of the Yangtze River in China, Southeast Asian and North and Northeast India), our model also identified other potential epidemic regions, inculding parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Our findings has redefined global talaromycosis, discovered hidden high-risk areas and prorvided insights about driving factors of talaromycosis distribution, which will help inform surveillance strategies and improve the effectiveness of public health interventions against Tm infections.

Author SummaryOur study aims to explore the spatial ecology of talaromycosis worldwhile. The diseases burden of Talaromycosis, a neglected zoonotic disease, is continuously rising in recent years because of the sheer size of susceptible population in the setting of increased globalization, rising HIV prevalence, and emerging iatrogenic immunodeficiency conditions. Here, we used historic reported talaromycosis cases from 1964 to 2017, combined with environmental factors, Rhizomys distribution and HIV/AIDS epidemic to build an maxent ecology model to define the ecological niche of talaromycosis, then predicting the potential distribution of the disease. The ecological niche of talaromycosis is characterized by a concentrated distribution, which can be cognitively divided into two regions: traditional talaromycosis epidemic areas (South of the Yangtze River in China, Southeast Asian and North and Northeast India), while other potential epidemic regions were predicted in parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Our model also identified 5 driving factors affecting talaromycosis distribution. These findings will help demonstrate the global distribution of talaromycosis, discover hidden high-risk areas, and improve the effectiveness of public health interventions against Tm infections.

category: ecology


title: Predictors of zoonotic potential in helminths

abstract: Helminths are parasites that cause disease at considerable cost to public health and present a risk for emergence as novel human infections. Although recent research has elucidated characteristics conferring a propensity to emergence in other parasite groups (e.g. viruses), the understanding of factors associated with zoonotic potential in helminths remains poor. We applied an investigator-directed learning algorithm to a global dataset of mammal helminth traits to identify factors contributing to spillover of helminths from wild animal hosts into humans. We characterized parasite traits that distinguish between zoonotic and non-zoonotic species with greater than 88% accuracy. Results suggest that helminth traits relating to transmission (e.g. definitive and intermediate hosts) and geography (e.g. distribution) are more important to predicting zoonotic species than morphological or epidemiological traits. Whether or not a helminth causes infection in companion animals (cats and dogs) is the most important predictor of propensity to cause human infection. Finally, we identified helminth species with high modeled propensity to cause zoonosis (over 70%) that have not previously been deemed to be of risk. This work highlights the importance of prioritizing studies on the transmission of helminths that infect pets and points to the risks incurred by close associations with these animals.


title: Fast model-based ordination with copulas

abstract:
O_LIVisualising data is a vital part of analysis, allowing researchers to find patterns, and assess and communicate the results of statistical modeling. In ecology, visualisation is often challenging when there are many variables (often for different species or other taxonomic groups) and they are not normally distributed (often counts or presence-absence data). Ordination is a common and powerful way to overcome this hurdle by reducing data from many response variables to just two or three, to be easily plotted.
C_LIO_LIOrdination is traditionally done using dissimilarity-based methods, most commonly non-metric multidimensional scaling (nMDS). In the last decade however, model-based methods for unconstrained ordination have gained popularity. These are primarily based on latent variable models, with latent variables estimating the underlying, unobserved ecological gradients.
C_LIO_LIDespite some major benefits, a major drawback of model-based ordination methods is their speed, as they typically taking much longer to return a result than dissimilarity-based methods, especially for large sample sizes.
C_LIO_LIWe introduce copula ordination, a new, scalable model-based approach to unconstrained ordination. This method has all the desirable properties of model-based ordination methods, with the added advantage that it is computationally far more efficient. In particular, simulations show copula ordination is an order of magnitude faster than current model-based methods, and can even be faster than nMDS for large sample sizes, while being able to produce similar ordination plots and trends as these methods.
C_LI


title: An additional area of apple domestication with crop-wild gene flow, and also cultivation of the local wild apple, in the Caucasus

abstract: Anthropogenic and natural divergence processes remain poorly studied in crop-wild fruit tree complexes, especially in the Caucasus, a pivotal region for plant domestication. We investigated anthropogenic and natural divergence processes in apples in the Caucasus using 26 microsatellite markers amplified in 550 wild and cultivated samples. We found two genetically distinct cultivated populations in Iran that are differentiated from Malus domestica, the standard cultivated apple worldwide. Coalescent-based inferences showed that these two cultivated populations originated from specific domestication events of M. orientalis in Iran. One of the Iranian clusters comprised both cultivated and forest trees, suggesting that either farmers use local wild apple for cultivation or that some forest trees are feral cultivars. We found evidence of substantial wild-crop and crop-crop gene flow in the Caucasus, as has been described in apple in Europe. In the Caucasus, we identified seven genetically differentiated populations of wild apple (Malus orientalis). Niche modeling combined with genetic diversity estimates indicated that these populations likely resulted from range changes during past glaciations. This study identifies Iran as a key region in the domestication of apple and M. orientalis as an additional contributor to the cultivated apple gene pool. Domestication of the apple tree therefore involved multiple origins of domestication in different geographic locations and substantial crop-wild hybridization, as found in other fruit trees. This study also highlights the impact of climate change on the natural divergence of a wild fruit tree and provides a starting point for apple conservation and breeding programs in the Caucasus.

category: evolutionary biology


title: Evidence of an additional center of apple domestication in Iran, with contributions from the Caucasian crab apple Malus orientalis


title: Evidence of an additional wild contributor, Malus orientalis Uglitzk., to the genome of cultivated apple varieties in the Caucasus and Iran


title: Turnip crinkle virus targets host ATG8 proteins to attenuate antiviral autophagy

abstract: Autophagy has emerged as a central player in plant virus disease and resistance. In this study we have addressed the potential roles of autophagy in Turnip crinkle virus (TCV) infection. We found that autophagy attenuates disease severity and contributes to resistance against TCV by limiting virus accumulation. These autophagy-dependent disease phenotypes intensify further when combined with defects in RNA silencing, suggesting that these two major defence pathways are largely uncoupled in TCV disease. Intriguingly, as a counterdefence, TCV employs the viral silencing suppressor protein P38 to suppress antiviral autophagy, likely by directly sequestering ATG8 proteins. This strategy appears to be novel for plant viruses, yet resembles mechanisms described for other pathogen classes. Together, these results broaden our understanding of autophagy in plant virus disease, and strengthens our view of virus-specific adaptation to the autophagy pathway.

category: plant biology


title: Age-dependent appearance of SARS-CoV-2 entry cells in mouse chemosensory systems reflects COVID-19 anosmia and ageusia symptoms.

abstract: COVID-19 pandemic has given rise to a collective scientific effort to study its viral causing agent SARS-CoV-2. Research is focusing in particular on its infection mechanisms and on the associated-disease symptoms. Interestingly, this environmental pathogen directly affects the human chemosensory systems leading to anosmia and ageusia. Evidence for the presence of the cellular entry sites of the virus, the ACE2/TMPRSS2 proteins, has been reported in non-chemosensory cells in the rodents nose and mouth, missing a direct correlation between the symptoms reported in patients and the observed direct viral infection in human sensory cells. Here, mapping the gene and protein expression of ACE2/TMPRSS2 in the mouse olfactory and gustatory cells, we precisely identified the virus target cells to be of basal and sensory origin and we revealed their age-dependent appearance. Our results not only clarify human viral-induced sensory symptoms but also propose new investigative perspectives based on ACE2-humanized mouse models.


