title: Bioinformatic analysis of B and T cell epitopes from SARS-CoV-2 Spike, Membrane and Nucleocapsid proteins as a strategy to assess possible cross-reactivity between emerging variants, including Omicron, and other human coronaviruses

abstract:
The COVID-19 pandemic caused by SARS-CoV-2 produced a global health emergency since December 2019, that up to the end of January 2022 had caused the death of more than 5.6 million people worldwide. Despite emergence of new variants of concern, vaccination remains one of the most important tools to control the pandemic. All approved vaccines and most of the vaccine candidates use the spike protein of the virus as a target antigen to induce protective immune responses. Several variants of the virus present key mutations in this protein which render the virus, at different rates, to evade the neutralizing antibody response. Although experimental evidence suggests that cross-reactive responses between coronaviruses are present in the population, it is unknown which potential antigens shared between different coronaviruses could be responsible for these responses. This study provides predictions of new potential B and T cell epitopes within SARS-CoV-2 Spike (S), Membrane (M) and Nucleocapsid (N) proteins together with a review of the reported B epitopes of these proteins. We also analyse amino acid changes present in the epitopes of variants of concern (VOC) and variants being monitored (VBM), and how these might affect the immune response, as these changes may alter the peptides immunogenicity index and the antigen presentation by related HLA alleles. Finally, given these observations, we performed an identity analysis between the repertoire of potential epitopes of SARS-CoV-2 and other human coronaviruses to identify which are conserved among them.

The results shown here together with the published experimental evidence, allow us to support the hypothesis that antibody and T cell cross-reactive responses to common coronaviruses epitopes, could contribute to broaden the protective response to SARS-CoV-2 and its variants. This evidence could help not only to understand cross-reactive responses among coronaviruses but also contribute to elucidate their role in immunity to SARS-CoV-2 induced by infection and/or vaccination. Finally, these findings could promote targeted analysis of antigen-specific immune responses and might orient and drive the rational development of new SARS-CoV-2 vaccines including candidates that ideally provide "universal" protection against other coronaviruses relevant to human health.

category: bioinformatics


title: A call to rethink the cognitive benefits of physical exercise: An umbrella review of randomized controlled trials

abstract:
BackgroundMany meta-analytic reviews have examined the cognitive benefits of regular physical exercise across the lifespan, with a wide range of inclusion criteria and disparate analytic approaches. In fact, most global health organizations incorporate the purported cognitive benefits as part of their policies to promote regular physical exercise. We assess whether those claims are well supported by scientific evidence.

MethodsWe conducted a pre-registered (PROSPERO: CRD42020191357) umbrella review of meta-analyses limited to randomized controlled trials (RCTs) on exercise-cognition. Outcomes of each meta-analysis were reanalyzed following a multilevel model approach. Publication bias was also analyzed to correct final effect sizes.

FindingsMost of the 12 reviewed meta-analyses reported a positive small effect (d = 0.21, [0.07-0.40]) of physical exercise across all cognitive domains in all life stages. However, a critical assessment of the primary studies included in these reviews and the meta-analytic approaches used indicates that the evidence might not be reliable and therefore no strong conclusions should be drawn based on the existing evidence. We found that the literature has undergone an unusually rapid growth upon underpowered RCTs and potentially biased meta-analytic reviews.

InterpretationThe exponential accumulation of low-quality evidence on the exercise-cognition topic has led to stagnation in the field, hindering the discernment of the potential impact of exercise on cognition. We urge worldwide organizations committed to public health to reconsider their recommendations on the promotion of regular physical exercise to boost cognitive functions in the healthy population until high-quality empirical evidence confirms these exercise-induced cognitive benefits.

FundingSpanish Ministry of Science and Innovation (PID2019-105635GB-I00 and PID2020-118583GB-I00).

category: neuroscience


title: Functional connectivity of the inferior frontal gyrus: A meta-analytic connectivity modeling study

abstract:
BackgroundNeurocognitive models of language processing highlight the role of the left inferior frontal gyrus (IFG) in the functional network underlying language. Furthermore, neuroscience research has shown that IFG is not a uniform region anatomically, cytoarchitectonically or functionally. However, no previous study explored the language-related functional connectivity patterns of different IFG subdivisions using a meta-analytic connectivity approach.

PurposeThe present meta-analytic connectivity modeling (MACM) study aimed to identify language-related coactivation patterns of the left and right IFG subdivisions.

MethodSix regions of interest (ROIs) were defined using a probabilistic brain atlas corresponding to pars opercularis (BA44), pars triangularis (BA45) and pars orbitalis (Fo6&7) of IFG in both hemispheres. The ROIs were used to search the BrainMap functional database to identify neuroimaging experiments with healthy, right-handed participants reporting language-related activations in each ROI. Activation likelihood estimation analyses were then performed on the foci extracted from the identified studies to compute functional convergence for each ROI, which was also contrasted with the other ROIs within the same hemisphere. In addition, a behavioral analysis was conducted to determine functional specificity for language subdomains within each ROI.