title: Sexually dimorphic placental responses to maternal SARS-CoV-2 infection

abstract: There is a persistent male bias in the prevalence and severity of COVID-19 disease. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of disease in adults, and play a key role in the placental anti-viral response. Moreover, the interferon response has been shown to alter Fc-receptor expression, and therefore may impact placental antibody transfer. Here we examined the intersection of viral-induced placental interferon responses, maternal-fetal antibody transfer, and fetal sex. Placental interferon stimulated genes (ISGs), Fc-receptor expression, and SARS-CoV-2 antibody transfer were interrogated in 68 pregnancies. Sexually dimorphic placental expression of ISGs, interleukin-10, and Fc receptors was observed following maternal SARS-CoV-2 infection, with upregulation in males. Reduced maternal SARS-CoV-2-specific antibody titers and impaired placental antibody transfer were noted in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.

category: immunology


title: SARS-CoV-2 variant B.1.1.7 caused HLA-A2+ CD8+ T cell epitope mutations for impaired cellular immune response

abstract:
The rapid spreading of the newly emerged SARS-CoV-2 variant, B.1.1.7, highlighted the requirements to better understand adaptive immune responses to this virus. Since CD8+ T cell responses play an important role in disease resolution and modulation in COVID-19 patients, it is essential to address whether these newly emerged mutations would result in altered immune responses. Here we evaluated the immune properties of the HLA-A2 restricted CD8+ T cell epitopes containing mutations from B.1.1.7, and furthermore performed a comprehensive analysis of the SARS-CoV-2 specific CD8+ T cell responses from COVID-19 convalescent patients and SARS-CoV-2 vaccinees recognizing the ancestral Wuhan strain compared to B.1.1.7. First, most of the predicted CD8+ T cell epitopes showed proper binding with HLA-A2, while epitopes from B.1.1.7 had lower binding capability than those from the ancestral strain. In addition, these peptides could effectively induced the activation and cytotoxicity of CD8+ T cells. Our results further showed that at least two site mutations in B.1.1.7 resulted in a decrease in CD8+ T cell activation and a possible immune evasion, namely A1708D mutation in ORF1ab1707-1716 and I2230T mutation in ORF1ab2230-2238. Our current analysis provides information that contributes to the understanding of SARS-CoV-2-specific CD8+ T cell responses elicited by infection of mutated strains or vaccination.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/437363v2_ufig1.gif" ALT="Figure 1">
View larger version (29K):
org.highwire.dtl.DTLVardef@b9f2cdorg.highwire.dtl.DTLVardef@1f39e3dorg.highwire.dtl.DTLVardef@119c313org.highwire.dtl.DTLVardef@565388_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Preliminary report on SARS-CoV-2 Spike mutation T478K

abstract: Several SARS-CoV-2 variants have emerged, posing a renewed threat to COVID-19 containment and to vaccine and drug efficacy. In this study, we analyzed more than 1,000,000 SARS-CoV-2 genomic sequences deposited up to April 27, 2021 on the GISAID public repository, and identified a novel T478K mutation located on the SARS-CoV-2 Spike protein. The mutation is structurally located in the region of interaction with human receptor ACE2 and was detected in 11,435 distinct cases. We show that T478K has appeared and risen in frequency since January 2021, predominantly in Mexico and USA, but we could also detect it in several European countries.


title: Modeling Substrate Coordination to Zn-Bound Angiotensin Converting Enzyme 2

abstract: The spike protein in the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with the receptor Angiotensin Converting Enzyme 2 (ACE2) on the host cell to facilitate the viral uptake. Angiotensin II (Ang II) peptide, which has a naturally high affinity for ACE2, may be useful in inhibiting this interaction. In this study, we computationally designed several Ang II mutants to find a strong binding sequence to ACE2 receptor and examined the role of ligand substitution in the docking of native as well as mutant Ang II to the ACE2 receptor. The peptide in the ACE2-peptide complex was coordinated to zinc in the ACE2 cleft. Exploratory molecular dynamics (MD) simulations were used to measure the time-based stability of the native and mutant peptides and their receptor complexes. The MD-generated root-mean-square deviation (RMSD) values are mostly similar between the native and seven mutant peptides considered in this work, although the values for free peptides demonstrated higher variation, and often were higher in amplitude than peptides associated with the ACE2 complex. An observed lack of a strong secondary structure in the short peptides is attributed to the latters greater flexibility and movement. The strongest binding energies within the ACE2-peptide complexes were observed in the native Ang II and only one of its mutant variants, suggesting ACE2 cleft is designed to provide optimal binding to the native sequence. An examination of the S1 binding site on ACE2 suggests that complex formation alone with these peptides may not be sufficient to allosterically inhibit the binding of SARS-CoV-2 spike proteins. However, it opens up the potential for utilizing AngII-ACE2 binding in the future design of molecular and supramolecular structures to prevent spike protein interaction with the receptor through creation of steric hindrance.

category: biophysics


title: Ultra-low Input Circulating Tumor DNA Detection by MED-Amp in Early-Stage Pancreatic Cancer

abstract:
PurposeThe clinical utility of circulating tumor DNA (ctDNA) has been shown in advanced pancreatic ductal adenocarcinoma (PDA). However, diagnostic sensitivity of many ctDNA assays is low in resectable and locally advanced disease, where tumor burden is substantially lower. We have previously described Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp), a multiplexed panel for the detection of the most common oncogenic KRAS mutations in PDA. In this study, we aimed to assess the diagnostic sensitivity of MED-Amp for detection of rare mutant alleles present in the plasma of patients with localized PDA.

Experimental DesignWe retrospectively analyzed ninety-eight plasma samples from 51 patients with various stages of localized disease. For comparison, we measured ctDNA levels in 20 additional patients with metastatic PDA. The MED-Amp assay was used to measure the abundance of the four most common KRAS codon 12 mutations (G12C/D/R/V). We correlated the presence and quantity of ctDNA with overall survival (OS) as well as progression-free survival (PFS). Using serial plasma draws, we also assessed the relationship between changes in ctDNA allelic frequency and progression.

ResultsKRAS-positive ctDNA was detected in 52.9% of localized PDA and 75% of metastatic samples tested using DNA inputs as low as 2 ng. As previously reported, the presence of KRAS mutant ctDNA was correlated with worse OS for all disease stages (p = 0.02). In patients with localized PDA high ctDNA levels also correlated with significantly worse median OS (533 days vs 1090 days) and PFS (192 days vs 787 days). We also studied a small cohort of serial plasma draws to observe the relationship between ctDNA fold change and PFS. We found 83% of patients with increased fold change in mutant KRAS experienced disease progression (n=6). In contrast, 75% (n=4) of patients with decreased fold change remained disease-free (p=0.03).

ConclusionsMED-Amp is a flexible and cost-effective approach for measurement of ctDNA in patients with localized cancer. Though this study focused on KRAS mutation detection, this assay could be adapted for a number of common oncogenic alterations.