ResultsA primarily left-lateralized functional network was revealed for the left and right IFG subdivisions. The left IFG ROIs exhibited a more robust coactivation pattern than the right IFG ROIs. In particular, the left posterior-dorsal IFG (BA44) was associated with the most extensive coactivation pattern involving bilateral frontal, bilateral parietal, left temporal, left subcortical (thalamus and putamen), and right cerebellar regions, while the left anterior-ventral IFG (BA45 and Fo6&7) revealed a predominantly left-lateralized involvement of frontotemporal regions.

ConclusionThe findings align with the neurocognitive models of language processing that propose a division of labor among the left IFG subdivisions and their respective functional networks. Also, the opercular part of left IFG (BA44) stands out as a major hub in the language network with connections to diverse cortical, subcortical and cerebellar structures.


title: The Amidella clade in Europe (Amanita Pers., Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens and the importance of taxon-specific PCR primers for identification

abstract: The species in genus Amanita section Amidella form a well-defined clade, but some taxa remain difficult to discriminate. In particular, the concept of Amanita valens (E.-J. Gilbert) Bertault remains controversial. To understand the phylogenetic placement of a set of collections from South Portugal with a novel nrDNA barcode, we have obtained nrDNA sequences for previously unassessed type collections. The taxon formerly described as Amanita curtipes f. pseudovalens Neville & Poumarat is interpreted as a separate species, Amanita pseudovalens (Neville & Poumarat) Arraiano-Castilho et al. comb. et stat. nov., and is genetically indistinct from the Portuguese collections, thus clarifying their taxonomic context. However, our collections are morphologically and ecologically distinct (respectively, ellipsoid to oblong basidiospores and association with Cistus on acidic schist soils), and are proposed as a new variety, Amanita pseudovalens var. tartessiana Arraiano-Castilho et al.. These developments also enable a better diagnosis of Amidella taxa in Europe, a progress that is most decisive for the late Winter to Spring season collections, and identification keys are proposed. However, the co-occurrence and morphological similarity of the new variety, in comparison with the prized edible Amanita ponderosa Malencon & Heim, could leave some collections of either taxa unresolved. Thus, a molecular marker approach was developed, to provide a clear and cost-effective identification aid to complement the keys. The proposed diagnostic tools can be applied toward a review of European Amidella taxa chorology from existing records, conserved materials, and future collections. Evolutionary convergence may contribute to the determination difficulties in the Amidella clade.

category: evolutionary biology


title: Locally adaptive temperature response of vegetative growth in Arabidopsis thaliana

abstract: We investigated early vegetative growth of natural Arabidopsis thaliana accessions in cold, non-freezing temperatures, similar to temperatures these plants naturally encounter in fall at northern latitudes. We found that accessions from northern latitudes produced larger seedlings than accessions from southern latitudes, partly as a result of larger seed size. However, their subsequent vegetative growth when exposed to colder temperatures was slower. The difference was too large to be explained by random population differentiation, and is thus suggestive of local adaptation, a notion that is further supported by substantial transcriptome and metabolome changes in northern accessions. We hypothesize that the reduced growth of northern accessions is an adaptive response, and a consequence of reallocating resources towards cold acclimation and winter survival.

category: plant biology


title: Factors that affected the qualification of standardized residency training of internal medicine: a prospective longitudinal study

abstract:
IntroductionSince the standardized residency training (SRT) was launched in 2014, there were very few data on evaluation of the factors that could influence the residents competency in China.

MethodsAll residents who started their SRT programme from September 2015 to September 2018 were enrolled in this study. During the following years, they finished the programme and took examinations. The demographic information and exam scores of each subject were collected and analyzed.

ResultsWe found that the qualification rates of SRT programme differed significantly annually. Age, gender, the highest medical education degree, residents from different places, training duration and years of medical school study were also different significantly year by year. According to the scores of examinations, residents were divided into two groups: the qualified group and the non-qualified group. Age, the highest medical education degree, residents from different places, training duration, years of medical school study and prior medical work experience differed significantly between the two groups. Multivariate Logistic regression analysis showed the residents from different places and the training duration were factors that affected the qualification rate of SRT programme independently (P = 0.000 and 0.005, respectively).