Statement of translational relevanceOnly 25% of pancreatic ductal adenocarcinoma (PDA) patients with localized disease survive five years post-resection. It is hypothesized PDA undergoes dissemination at the earliest stages of tumor formation, driving formation of occult metastases which go undetected using conventional screening methods. Development of high specificity, high sensitivity biomarkers is critical to improving patient mortality. Circulating tumor DNA (ctDNA) has gained increasing acceptance as a non-invasive prognostic in metastatic disease. However, the sensitivity of most targeted ctDNA assays precludes reliable detection of localized and resected disease. Here, we present a digital droplet PCR assay for multiplexed enrichment and detection of KRAS mutations, the most commonly mutated oncogene in PDA. This assay preserves ctDNA allelic frequency in the original sample, while increasing the molecular signal over 50-fold. This study shows the ctDNA has potential diagnostic value in early-stage PDA, and that digital preenrichment of cell-free DNA increases overall assay sensitivity without sacrificing specificity.

category: genomics


title: Phase separation of RNA-binding protein promotes polymerase engagement and transcription

abstract: An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA binding to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs--such as PSPC1 and PTBP1--show that the paraspeckle protein PSPC1 not only prevents the RNA-induced premature release of Pol II, and also makes use of RNA as multivalent molecules to promote Pol II engagement and activity, by enhancing the phase separation and subsequent phosphorylation and release of polymerase condensates. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II phosphorylation and chromatin-binding and nascent transcription. We propose that the synergistic interplay of RBPs and RNA aids in the rate-limiting step of polymerase condensate formation to promote active transcription.

category: biochemistry


title: Benchmarking small-variant genotyping in polyploids

abstract: Genotyping from sequencing is the basis of emerging strategies in the molecular breeding of polyploid plants. However, compared with the situation for diploids, where genotyping accuracies are confidently determined with comprehensive benchmarks, polyploids have been neglected; there are no benchmarks measuring genotyping error rates for small variants using real sequencing reads. We previously introduced a variant calling method - Octopus - that accurately calls germline variants in diploids and somatic mutations in tumors. Here, we evaluate Octopus and other popular tools on whole-genome tetraploid and hexaploid datasets created using in silico mixtures of diploid Genome In a Bottle samples. We find that genotyping errors are abundant for typical sequencing depths, but that Octopus makes 25% fewer errors than other methods on average. We supplement our benchmarks with concordance analysis in real autotriploid banana datasets.

category: bioinformatics


title: The Arabidopsis WRR4A and WRR4B paralogous NLR proteins both confer recognition of multiple Albugo candida effectors

abstract:
The oomycete Albugo candida causes white blister rust, an important disease of Brassica crops. Distinct races of A. candida are defined by their specificity for infecting different host species.

The White Rust Resistance 4 (WRR4) locus in Col-0 accession of Arabidopsis thaliana contains three genes that encode TIR-NLR resistance proteins. The Col-0 alleles of WRR4A and WRR4B confer resistance to at least four A. candida races (2, 7 and 9 from B. juncea, B. rapa and B. oleracea, respectively, and Race 4 from Capsella bursa-pastoris). Resistance mediated by both paralogs can be overcome by Col-0-virulent isolates of Race 4.

After comparing repertoires of candidate effectors in resisted and resistance-breaking strains, we used transient co-expression in tobacco or Arabidopsis to identify effectors recognized by WRR4A and WRR4B. A library of CCG effectors from four A. candida races was screened for WRR4A- or WRR4B- dependent elicitation of hypersensitive response (HR). These CCG genes were validated for WRR-dependent HR by bombardment assays in wild type Col-0, wrr4A or wrr4B mutants.

Our analysis revealed eight WRR4A-recognized CCGs and four WRR4B-recognized CCGs. Remarkably, the N-terminal region of 100 amino acids after the secretion signal is sufficient for WRR4A recognition of these eight recognized effectors. This multiple recognition capacity potentially explains the broad-spectrum resistance to many A. candida races conferred by WRR4 paralogs.


title: A synthetic receptor platform enables rapid and portable monitoring of liver dysfunction via engineered bacteria.

abstract: Bacterial biosensors, or bactosensors, are promising field-deployable agents for medical and environmental diagnostics. However, the lack of scalable frameworks to systematically program ligand detection limits their applications. Here we present a synthetic receptor platform, termed EMeRALD (Engineered Modularized Receptors Activated via Ligand-induced Dimerization) which supports the modular assembly of sensing modules onto a high-performance, generic signaling scaffold controlling gene expression in E. coli. We applied EMeRALD to detect bile salts, a biomarker of liver dysfunction, by repurposing sensing modules from enteropathogenic Vibrio species. We improved the sensitivity and lowered the limit-of-detection of the sensing module by directed evolution. We then engineered a colorimetric bactosensor detecting pathological bile salt levels in serum from patients having undergone liver transplant, providing an output detectable by the naked-eye. The EMeRALD technology enables functional exploration of natural sensing modules and rapid engineering of synthetic receptors for diagnostics, environmental monitoring, and control of therapeutic microbes.

category: synthetic biology


title: Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

abstract: Sulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene-microbe-environment interactions through the lens of enzyme kinetics.

category: microbiology


title: OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

abstract: Elucidating the wiring diagram of the human cell is a central goal of the post-genomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery, and uncovers that RNA-binding proteins form a specific sub-group defined by unique interaction and localization properties. Furthermore, we discover that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions. Paired with a fully interactive website opencell.czbiohub.org, we provide a resource for the quantitative cartography of human cellular organization.

category: cell biology


title: Bayesian metamodeling of complex biological systems across varying representations

abstract:
Comprehensive modeling of a whole cell requires an integration of vast amounts of information on various aspects of the cell and its parts. To divide-and-conquer this task, we introduce Bayesian metamodeling, a general approach to modeling complex systems by integrating a collection of heterogeneous input models. Each input model can in principle be based on any type of data and can describe a different aspect of the modeled system using any mathematical representation, scale, and level of granularity. These input models are (i) converted to a standardized statistical representation relying on Probabilistic Graphical Models, (ii) coupled by modeling their mutual relations with the physical world, and (iii) finally harmonized with respect to each other. To illustrate Bayesian metamodeling, we provide a proof-of-principle metamodel of glucose-stimulated insulin secretion by human pancreatic {beta}-cells. The input models include a coarse-grained spatiotemporal simulation of insulin vesicle trafficking, docking, and exocytosis; a molecular network model of glucose-stimulated insulin secretion signaling; a network model of insulin metabolism; a structural model of glucagon-like peptide-1 receptor activation; a linear model of a pancreatic cell population; and ordinary differential equations for systemic postprandial insulin response. Metamodeling benefits from decentralized computing, while often producing a more accurate, precise, and complete model that contextualizes input models as well as resolves conflicting information. We anticipate Bayesian metamodeling will facilitate collaborative science by providing a framework for sharing expertise, resources, data, and models, as exemplified by the Pancreatic {beta}-Cell Consortium.

Significance StatementCells are the basic units of life, yet their architecture and function remain to be fully characterized. This work describes Bayesian metamodeling, a modeling approach that divides-and-conquers a large problem of modeling numerous aspects of the cell into computing a number of smaller models of different types, followed by assembling these models into a complete map of the cell. Metamodeling enables a facile collaboration of multiple research groups and communities, thus maximizing the sharing of expertise, resources, data, and models. A proof-of-principle is provided by a model of glucose-stimulated insulin secretion produced by the Pancreatic {beta}-Cell Consortium.


title: A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis

abstract:
RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first processing-specific human disorder to be described.

HighlightsO_LIMutations in RMRP lncRNA impair pre-rRNA processing and T cell activation
C_LIO_LIPatient derived fibroblasts show impaired pre-rRNA processing
C_LIO_LICells with the most common disease-linked mutation have specific processing defects
C_LIO_LICytoplasmic ribosomes and intact RNase MRP complexes are also reduced in these cells
C_LI


title: Self-Supervised Deep-Learning Encodes High-Resolution Features of Protein Subcellular Localization

abstract: Elucidating the diversity and complexity of protein localization is essential to fully understand cellular architecture. Here, we present cytoself, a deep-learning approach for fully self-supervised protein localization profiling and clustering. cytoself leverages a self-supervised training scheme that does not require pre-existing knowledge, categories, or annotations. Training cytoself on images of 1,311 endogenously labeled proteins from the OpenCell database reveals a highly resolved protein localization atlas that recapitulates major scales of cellular organization, from coarse classes such as nuclear, cytoplasmic and vesicular, to the subtle localization signatures of individual protein complexes. We quantitatively validate cytoselfs ability to cluster proteins into organelles and protein complex clusters using a clustering score, and show that cytoself attains higher scores than previous unsupervised or self-supervised approaches. Finally, to better understand the inner workings of our model, we dissect the emergent features from which our clustering is derived, interpret these features in the context of the fluorescence images, and analyze the performance contributions of the different components of our approach.


title: An ERK5-KLF2 signalling module regulates early embryonic gene expression dynamics and stem cell rejuvenation

abstract: The ERK5 MAP kinase signalling pathway drives transcription of naive pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5-dependent ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative-feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern dynamic 2C gene transcription and mESC rejuvenation.


title: Intergenerational metabolic priming by sperm piRNAs

abstract: SummaryPreconception parental environment can reproducibly program offspring phenotype without altering the DNA sequence, yet the mechanisms underpinning this  epigenetic inheritance remains elusive. Here, we demonstrate the existence of an intact piRNA-pathway in mature Drosophila sperm and show that pathway modulation alters offspring gene transcription in a sequence-specific manner. We map a dynamic small RNA content in developing sperm and find that the mature sperm carry a highly distinct small RNA cargo. By biochemical pulldown, we identify a small RNA subset bound directly to piwi protein. And, we show that piRNA-pathway controlled sperm small RNAs are linked to target gene repression in offspring. Critically, we find that full piRNA-pathway dosage is necessary for the intergenerational metabolic and transcriptional reprogramming events triggered by high paternal dietary sugar. These data provide a direct link between regulation of endogenous mature sperm small RNAs and transcriptional programming of complementary sequences in offspring. Thus, we identify a novel mediator of paternal intergenerational epigenetic inheritance.


title: Degradation of visible autumn icons and conservation opportunities: trends in deciduous forest loss in the contiguous US

abstract:
Temperate deciduous forests are one of the most visible biomes on Earth because of their autumn aesthetics and because they harbor some of the most heavily populated regions. Their ability to attract visitors may increase opportunities for people to experience nature, which has been linked to greater conservation action. Identifying regions with high leaf-peeping opportunities and regions where color has been lost to landscape conversion may help to inform these connections. We use spatial overlay analyses to quantify temperate deciduous forest coverage, disturbance, and protections in each U.S. ecoregion. We evaluated recent (1984-2016) and predicted (2016-2050) disturbance under extreme future scenarios. Almost all ecoregions saw a decline in deciduous forest cover between 1985 and 2016. Some ecoregions with the greatest opportunities for leaf-peeping are also underrepresented in the protected areas network and vulnerable to additional losses. Under economic-growth forecasting scenarios, losses are predicted to continue. However, environmentally focused scenarios suggest there is still opportunity to reverse deciduous forest loss in some ecoregions. Differences in forest loss between predictions scenarios emphasize the importance of human approaches in securing environmental stability. Increasing public exposure to temperate forests may help ensure conservation of more natural areas and preserve the quantity and quality of autumn forest viewing.

Key PointsO_LITemperate deciduous forests aesthetics attract visitors to experience nature, but degradation and loss can hinder connections.
C_LIO_LIUS ecoregions with the greatest leaf-peeping opportunities are underrepresented in the protected areas and vulnerable to additional losses.
C_LIO_LIDifferences in predictions scenarios emphasize the importance of conservation action, which may be linked to human connections with nature.
C_LI


title: Proteasome autophagy is specifically regulated and requires factors dispensible for general autophagy

abstract: Changing physiological conditions can increase the need for protein degradative capacity in eukaryotic cells. Both the ubiquitin-proteasome system and autophagy contribute to protein degradation. However, proteasomes are also an autophagy substrate. Thus, these processes must be differentially regulated depending on the physiological conditions presented. The signals and molecular mechanisms that govern proteasome autophagy are only partly elucidated. Our data indicate that chemical inhibition of TORC1 with rapamycin induces a bi-phasic response where proteasome levels are upregulated followed by an autophagy-dependent reduction. Surprisingly, several conditions that result in inhibited TORC1 exclusively induce proteasome autophagy (i.e. without any proteasome upregulation), suggesting a convergence of signals upstream of proteasome autophagy under different physiological conditions. Indeed, several conditions that activate general autophagy did not induce proteasome autophagy further distinguishing between proteasome autophagy and general autophagy. Consistent with this, we found that Atg11, the receptor for selective autophagy, and the map kinases Mpk1, Mkk1, and Mkk2, all play a role in autophagy of proteasomes, while they are dispensible for general autophagy. In all, our data provide new insights into the molecular regulation of proteasome autophagy by demonstrating that these complexes are specifically regulated under different autophagy inducing conditions.


title: Competitive SNP-LAMP probes for rapid and robust single-nucleotide polymorphism detection

abstract: Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are typically detected using DNA sequencing, which requires advanced sample preparation and instrumentation, and thus cannot be deployed for on-site testing or in low-resource settings. In this work we have developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines LAMP-based target amplification with fluorescent probes to detect SNPs with high specificity in a one-pot reaction format. A competitive "sink" strand preferentially binds to off-target products and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible SARS-CoV-2 variants. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity.

category: bioengineering


title: Multi-omic meta-analysis of transcriptomes and the bibliome uncovers GPR146 as the novel hypoxia-inducible gene

abstract: Hypoxia is a condition in which cells, tissues, or organisms are deprived of sufficient oxygen supply. Aerobic organisms have a hypoxic response system, represented by hypoxia-inducible factor 1- (HIF1A), to adapt to this condition. Due to publication bias, there has been little focus on genes other than well-known signature hypoxia-inducible genes. Therefore, in this study, we performed a meta-analysis to identify novel hypoxia-inducible genes. We searched publicly available transcriptome databases to obtain hypoxia-related experimental data, retrieved the metadata, and manually curated it. We selected the genes that are differentially expressed by hypoxic stimulation, and evaluated their relevance in hypoxia by performing enrichment analyses. Next, we performed a bibliometric analysis using gene2pubmed data to examine genes that have not been well studied in relation to hypoxia. Gene2pubmed data that provides information about the relationship between gene and publication. We calculated and evaluated the number of reports and similarity coefficients of each gene to HIF1A, which is a representative gene in hypoxia studies. In this data-driven study, we report that several genes that were not known to be associated with hypoxia, including the G protein-coupled receptor 146 gene, are upregulated by hypoxic stimulation.


title: Multi-omic meta-analysis of transcriptomes and the bibliome uncovers novel hypoxia-inducible genes


title: Phase Resolution of Heterozygous Sites in Diploid Genomes is Important to Phylogenomic Analysis under the Multispecies Coalescent Model

abstract: AO_SCPLOWBSTRACTC_SCPLOWGenome sequencing projects routinely generate haploid consensus sequences from diploid genomes, which are effectively chimeric sequences with the phase at heterozygous sites resolved at random. The impact of phasing errors on phylogenomic analyses under the multispecies coalescent (MSC) model is largely unknown. Here we conduct a computer simulation to evaluate the performance of four phase-resolution strategies (the true phase resolution, the diploid analytical integration algorithm which averages over all phase resolutions, computational phase resolution using the program PHASE, and random resolution) on estimation of the species tree and evolutionary parameters in analysis of multi-locus genomic data under the MSC model. We found that species tree estimation is robust to phasing errors when species divergences were much older than average coalescent times but may be affected by phasing errors when the species tree is shallow. Estimation of parameters under the MSC model with and without introgression is affected by phasing errors. In particular, random phase resolution causes serious overestimation of population sizes for modern species and biased estimation of cross-species introgression probability. In general the impact of phasing errors is greater when the mutation rate is higher, the data include more samples per species, and the species tree is shallower with recent divergences. Use of phased sequences inferred by the PHASE program produced small biases in parameter estimates. We analyze two real datasets, one of East Asian brown frogs and another of Rocky Mountains chipmunks, to demonstrate that heterozygote phase-resolution strategies have similar impacts on practical data analyses. We suggest that genome sequencing projects should produce unphased diploid genotype sequences if fully phased data are too challenging to generate, and avoid haploid consensus sequences, which have heterozygous sites phased at random. In case the analytical integration algorithm is computationally unfeasible, computational phasing prior to population genomic analyses is an acceptable alternative.