DiscussionThe "homogeneity" of SRT had been achieved regardless of the individual discrepancy, while the resident from different places and the training duration were obstacles we need to overcome further. Therefore, we should further advance the process of the transition from "Unit Persons" to "Society Persons". And extending training duration will facilitate their qualification and be benefit to improve their competency.

category: scientific communication and education


title: Multisensory integration of social signals by a pathway from the basal amygdala to the auditory cortex in maternal mice

abstract: Social encounters are inherently multimodal events, yet how and where social cues of distinct modalities merge and interact in the brain is poorly understood. For example, when their pups wander away from the nest, mother mice use a combination of vocal and olfactory signals emitted by the pups to locate and retrieve them. Previous work revealed the emergence of multisensory interactions in the auditory cortex (AC) of both dams and virgins who co-habitate with pups ( surrogates). Here we identify a neural pathway that integrates information about odors with responses to sound. We found that a scattered population of glutamatergic neurons in the basal amygdala (BA) projects to the AC and responds to odors, including the smell of pups. These neurons also exhibit increased activity when the surrogate female is searching for pups. Finally, we show that selective optogenetic activation of BA-AC neurons modulates responses to pup calls, and that this modulation switches from predominantly suppressive to predominantly excitatory after maternal experience. This supports an underappreciated role for the amygdala in directly shaping sensory representations in an experience-dependent manner. We propose that the BA-AC pathway integrates olfaction and audition to facilitate maternal care, and speculate that it may carry valence information to the AC.


title: A natural broad-spectrum inhibitor of enveloped virus entry, effective against SARS-CoV-2 and Influenza A Virus in preclinical animal models.

abstract: The COVID-19 pandemic has highlighted the need for novel antivirals for pandemic management and preparedness. Targeting host processes that are co-opted by viruses is an attractive strategy for developing antivirals with a high resistance barrier. Picolinic acid (PA) is a byproduct of tryptophan metabolism, endogenously produced in humans and other mammals. Here we report broad-spectrum antiviral effects of PA against enveloped viruses, including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), Influenza A virus (IAV), Flaviviruses, Herpes Simplex Virus, and Human Parainfluenza Virus. We further demonstrate using animal models that PA is effective against SARS-CoV-2 and IAV, especially as an oral prophylactic. The mode of action studies revealed that PA inhibits viral entry of enveloped viruses, primarily by interfering with viral-cellular membrane fusion, inhibiting virus-mediated syncytia formation, and dysregulating cellular endocytosis. Overall, our data establish PA as a broad-spectrum antiviral agent, with promising preclinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

category: microbiology


title: An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

abstract:
The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity.

One sentence summaryA human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants


title: Transmission risk assessment of Aedes-borne arboviral diseases in Accra, Ghana

abstract:
BackgroundDengue, Zika and Chikungunya are Aedes-borne viral diseases that have risen to become great global health concerns in the past years. Several countries in Africa have reported outbreaks of these diseases and despite Ghana sharing borders with some of such countries, it remains free of these outbreaks.

Recent studies in Ghana have revealed that there are antibodies and viral RNA of the Dengue virus serotype-2 among individuals in some localities in the Greater Accra Region. This is an indication of a possible silent transmission ongoing in the population, hence the need to assess the risk of transmission of these viruses within the country. This cross-sectional study, therefore, assessed the risk of transmission of Dengue, Zika and Chikungunya viruses in a domestic/peri-domestic (Madina) and a forest (Achimota Forest) population in the Greater Accra Region, Ghana.

Methodology/FindingsAll stages of the Aedes mosquito (egg, larvae, pupae and adults) were collected around homes and in the forest area for estimation of risk indices. All eggs and immature stages were reared to adults and morphologically identified. The predominant species of Aedes mosquitoes identified from both sites were Aedes aegypti (98 % in Madina and 98.1% in Achimota forest). Aedes albopictus, an important arbovirus vector, was identified only in Madina at a prevalence of 1.5% but Achimota forest had the higher species diversity. Both study sites recorded high risk indices; Madina: Positive Ovitrap Index = 26.6%, Container Index = 36.8%, House Index = 19.8%, Breteau Index = 70.4%; Achimota: Positive Ovitrap Index = 34.2% and Container Index = 67.9%. RT-PCR to detect the presence of Dengue, Chikungunya and Zika viruses was negative for all pools tested.

ConclusionAll entomological risk indicators estimated showed that both sites had a high potential of an outbreak of arboviral diseases following the introduction of these viruses.

Author SummaryThe detection of antibodies and viral RNA of the dengue virus serotype 2 in some communities in the urban city of Accra, suggested the possibility of silent transmission of arboviral disease within the city. We assessed the risk of arboviral disease transmission using entomological risk indices. The study was a cross-sectional study conducted in a forest and peri domestic setting located in the southern urban city of Accra.

The different stages of the Aedes mosquito were collected and, houses and containers positive for Aedes mosquitoes were also noted. The Breteau (BI), House (HI), Container (CI) and Positive ovitrap (POI) indices were determined. Real Time-PCR was conducted to determine the presence of Dengue, Zika and Chikungunya viruses in the larvae and adults collected.

Aedes aegypti was the most common species identified from both sites. Aedes albopictus another competent arbovirus vector was identified in the peri-domestic site. Almost all risk indices recorded for both sites were higher than the WHO thresholds allowed for these indices. However, real time-PCR to detect the presence of Dengue, Chikungunya and Zika viruses was negative.