title: Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

abstract: Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.

category: genetics


title: Bacterial retrons enable precise gene editing in human cells

abstract: Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve editing efficiency of precise genome editing via homology-directed repair (HDR). HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.3%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.


title: Somatic DNA demethylation generates tissue-specific methylation states and impacts flowering time

abstract: Cytosine methylation is a reversible epigenetic modification to DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DME family of 5-methylcytosine DNA glycosylases. Demethylation by DME is critical for seed development. Consequently, determining the function of the entire gene family in somatic tissues by mutant analysis has not been possible. Here, we bypassed the reproductive defects of dme mutants to create somatic quadruple homozygous mutants of the entire DME family. dme; ros1; dml2; dml3 (drdd) leaves exhibit hypermethylated genomes compared to both wild-type plants and rdd triple mutants, indicating functional redundancy among all four demethylases. Targets of demethylation include regions co-targeted by RNA-directed DNA methylation and, surprisingly, CG gene body methylation, indicating dynamic methylation at these little-understood sites. Additionally, many tissue-specific methylation differences are absent in drdd, suggesting a role for active demethylation in generating divergent epigenetic states across wild-type tissues. Furthermore, drdd plants display a striking early flowering phenotype, which is associated with 5 hypermethylation and transcriptional down-regulation of FLOWERING LOCUS C. Active DNA demethylation is therefore required for proper methylation patterning across somatic tissues and defines the epigenetic landscape of both intergenic and coding regions.


title: An improved assembly of the Albugo candida Ac2V genome reveals the expansion of the "CCG" class of effectors

abstract: Albugo candida is an obligate oomycete pathogen that infects many plants in the Brassicaceae family. We re-sequenced the genome of isolate Ac2V using PacBio long reads and constructed an assembly augmented by Illumina reads. The Ac2VPB genome assembly is 10% larger and more contiguous compared to a previous version. Our annotation of the new assembly, aided by RNASeq information, revealed a dramatic 250% expansion (40 to 110) in the CHxC effector class, which we redefined as "CCG" based on motif analysis. This class of effectors consist of arrays of phylogenetically related paralogs residing in gene sparse regions, and shows signatures of positive selection and presence/absence polymorphism. This work provides a resource that allows the dissection of the genomic components underlying A. candida adaptation and particularly the role of CCG effectors in virulence and avirulence on different hosts.


title: Alpha-synuclein alters the faecal viromes of rats in a gut-initiated model of Parkinson's disease

abstract: Parkinsons disease (PD) is a chronic neurological disorder associated with the misfolding of alpha-synuclein (-syn) into Lewy body aggregates within nerve cells that contribute to their neurodegeneration. Recent evidence suggests -syn aggregation may begin in the gut and travel to the brain along the vagus nerve, with microbes a potential trigger initiating the misfolding of -syn. However, changes in the gut virome in response to -syn alterations have not been investigated. In this study, we show longitudinal changes in the faecal virome of rats administered either monomeric or preformed fibrils (PFF) of -syn directly into their enteric nervous system. Differential changes in rat viromes were observed when comparing monomeric and PFF -syn. The virome {beta}-diversity changes after -syn treatment were compounded by the addition of LPS as an adjunct. Changes in the diversity of rat faecal viromes were observed after one month and did not resolve within the studys five month observational period. Overall, these results suggest that microbiome alterations associated with PD may, partially, be reactive to host -syn associated changes.


title: Plant phenology influences rhizosphere microbial community and is accelerated by serpentine microorganisms in Plantago erecta

abstract: Serpentine soils are drought-prone and rich in heavy metals, and plants growing on serpentine soils host distinct microbial communities that may affect plant survival and phenotype. However, whether the rhizosphere communities of plants from different soil chemistries are initially distinct or diverge over time may help us understand drivers of microbial community structure and function in stressful soils. Here, we test the hypothesis that rhizosphere microbial communities will converge over time (plant development), independent of soil chemistry and microbial source. We grew Plantago erecta in serpentine or nonserpentine soil, with serpentine or nonserpentine microbes and tracked plant growth and root phenotypes. We used 16S rRNA barcoding to compare bacterial species composition at seedling, vegetative, early-, and late-flowering phases. Plant phenotype and rhizosphere bacterial communities were mainly structured by soil type, with minor contributions by plant development, microbe source and their interactions. Serpentine microorganisms promoted early flowering in plants on non-serpentine soils. Despite strong effects of soil chemistry, the convergence in bacterial community composition across development demonstrates the importance of the plant-microbe interactions in shaping microbial assembly processes across soil types.


title: A lipid nanoparticle RBD-hFc mRNA vaccine protects hACE2 transgenic mice against lethal SARS-CoV-2 infection

abstract: The current global COVID-19 pandemic led to an unprecedented effort to develop effective vaccines against SARS-CoV-2. mRNA vaccines were developed very rapidly during the last year, and became the leading immunization platform against the virus, with highly promising phase-3 results and remarkable efficacy data. Since most animal models are not susceptible to SARS CoV-2 infection, pre-clinical studies are often limited to infection-prone animals such as hamsters and non-human primates. In these animal models, SARS-CoV-2 infection results in viral replication and a mild disease disease. Therefore, the protective efficacy of the vaccine in these animals is commonly evaluated by its ability to elicit immunologic responses, diminish viral replication and prevent weight loss. Our lab recently reported the design of a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). These experiments demonstrated the development of a robust and specific immunologic response in RBD-hFc mRNA-vaccinated BALB/c mice. In the current study, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18-hACE2 mouse model. We report that administration of RBD-hFc mRNA vaccine to K18-hACE2 mice led to a robust humoral response comprised of both binding and neutralizing antibodies. In accordance with the recorded immunologic immune response, 70% of vaccinated mice were protected against a lethal dose (3000 plaque forming units) of SARS-CoV-2, while all control animals succumbed to infection. To the best of our knowledge, this is the first non-replicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.


title: The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation.

abstract:
Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main /{beta} SASPs, SspA and SspB, and two annotated SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated SASPs do not contribute to UV resistance. In addition, none of these SASPs contribute to the resistance of tested chemicals. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate / serve as a checkpoint for spore formation, a previously unreported function of SASPs.

ImportanceC. difficile infections remain problematic and elimination of spores within an environment is essential to limit person-to-person spread. A deeper understanding of how spores resist cleaning efforts could lead to better strategies to eradicate the spores in a contaminated environment. The small acid-soluble proteins (SASPs), found in all endospore-forming organisms, are one mechanism that allows for spore resilience. Here, we find that C. difficile SspA and SspB protect against UV light. Unexpectedly, these SASPs also regulate spore formation, a role not described for any SASP to date.


title: High-resolution single-molecule long-fragment rRNA gene amplicon sequencing for uncultured bacterial and fungal communities

abstract: Although several large-scale environmental microbial projects have been initiated in the past two decades, understanding of the role of complex microbiotas is still constrained by problems of detecting and identifying unknown microorganisms1-6.Currently, hypervariable regions of rRNA genes as well as internal transcribed spacer regions are broadly used to identify bacteria and fungi within complex communities7,8, but taxonomic and phylogenetic resolution is hampered by insufficient sequencing length9-11. Direct sequencing of full length rRNA genes is currently limited by read length using second generation sequencing or sacrificed quality and throughput by using single molecule sequencing. We developed a novel method to sequence and assemble nearly full length rRNA genes using second generation sequencing.Benchmarking was performed on mock bacterial and fungal communities as well as two forest soil samples. The majority of rRNA gene sequences of all species in the mock community samples were successfully recovered with identities above 99.5% compared to the reference sequences. For soil samples we obtained exquisite coverage with identification of a large number of putative new species, as well as high abundance correlation between replicates. This approach provides a cost-effective method for obtaining extensive and accurate information on complex environmental microbial communities.


title: HPMCP-coated microcapsules containing the Ctx(Ile21)-Ha antimicrobial peptide reduces the mortality rate caused by resistant Salmonella Enteritidis in poultry

abstract: The constant use of synthetic antibiotics as growth promoters can cause great bacterial resistance in chicks. Consequently, the use of these drugs has been restricted in different countries. Antimicrobial peptides have gained relevance in recent years due to their minimal capacity for bacterial resistance and does not generate toxic residues that harm the environment and human health. In this work, Ctx(Ile21)-Ha antimicrobial peptide was employed, due to its previously reported largely antimicrobial potential, to evaluate its application effect in laying chicks challenged with Salmonella Enteritidis, resistant to nalidixic acid and spectinomycin. For this, Ctx(Ile21)-Ha was synthesized, microencapsulated and coated with hypromellose phthalate (HPMCP) to be release in the intestine. Two different doses (3.2 and 6.4 {micro}g of Ctx(Ile21)-Ha per kg of isoproteic and isoenergetic poultry feed) were included in the chicks food and administered for 28 days. Antimicrobial activity, effect and response as treatment were evaluated. Statistical results were analyzed in detail and indicate that the formulated Ctx(Ile21)-Ha peptide had a positive and significant effect in relation to the reduction of chick mortality. There was a significant difference in laying chick weight between the control and microencapsulation treatment groups as a function of time. Therefore, the microencapsulated Ctx(Ile21)-Ha antimicrobial peptide may be an interesting and promising option in the substitution of conventional antibiotics.


title: The CTEXT complex in Saccharomyces cerevisiae plays a crucial role in degrading distinct sets of aberrant mRNAs by the nuclear exosome

abstract: In Saccharomyces cerevisiae, DRN (Decay of RNA in the Nucleus) requiring Cbc1/2p, Tif4631p, and Upf3p promotes the exosomal degradation of aberrantly long 3'-extended-, export-defective transcripts and a small group of normal (special) mRNAs. In this study, using a systematic proteomic analysis we show that each of the known components interacts with one another and they exist as a separate complex, which was dubbed CTEXT (CBC-Tif4631p-dependent EXosome Targeting). We also identified a DEAD-box RNA helicase Dbp2p as an additional novel component of CTEXT during this analysis which was further bolstered by the finding that genomic deletions of Dbp2p led to the stabilization of all the signature nuclear messages. Interestingly, the RRM domain of Tif4631p located at the extreme N-termini of this polypeptide was found to play a vital role in in mediating the interaction of the CTEXT with the core exosome complex. These inferences were substantiated by the finding that deletion of this domain led to the functional impairment of the CTEXT complex. Thus, the CTEXT constitutes an independent complex that assists the nuclear exosome in degrading the select classes of nuclear transcripts in Saccharomyces cerevisiae.


title: Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA

abstract: Base-pairing of the seed region (g2-g8) is essential for microRNA targeting, however, the in vivo function of the 3 non-seed region (g9-g22) are less well understood. Here we report the first systematic investigation of the in vivo roles of 3 non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We found that the 3 non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets, including evolutionarily conserved lin-41, daf-12 and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, the 3 non-seed pairing of let-7a can be critically required with certain minimal complementarity for sites with perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3 non-seed base-pairing can critically influence microRNA-mediated gene regulation in vivo.


title: Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori

abstract: Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, it is still inconclusive how cholinergic genes express only in cholinergic neurons in insects. In this study, we analyzed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. We found multiple enhancer regions that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. We found that dibutyryl-cAMP, an analog of cAMP, influences the expression of ChAT. Tissue-specific expression analysis of transcriptional factors showed potential candidates that control the cholinergic gene locus expression. Our investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.


title: Phosphorylation of myosin A regulates Plasmodium sporozoite motility and is essential for efficient malaria transmission

abstract:
Malaria-causing parasites rely on an actin-myosin based motor for the invasion of different host cells as well as tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that serine 19 is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry and migration of salivary gland sporozoites in both 2D and 3D environments. Our data suggests that entry into salivary glands represents the strongest barrier in parasite transmission and hence is the key determinant for evolution of the motility and invasion machinery of these parasites.

HighlightsThe unusual N-terminal extension of Plasmodium myosin A is important for efficient gliding motility

Altering the kinetics of the myosin A power stroke impacts Plasmodium life cycle progression and sporozoite motility

Myosin A phosphorylation at serine 19 is important for malaria transmission by mosquitoes

Salivary gland invasion emerges as key selection step for evolution of the parasite motor


title: Clinically relevant mutations of mycobacterial GatCAB inform regulation of translational fidelity

abstract: Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error-prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampicin tolerance. However, the mechanisms by which these clinically-derived mutations in gatA impact GatCAB function was unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains, but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.


title: A novel domain within the CIL regulates egress of IFITM3 from the Golgi and prevents its deleterious accumulation in this apparatus

abstract:
The InterFeron-Induced TransMembrane proteins (IFITMs) are broad viral inhibitors that protect cells by preventing viral-to-cellular membrane fusion and they belong to the dispanin/CD225 family that includes vesicle trafficking regulators and proteins of unknown functions into four subfamilies (A-D). In this study, we uncover a novel domain that regulates the egress of IFITM3 from the Golgi and that is required to prevent IFITM3-driven v- to t-SNAREs membrane fusion inhibition and Golgi dysfunctions.

The S-x-K-x-R-D domain is conserved among vertebrate members of the dispanin/CD225 A subfamily that regroups all IFITMs and through the study of mutations identified in patients affected by paroxysmal kinesigenic dyskinesia (PKD), we determine that it is functionally conserved also in PRRT2, member of the B subfamily.