The high entomological risk indicators estimated showed that both sites had a great potential of an outbreak following the introduction of these viruses, and a well-structured surveillance for these vectors is highly recommended. The detection of the presence of Ae. albopictus, an invasive species is also of great concern.

category: ecology


title: An efficient early-pooling protocol for environmental DNA metabarcoding

abstract: Environmental DNA (eDNA) metabarcoding, a method that applies high-throughput sequencing and universal primer sets to eDNA analysis, has been a promising approach for efficient, comprehensive biodiversity monitoring. However, significant money-, labor-, and time-costs are still required for performing eDNA metabarcoding. In the present study, we assessed the performance of an "early-pooling" protocol (a protocol based on the 1st PCR indexing) to reduce the experimental costs of the library preparation for eDNA metabarcoding. Specifically, we performed three experiments to test the effects of 1st PCR and 2nd PCR indexing protocols on the community composition revealed by eDNA metabarcoding, of post-1st-PCR exonuclease purification on index-hopping, and of the number of PCR replicates and eDNA template volume on the number of detected OTUs. By analyzing 204 eDNA libraries from 3 natural aquatic ecosystems and 1 mock eDNA sample, we show that (i) the 1st PCR indexing does not cause clear biases in the outcomes of eDNA metabarcoding, (ii) post-1st-PCR exonuclease purification reduces the risk of index-hopping, and (iii) increasing the eDNA template volume can increase the number of detected OTUs and reduce the variations in detected community compositions, as can increasing the number of the 1st PCR replicates. Our results show that an early-pooling protocol with post-1st-PCR exonuclease purification and an increased amount of DNA template will reduce the risk of index-hopping, the costs for consumables and reagents, and the handling time in the library preparation, and that it produces comparable results to a 2nd-PCR-indexing protocol. Therefore, once a target metabarcoding region is determined and a set of indexed-1st-PCR primers is prepared, the early-pooling protocol provides a cost-, labor-, and time-efficient way to process a large number of samples.


title: Age and spatio-temporal variations in food resources modulate stress-immunity relationships in three populations of wild roe deer

abstract: Living in variable and unpredictable environments, organisms face recurrent stressful situations. The endocrine stress response, which includes the secretion of glucocorticoids, helps organisms to cope with these perturbations. Although short-term elevations of glucocorticoid levels are often associated with immediate beneficial consequences for individuals, long-term glucocorticoid elevation can compromise key physiological functions such as immunity. While laboratory works highlighted the immunosuppressive effect of long-term elevated glucocorticoids, it remains largely unknown, especially in wild animals, whether this relationship is modulated by individual and environmental characteristics. In this study, we explored the co-variation between baseline cortisol levels, assessed non-invasively using faecal cortisol metabolites (FCMs), and 12 constitutive indices of innate, inflammatory, and adaptive immune functions, in wild roe deer living in three populations with contrasting environmental conditions. Using longitudinal data on 564 individuals, we further investigated whether age and spatio-temporal variations in the quantity and quality of food resources affect the relationship between FCMs and immunity. Negative covariation with glucocorticoids was evident only for innate and inflammatory markers of immunity, while adaptive immunity appeared to be positively or not linked to glucocorticoids. In addition, the negative covariations were generally exacerbated, or revealed, in individuals facing harsh environmental constraints and in old individuals. Therefore, our results highlight the importance of measuring multiple immune markers of immunity in individuals from contrasted environments to unravel the complex relationships between glucocorticoids and immunity in wild animals. Our results also help explain conflicting results found in the literature and could improve our understanding of the long-term consequences of elevated glucocorticoid levels on disease spread and population dynamics.


title: Finding coarse and fine scale population structure in a coastal species: population demographics meets genomics

abstract: Population genetic studies often focus on patterns at a regional scale and use spatially aggregated samples to draw inferences about population structure and drivers, potentially masking ecologically relevant population sub-structure and dynamics. In this study we use a multidisciplinary approach combining genomic, demographic, and habitat data with an oceanographic particle drift model, to unravel the patterns of genetic structure at different scales in the black goby (Gobius niger) along the Norwegian coast. Using a high-density sampling protocol, we observed restricted gene flow both at a surprisingly fine (kms) and large (100s km) scale. Our results showed a pattern of isolation by distance related to the level of exposure along the Skagerrak coast, where sheltered sampling stations had an overall level of genetic divergence about three times higher (FST =0.0046) than levels observed among exposed samples (FST =0.0015). These results were corroborated by demographic analyses which showed that population-fluctuations decrease in synchrony with distance at much smaller scales for sheltered samples (20 km) than for exposed sites (80 km), suggesting higher population connectivity among exposed sites. We also found a pronounced genetic discontinuity between populations along the Norwegian west and east coasts, with a sharp "break" around the southern tip of Norway, likely driven both by lack of habitat and by oceanographic features.