Overall, our study defines a novel domain that regulates the egress of dispanin/CD225 members from the Golgi and stresses the importance that regulation of this process bears to preserve the functions of this apparatus.


title: Dual control exerted by dopamine in blood-progenitor cell cycle regulation in Drosophila

abstract: In Drosophila, definitive hematopoiesis occurs in a specialized organ termed "lymph gland", where multi-potent stem-like blood progenitor cells reside and their homeostasis is central to growth of this organ. Recent findings have implicated a reliance on neurotransmitters in progenitor development and function however, our understanding of these molecules is still limited. Here, we extend our analysis and show that blood-progenitors are self-sufficient in synthesizing dopamine, a well-established neurotransmitter and have modules for its sensing through receptor and uptake via, transporter. Modulating their expression in progenitor cells affects lymph gland growth. Progenitor cell cycle analysis revealed an unexpected requirement for intracellular dopamine in the progression of early progenitors from S to G2 phase of the cell cycle, while activation of dopamine-receptor later in development regulated progression from G2 to entry into mitosis. The dual capacity in which dopamine operates, both intra-cellular and extra-cellular, controls lymph gland growth. These data highlight a novel and non-canonical use of dopamine as a proliferative cue by the myeloid-progenitor system and reveals a functional requirement for intracellular dopamine in cell-cycle progression.


title: Revisiting the evolutionary history of pigs via de novo mutation rate estimation by deep genome sequencing on a three-generation pedigree

abstract: The mutation rate used in the previous analyses of pig evolution and demographics was cursory and brought potential bias in inferring its history. Herein, we estimated de novo mutation rate of pigs using high-quality whole-genome sequencing data from nine individuals in a three-generation pedigree through stringent filtering and validation. The estimated mutation rate was 3.6 x 10-9 per generation, corresponding to 1.2 x 10-9 per site per year. Using this mutation rate, we re-investigated the evolutionary history of pigs. Our estimates agreed to the divergence time of ~10 kiloyears ago (Kya) between European wild and domesticated pigs, consistent with the domestication time of European pigs based on archaeological evidence. However, other divergence events inferred here were not as ancient as previously described. Our estimates suggested that: Sus speciation occurred ~1.36 Million years ago (Mya); European pigs split up with Asian ones only ~219 Kya; South and North Chinese wild pig split ~25 Kya. Meanwhile, our results showed that the most recent divergence event between Chinese wild and domesticated pigs occurred in the Hetao plain, North China, approximately 20 Kya, supporting the possibly independent domestication in North China along the middle Yellow River. We also found the maximum effective population size of pigs was ~6 times larger than the previous estimate. Notably by simulation, we confirmed an archaic migration from other Sus species originating ~ 2 Mya to European pigs during pigs western colonization, which possibly interfered with the previous demographic inference. Our findings advance the understanding of pig evolutionary history.


title: Gene expression in the developing nemertean brain indicates convergent evolution of complex brains in Spiralia

abstract:
BackgroundNemertea is a clade of worm-like animals, which belongs to a larger animal group called Spiralia (together with e.g. annelids, flatworms and mollusks). Many of the nemertean species possess a complex central nervous system (CNS) with a prominent brain, and elaborated chemosensory and neuroglandular cerebral organs, which have been suggested as homologues to the annelid mushroom bodies. In order to understand the developmental and evolutionary origins of complex nemertean brain, we investigated details of neuroanatomy and gene expression in the brain and cerebral organs of the juveniles of nemertean Lineus ruber.

ResultsIn the hatched juveniles the CNS is already composed of all major elements present in the adults, including the brain (with dorsal and ventral lobes), paired longitudinal lateral nerve cords and an unpaired dorsal nerve cord. The TEM investigation of the juvenile cerebral organ revealed that the structure is already composed of several distinct cell types present also in the adults. We further investigated the expression of twelve transcription factors commonly used as brain and cell type markers in bilaterian brains, including genes specific for annelid mushroom bodies. The expression of the investigated genes in the brain is region-specific and divides the entire organ into several molecularly distinct areas, partially overlapping with the morphological compartments. Additionally, we detected expression of mushroom body specific genes in the developing cerebral organs.

ConclusionsAt the moment of hatching, the juveniles of L. ruber already have a similar neuroarchitecture as adult worms, which suggests that further neural development is mostly related with increase in the size but not in complexity. Comparison in the gene expression between L. ruber and the annelid Platynereis dumerilii and other spiralians, indicates that the complex brains present in those two species evolved convergently by independent expansion of non-homologues regions of the simpler brain present in their common ancestor. The similarities in gene expression in mushroom bodies and cerebral organs might be a result of the convergent recruitment of the same genes into patterning of non-homologues organs or the results of more complicated evolutionary processes, in which conserved and novel cell types contribute to the non-homologues structures.


title: Distal and proximal cis-regulatory elements sense X-chromosomal dosage and developmental state at the Xist locus

abstract: Developmental genes such as Xist, the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist during the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of the Xist promoter. The strongest distal element is part of an enhancer cluster [~]200 kb upstream of the Xist gene which is associated with a previously unannotated Xist-enhancing regulatory transcript, we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.


title: Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens

abstract: Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-O_SCPLOWDC_SCPLOW-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. In this study, we observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties, as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.


title: Intrinsic Degradation of the Type-II Antitoxin ParD from Pseudomonas aeruginosa

abstract: Type-II Toxin Antitoxin (TA) systems are regulated by differential half-lives of the resulting non-secreted proteins, such that the neutralizing antitoxin undergoes continual degradation and replenishment to maintain neutralization of its cognate toxin. Antitoxin proteins are widely reported as labile, including upon purification and in vitro storage. During the course of studies on a ParDE TA system we noted a prevalent in vitro degradation of the ParD antitoxin. In efforts to combat this for practical use in assays, we characterized parameters impacting the degradation as well as the resulting products. These revealed a mechanism likely mediated by a serine or metal-dependent protease. Using Direct Infusion Mass Spectrometry, the cleavage products were identified as an essentially intact DNA binding region of the antitoxin and with the toxin binding domain completely removed. No other species were identified in the solution, such as a contaminant that may mediate such cleavage. Therefore, while our studies revealed viable strategies to mitigate the in vitro degradation they did not identify any protease, leaving open the possibility of a potential auto-catalytic proteolytic activity of the antitoxin proteins.


title: COVID-19 dominant D614G mutation in the SARS-CoV-2 spike protein desensitizes its temperature-dependent denaturation

abstract: The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, making it the dominant form in the COVID-19 pandemic. Here we demonstrated by cryo-electron microscopy that the D614G mutation does not significantly perturb the structure of the spike protein, but multiple receptor binding domains are in an upward conformation poised for host receptor binding. The impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics, and to significantly increase the thermal stability under physiological pH. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation, and suggest an approach for better design of spike-protein based conjugates for vaccine development.


title: Enrichment of SARS-CoV-2 entry factors and interacting intracellular genes in peripheral immune cells

abstract: SARS-CoV-2 uses ACE2 and TMPRSS2 to gain entry into the cell. However, recent studies have shown that SARS-CoV-2 may use additional host factors that are required for the viral lifecycle. Here we used publicly available datasets, CoV associated genes and machine learning algorithms to explore the SARS-CoV-2 interaction landscape in different tissues. We find that in general a small fraction of cells expresses ACE2 in the different tissues including nasal, bronchi and lungs. We show that a small fraction of immune cells (including T-cells, macrophages, dendritic cells) found in tissues also express ACE2. We show that healthy circulating immune cells do not express ACE2 and TMPRSS2. However, a small fraction of circulating immune cells (including dendritic cells, monocytes, T-cells) in the PBMC of COVID-19 patients express ACE2 and TMPRSS2. Additionally, we found that a large spectrum of cells (in circulation and periphery) in both healthy and COVID-19 positive patients were significantly enriched for SARS-CoV-2 factors. Thus, we propose that further research is needed to explore if SARS-CoV-2 can directly infect peripheral immune cells to better understand the virus mechanism of action.


title: SARS-CoV-2, a threat to marine mammals? A study from Italian seawaters

abstract:
Zoonotically transmitted coronaviruses were responsible for three disease outbreaks since 2002, with the "Severe Acute Respiratory Syndrome Coronavirus-2" (SARS-CoV-2) causing the dramatic "Coronavirus Disease-2019" (CoViD-19) pandemic, which affected public health, economy, and society on a global scale. The impacts of the SARS-CoV-2 pandemic permeate into our environment and wildlife as well; in particular, concern has been raised about the viral occurrence and persistence in aquatic and marine ecosystems. The discharge of untreated wastewaters carrying infectious SARS-CoV-2 into natural water systems that are home of sea mammals may have dramatic consequences on vulnerable species.

The efficient transmission of coronaviruses raise questions regarding the contributions of virus-receptors interactions. The main receptor of SARS-CoV-2 is Angiotensin Converting Enzyme-2 (ACE-2), serving as a functional receptor for the viral spike (S) protein. This study was aimed, through the comparative analysis of the ACE-2 receptor with the human one, at assessing the susceptibility to SARS-CoV-2 of the different species of marine mammals living in Italian waters. We also determined, by means of immunohistochemistry, ACE-2 receptor localization in the lung tissue from different cetacean species, in order to provide a preliminary characterization of ACE-.2 expression in the marine mammals respiratory tract.

Furthermore, in order to evaluate if and how wastewater management in Italy may lead to susceptible marine mammal populations being exposed to the virus, geo-mapping data of wastewater plants, associated to the identification of specific stretches of coast more exposed to extreme weather events, overlapped to marine mammal population data, were carried out. Results showed the SARS-CoV-2 exposure for marine mammals inhabiting Italian coastal waters. Thus, we highlight the potential hazard of reverse zoonotic transmission of SARS-CoV-2 infection, along with its impact on marine mammals regularly inhabiting the Mediterranean Sea, whilst also stressing the need of appropriate action to prevent further damage to specific vulnerable populations.

Significance StatementGrowing concern exists that SARS-CoV-2, as already ascertained for its SARS-CoV and MERS-CoV "predecessors", originated from an animal "reservoir", performing thereafter its spillover into mankind, that was possibly anticipated by viral "passage" into a secondary animal host. Within the dramatic SARS-CoV-2 pandemic context, hitherto characterized by over 110 million cases and almost 2,500,000 deaths on a global scale, several domestic and wild animal species have been reported as susceptible to natural and/or experimental SARS-CoV-2 infection. In this respect, while some marine mammal species are deemed as potentially susceptible to SARS-CoV-2 infection on the basis of the sequence homology of their ACE-2 viral receptor with the human one, this study addresses such a critical issue also in stranded sea mammal specimens.

category: pathology


title: Chromatin accessibility dynamics of neurogenic niche cells reveal a reversible decline in neural stem cell migration during aging

abstract: Aging is accompanied by a deterioration in the regenerative and repair potential of stem cell regions in the brain. However, the mechanisms underlying this decline are largely unknown. Here we profile the chromatin landscape of five different cell types freshly isolated from the subventricular zone neurogenic niche of young and old mice. We find that chromatin states exhibit distinct changes with aging in different cell types. Notably, the chromatin of quiescent neural stem cells (NSCs) becomes more repressed with age whereas that of proliferative, activated NSCs becomes more open. Surprisingly, these opposing age-related chromatin changes involve cell adhesion and migration pathways. We experimentally validate that quiescent and activated NSCs exhibit opposite migratory deficits during aging. Quiescent NSCs become more migratory during aging, whereas activated NSCs and progeny become less migratory and less able to mobilize out of the niche in vivo during aging. The cellular mechanism by which aging impairs the migration of activated NSCs and progeny involves increased occurrence of force-producing focal adhesions. Inhibiting the cytoskeletal-regulating kinase ROCK in old activated NSCs and progenitors eliminates cell adhesive forces and boosts migration speed, reverting these cells to a more youthful migratory state. Our work has important implications for restoring the migratory potential of NSCs during aging and brain injury.

category: neuroscience


title: Astrocyte-neuron interplay is critical for Alzheimer's disease pathogenesis and is rescued by TRPA1 channel blockade

abstract:
BackgroundThe sequence of cellular dysfunctions in preclinical Alzheimers disease must be understood if we are to plot new therapeutic routes. Hippocampal neuronal hyperactivity is one of the earliest events occurring during the preclinical stages of Alzheimers disease in both humans and mouse models. The most common hypothesis describes amyloid {beta} accumulation as the triggering factor of the disease but the effects of such accumulation and the cascade of events leading to cognitive decline remain unclear. In mice, we previously showed that amyloid {beta}-dependent TRPA1 channel activation triggers hippocampal astrocyte hyperactivity, subsequently inducing hyperactivity in nearby neurons. In this work, we investigated the potential protection brought by an early chronic pharmacological inhibition of TRPA1 channel on Alzheimers disease progression.

MethodsWe administered a specific inhibitor of TRPA1 channel (HC030031) intraperitoneally from the onset of amyloid {beta} overproduction in the APP/PS1-21 mouse model of Alzheimers disease. We characterized short-, medium-, and long-term effects of this chronic pharmacological TRPA1 blockade on Alzheimers disease progression at functional (astrocytic and neuronal activity), structural, biochemical, and behavioural levels.

ResultsOur results revealed that the first observable disruptions in the Alzheimers disease transgenic mouse model used correspond to aberrant hippocampal astrocyte and neuron hyperactivity. We showed that chronic TRPA1 blockade normalizes astrocytic activity, avoids perisynaptic astrocytic process withdrawal, prevents neuronal dysfunction, preserves structural synaptic integrity and strengthens the glial plaque barrier. These protective effects preserved spatial working-memory in this Alzheimers disease mouse model.

ConclusionThe toxic effect of amyloid {beta} on astrocytes triggered by TRPA1 channel activation is pivotal to Alzheimers disease progression. TRPA1 blockade prevent irreversible neuronal dysfunction, making this channel a potential therapeutic target to promote neuroprotection.


title: Systematic analysis of CD39, CD103, CD137 and PD-1 as biomarkers for naturally occurring tumor antigen-specific TILs

abstract: The detection of tumor-specific T cells in solid tumors is integral to the interrogation of endogenous antitumor responses and to the advancement of downstream therapeutic applications, such as checkpoint immunotherapy and adoptive cell transfer. A number of biomarkers are reported to identify endogenous tumor-specific tumor infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39, however a direct comparison of these molecules has yet to be performed. Here, we evaluate these biomarkers in primary human high-grade serous ovarian tumor samples using single-cell mass cytometry to characterize and compare their relative phenotypic profiles, as well as their response to autologous tumor cells ex vivo. CD137+, PD-1+, CD103+, and CD39+ TILs are all detectable in tumor samples with CD137+ TILs being the least abundant. PD-1+, CD103+, and CD39+ TILs all express a subset of CD137+ cells, while CD137+ TILs highly co-express the aforementioned markers. CD137+ TILs exhibit the highest expression of cytotoxic effector molecules, such as IFN{gamma} and Granzyme B, compared to PD-1+, CD103+ or CD39+ TILs. Removal of CD137+ TILs from PD-1+, CD103+, or CD39+ TILs results in lower secretion of IFN{gamma} in response to autologous tumor stimulation, while CD137+ TILs highly secrete IFN{gamma} in an HLA-dependent manner. CD137+ TILs exhibited an exhausted phenotype with CD28 co-expression, suggestive of antigen recognition and receptiveness to reinvigoration via immune checkpoint blockade. Together, our findings demonstrate that the antitumor abilities of PD-1+, CD103+, and CD39+ TILs are mainly derived from a subset of TILs expressing CD137, implicating CD137 is a more selective biomarker for naturally occurring tumor-specific TILs.


title: Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells

abstract: In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1B, tumor necrosis factor (TNF) and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naive T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFNg production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.