category: genomics


title: Genome-wide in vivo CRISPR screens identify GATOR1 as a TP53 induced tumour suppressor

abstract: The tumour suppressor TP53 (called TRP53 in mice) protects cells from neoplastic transformation by activating diverse cellular processes 1,2. While tumour suppression by TRP53-mediated induction of apoptosis, cell cycle arrest, cell senescence and DNA repair has been well characterised 1-4, there is so far no example of the loss of a TP53 activated regulator of cellular metabolism promoting tumour development. Using in vivo genome-wide CRISPR knockout screens we identified Nprl3 and Depdc5, encoding components of the GATOR1 complex which inhibits mTORC1, as novel tumour suppressors that inhibit c-MYC-driven lymphomagenesis. In a parallel in vivo CRISPR knockout screen using a focused library of sgRNAs targeting TRP53 binding sites in gene promoters/enhancers, we discovered that loss of a TRP53 binding site in the Nprl3 promoter accelerated c-MYC-driven lymphomagenesis to a similar extent as loss of the Nprl3 gene itself. These findings along with the observations that (i) Nprl3 is upregulated in response to DNA damaging drugs in wild-type (wt) but not TRP53 knockout cells and that (ii) GATOR1 deficient E-Myc lymphomas all retained wt TRP53 function, whereas [~]30% of control E-Myc lymphomas had selected for mutations in Trp53, suggested that direct transcriptional inducton of GATOR1 by TRP53 is a critical tumour suppressive mechanism. Additionally, we found that GATOR1 deficient tumours exhibited abnormally increased mTORC1 signalling, which rendered them highly sensitive to the mTORC1 inhibitor rapamycin, both in vitro and in vivo. Collectively, these findings identify the first mechanism by which TRP53 suppresses tumourigenesis by transcriptional activation of a regulator of metabolism and they also reveal a potential bio-marker to predict responses to mTORC1 inhibitors in the clinic.

category: cancer biology


title: Rock inhibitors target SRSF2 leukemia by disrupting cell mitosis and nuclear morphology

abstract: Spliceosome machinery mutations are common early mutations in myeloid malignancies, however effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified ROCK inhibitors (ROCKi) as selective inhibitors of SRSF2 mutants. ROCKi targeted SRSF2 Mut primary human samples in a xenografts model and were not toxic to mice nor human cells. ROCKi induced mitotic catastrophe through their apparent effects on microtubules and nuclear organization. Transmission electron microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation, driven by microtubule-rich cytoplasmic intrusions, which were exacerbated by ROCKi. The severe nuclear deformation driven by the combination of SRSF2 Mut and ROCKi prevent cells from completing mitosis. These findings shed light on new ways to target SRSF2 and on the role of the microtubule system in SRSF2 Mut cells.


title: Reconstitution and Expression of mcy Gene Cluster in The Model Cyanobacterium Synechococcus 7942 Reveals a Roll of MC-LR in Cell Division

abstract:
Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. The most common cyanotoxins, microcystins (MCs), can cause acute poisoning at high concentrations and hepatocellular carcinoma following chronic exposure. Among all MC variants, MC-LR produced by Microcystis aeruginosa PCC 7806 is the most common toxic MC. Although the biosynthetic pathway for MC-LR has been proposed, experimental support of this pathway is lacking. In an effort to experimentally validate this pathway, we expressed the 55 kb microcystin biosynthetic gene cluster (mcy cluster) (mcyA-J) and produced MC-LR in the model cyanobacterium Synechococcus 7942. We designed and constructed the strong bidirectional promoter biPpsbA2 between mcyA and mcyD, reassembled the mcy cluster in yeast by transformation-associated recombination (TAR cloning), transformed the gene cluster into the NSII site of Synechococcus 7942, and successfully expressed MC-LR at a level of 0.006-0.018 fg cell-1 day-1. The expression of MC-LR led to abnormal cell division and the filamentation of Synechococcus 7942 cells, further analysis proved a role of MC-LR in functional assembly of the cell division protein FtsZ, by competing its GTP binding site. These results represent the first synthetic biological expression of the mcy cluster and the autotrophic production of MC-LR in a photosynthetic model organism, which lays the foundation for resolving the MC biosynthesis pathway. The suggested role of MC-LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their non-blooming counterparts.

HighlightsO_LIWe expressed the 55 kb mcy cluster and produced MC-LR in the model cyanobacterium Synechococcus 7942.
C_LIO_LIThis is the first realized production of MC in the model non-toxin-production cyanobacteria from CO2 by photosynthesis.
C_LIO_LICompared with the self-replicating plasmid, the recombination of the mcy cluster into the genome of Synechococcus 7942 is more suitable for the heterologous production of microcystin.
C_LIO_LIMC-LR inhibits cell division by irreversibly competing the GTP binding domain of the cell division protein FtsZ.
C_LIO_LIThe newly discovered effect of MC-LR on cell division reveals a mechanism of how blooming cyanobacteria gain competitive edge over their non-blooming counterparts.
C_LI

category: synthetic biology


title: A Random Forest Classifier Uses Antibody Responses to Plasmodium Antigens to Reveal Candidate Biomarkers of the Intensity and Timing of Past Exposure to Plasmodium falciparum

abstract: 1Important goals of malaria surveillance efforts include accurately quantifying the burden of malaria over time, which can be useful to target and evaluate interventions. The majority of malaria surveillance methods capture active or recent infections which poses several challenges to achieving malaria surveillance goals. In high transmission settings, asymptomatic infections are common and therefore accurate measurement of malaria burden often demands active surveillance; in low transmission regions where infections are rare accurate surveillance requires sampling a large subset of the population; and in any context monitoring malaria burden over time necessitates serial sampling. Antibody responses to Plasmodium falciparum parasites persist after infection and therefore measuring antibodies has the potential to overcome several of the current difficulties associated with malaria surveillance. However, identifying which antibody responses are markers of the timing and intensity of past exposure to P. falciparum is challenging, particularly among adults who tend to be re-exposed multiple times over the course of their lifetime and therefore have similarly high antibody responses to many P. falciparum antigens. A previous analysis of 479 serum samples from individuals in three regions in southern Africa with different historical levels of P. falciparum malaria transmission (high, intermediate, and low) revealed regional differences in antibody responses to P. falciparum antigens among children under 5 years of age. Using a novel bioinformatic pipeline optimized for protein microarrays that minimizes between-sample technical variation, we used antibody responses to P. falciparum and P. vivax antigens as predictors in random forest models to classify adult samples into these three regions of differing historical malaria transmission with high accuracy. Many of the antigens that were most important for classification in these models do not overlap with previously published results and are therefore novel candidate markers for the timing and intensity of past exposure to P. falciparum. Measuring antibody responses to these antigens could potentially lead to improved malaria serosurveillance that captures the timing and intensity of past exposure.

category: immunology


title: Azithromycin possesses biofilm-inhibitory activity and potentiates non-bactericidal colistin methanesulfonate against Klebsiella pneumonia

abstract: Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.


title: Distinct physiological responses of Coccolithus braarudii life cycle phases to light intensity and nutrient availability

abstract:
Coccolithophores feature a haplo-diplontic life cycle comprised of diploid cells producing heterococcoliths and haploid cells producing morphologically different holococcoliths. These life cycle phases of each species appear to have distinct spatial and temporal distributions in the oceans, with the heavily-calcified heterococcolithophores (HET) often more prevalent in winter and at greater depths, whilst the lightly-calcified holococcolithophores (HOL) are more abundant in summer and in shallower waters. The haplo-diplontic life cycle may therefore allow coccolithophores to expand their ecological niche, switching between life cycle phases to exploit conditions that are more favourable. However, coccolithophore life cycles remain poorly understood and fundamental information on the physiological differences between life cycle phases is required if we are to better understand the ecophysiology of coccolithophores. In this study, we have examined the physiology of HET and HOL phases of the coccolithophore Coccolithus braarudii in response to changes in light and nutrient availability. We found that the HOL phase was more tolerant to high light than the HET phase, which exhibited defects in calcification at high irradiances. The HET phase exhibited defects in coccolith formation under both nitrate (N) and phosphate (P) limitation, whilst no defects in calcification were detected in the HOL phase. The HOL phase grew to a higher cell density under P-limitation than N-limitation, whereas no difference was observed in the maximum cell density reached by the HET phase at these nutrient concentrations. HET cells grown under a light:dark cycle divided primarily in the dark and early part of the light phase, whereas HOL cells continued to divide throughout the 24 h period. The physiological differences may contribute to the distinct biogeographical distributions observed between life cycle phases, with the HOL phase potentially better adapted to high light, low nutrient regimes, such as those found in seasonally stratified surface waters.

HighlightsO_LICoccolithus braarudii life cycle phases exhibit different physiological responses.
C_LIO_LIThe heavily-calcified heterococcolithophores (HET) life cycle phase is more sensitive to high light.
C_LIO_LIThe lightly-calcified holococcolithophores (HOL) life cycle phase may be better suited to growth under low phosphate availability.
C_LI


title: Cross-kingdom metagenomic profiling of Lake Hillier reveals pigment-rich polyextremophiles and wide-ranging metabolic adaptations

abstract:
Background Lake Hillier is a hypersaline lake known for its distinctive bright pink color. The cause of this phenomenon in other hypersaline sites has been attributed to halophiles, Dunaliella, and Salinibacter, however, a systematic analysis of the microbial communities, their functional features, and the prevalence of pigment-producing-metabolisms has not been previously studied.

Our results are evidence that Lake Hillier is composed of a diverse set of microorganisms including archaea, bacteria, algae, and viruses. Our data indicate a core microbiome in Lake Hillier composed of multiple pigment-producer microbes, many of which are cataloged as polyextremophiles. Additionally, we estimated the diversity of metabolic pathways in the lake and determined that many of these are related to pigment production. We reconstructed complete or partial genomes for 21 discrete bacteria (N = 14) and archaea (N = 7), only 2 of which could be taxonomically annotated to previously observed species.

Our findings provide the first metagenomic study to decipher the source of the pink color of Australias Lake Hillier. The study of this pink hypersaline environment is evidence of a microbial consortium of pigment producers, a repertoire of polyextremophiles, a core microbiome and potentially novel species.


title: Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold

abstract:
Hepatitis B virus (HBV) replicates by protein-primed reverse transcription. It chronically infects >250 million people, and the dominant anti-HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains, the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNaseH). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviral pathway, HBV P has resisted structural analysis for decades. Here, we exploited advances in protein structure prediction to model the structure of P. The predicted HBV RT and RNaseH domains aligned to the HIV RT-RNaseH at 3.75 [A] RMSD, had a nucleic acid binding groove spanning the two active sites, had DNA polymerase active site motifs in their expected positions, and accommodated two Mg++ ions in both active sites. Surprisingly, the TP domain wrapped around the RT domain, with the priming tyrosine poised over the RT active site. This model was validated using published mutational analyses, and by docking RT and RNaseH inhibitors, RNA:DNA heteroduplexes, and the HBV {varepsilon} RNA stem-loop that templates DNA priming into the model. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses from rodents to fish and in P from a fish nackednavirus, but not in other non-retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will guide drug development and mechanistic studies into Ps function.

SignificanceThe hepatitis B virus (HBV) polymerase (P) catalyzes an unusual reverse transcription pathway and is a major drug target. However, Ps insolubility and instability have long prevented its structural analyses. This work predicts the structure of P proteins from human to fish viruses, revealing an unanticipated conserved protein fold that is at least 400 million years old. The HBV P model was validated by mechanistically interpreting mutations with strong phenotypes and by computationally docking nucleic acids to P and inhibitors into both of its active sites. The model will advance mechanistic studies into the function of P and enable drug discovery against targets on P other than the reverse transcriptase and ribonuclease H active sites.


title: The Mycobacterium tuberculosis protein O-phosphorylation landscape

abstract: Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria remains unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated STPK loss-and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is an underexplored protein modification that affects >70% of the proteome. The substrate-STPK interactions show an extensive interface with the transcriptional machinery, resulting in regulation of gene expression of [~]30% of Mtb genes. Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.


title: CipA mediates complement resistance of Acinetobacter baumannii by formation of a Factor I-dependent quadripartite assemblage

abstract:
Multidrug-resistant Acinetobacter baumannii is known to be one of the leading pathogens that cause severe nosocomial infections. To overcome eradication by the innate immune system during infection, A. baumannii developed a number of immune evasion strategies. Previously, we identified CipA as a plasminogen-binding and complement-inhibitory protein. Here we show that CipA strongly inhibits all three complement activation pathways and interacts with key complement components C3, C3b, C4b, C5, Factor B, Factor D, and in particular Factor I. CipA also targets function of the C5 convertase as cleavage of C5 was impaired. Systematic screening of CipA variants identified two separate binding sites for C3b and a Factor I-interacting domain located at the C-terminus. Structure predictions using AlphaFold2 and binding analyses employing CipA variants lacking Factor I-binding capability confirmed that the orientation of the C-terminal domain is essential for the interaction with Factor I. Hence, our analyses point to a novel, Factor I-dependent mechanisms of complement inactivation mediated by CipA of A. baumannii. Recrutiment of Factor I by CipA initiates the assembly of a quadripartite complex following binding of either Factor H or C4b-binding protein to degrade C3b and C4b, respectively. Loss of Factor I binding in a CipA-deficient strain, or a strain producing a CipA variant lacking Factor I-binding capability, correlated with a higher susceptibility to human serum, indicating that recruitment of Factor I enables A. baumannii to resist complement-mediated killing.

Significance StatementThe Gram-negative bacterium Acinetobacter baumannii causes severe nosocomial infections and has developed various immune evasion strategies to overcome complement. The mechanisms how Acinetobacter baumannii successfully circumvents complement-mediated bacteriolysis are still poorly understood. Here, we show that the plasminogen-binding and complement inhibitory protein CipA terminates all three complement activation pathways. We describe a novel mechanism by which CipA cleaves the key complement components C3b and C4b in the presence of Factor H and C4b-binding protein by formation of a novel Factor I-dependent quadripartite complex. CipA, which has recently been successfully used for vaccination approaches, might represent an attractive target for the development of novel therapeutic interventions to block disorders with excessive hyperinflammatory complement activation.


title: Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA

abstract:
Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable  sterilizing immunity at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 [≤] 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting.

Significance statementWe unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.


title: A novel fluorescent multi-domain protein construct reveals the individual steps of the unfoldase action of Hsp70

abstract: A detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the detailed characterization of the aggregates, which are typically heterogeneous. To tackle this problem, we designed here a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase core, flanked by stress-resistant fluorescent mTFP and Venus domains, which upon denaturation formed a discrete stable population of small aggregates. Combining Forster Resonance Energy Transfer and enzymatic activity measurements provided unprecedent details on MLucV states, including native, aggregated, unfolded and chaperone-bound conformations. Using MLucV, we probed the various steps undertaken by bacterial Hsp70 to convert stable discrete aggregates into native proteins. The mechanism first involved an ATP-fuelled disaggregation and unfolding step of the stable pre-aggregated substrate, with a consequent stretching of MLucV beyond simply-unfolded conformations, followed, upon release, by native refolding. Furthermore, the ATP-fuelled unfolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures, highly adverse to the native state. These results unambiguously excluded binding and preventing aggregation from the non-equilibirum mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

category: molecular biology


title: Mitochondrial genomes in Perkinsus decode conserved frameshifts in all genes

abstract: Mitochondrial genomes of apicomplexans, dino-flagellates and chrompodellids, that collectively make up the Myzozoa, are uncommonly reduced in coding capacity and display divergent gene configuration and expression mechanisms. They encode only three proteins -- COB, COX1, COX3 -- contain rRNAs fragmented to [~]100-200 base pair elements, and employ extensive recombination, RNA trans-splicing, and RNA-editing for genome maintenance and expression. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remain poorly characterized. Previous reports of Perkinsus cox1 and cob partial gene sequences have indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts in both genes. To determine ancestral myzozoan mitochondrial genome features, as well as any novel ones in Perkinsozoa, we sequenced and assembled four Perkinsus species mitochondrial genomes. These data show a simple ancestral genome with the common reduced coding capacity, but one already prone to rearrangement. Moreover, we identified 75 frameshifts across the four species that are present in all genes, that are highly conserved in gene location, and that occur as four distinct types. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of the frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. COX3 is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in COX3 and COB. All genes also lack cysteine codons implying a further constraint on these genomes with reduction to only 19 different amino acids. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA, suggesting these may be imported into the organelle as for tRNAs. Perkinsus demonstrates additional remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.


title: FAM21 is critical for TLR2-CLEC4E-mediated dendritic cell function against Candida albicans

abstract: FAM21 (family with sequence similarity 21) is a component of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) protein complex that mediates actin polymerization at endosomal membranes to facilitate sorting of cargo-containing vesicles out of endosomes. To study the function of FAM21 in vivo, we generated conditional knockout (cKO) mice in the C57BL/6 background in which FAM21 was specifically knocked out of CD11c-positive dendritic cells. Bone marrow-derived dendritic cells (BMDC) from those mice displayed enlarged early endosomes, and altered cell migration and morphology relative to wildtype (WT) cells. FAM21-cKO cells were less competent in phagocytosis and antigen processing in vitro, though antigen presentation was not affected. More importantly, we identified the TLR2/CLEC4E signaling pathway as being downregulated in FAM21-cKO BMDCs when challenged with its specific ligand Candida albicans. Moreover, FAM21-cKO mice were more susceptible to C. albicans infection than WT mice. Reconstitution of WT BMDCs in FAM21-cKO mice rescued them from lethal C. albicans infection. Thus, our study highlights the importance of FAM21 in a host immune response against a significant pathogen.

category: cell biology


title: Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development

abstract:
Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, Magainin 2 and Melittin. This small genetic modification, capable of efficient non-autonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites while it simultaneously reduces the lifespan of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of endemic settings.

One sentence summaryWe developed a gene drive effector that retards Plasmodium development in transgenic Anopheles gambiae mosquitoes via the expression of antimicrobial peptides in the midgut and which is predicted to eliminate malaria under a range of transmission scenarios.

category: genetics


title: Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells

abstract: Induced pluripotent stem cells (iPSCs) may differentiate into any cell of the body and as such have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements (TEs) and prevent TE-mediated mutagenesis. Here we applied short- or long-read genome sequencing to 30 bulk miPSC lines reprogrammed from 10 parental cell types, as well as 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs were rare, we found 55 de novo TE insertions, including examples intronic to Brca1 and Dmd. LINE-1 (L1) retrotransposon families were profoundly hypomethylated in miPSCs, beyond other TEs and the genome overall, and harbored alternative promoters for protein-coding genes. Treatment with the L1 reverse transcriptase inhibitor lamivudine did not hinder reprogramming, pointing to a viable strategy to block retrotransposition. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.


title: Accelerating the Validation of Endogenous On-Target Engagement and In-cellulo Kinetic Assessment for Covalent Inhibitors of KRASG12C in Early Drug Discovery

abstract: Covalent inhibition is a valuable modality in drug-discovery due to its potential ability in decoupling pharmacokinetics from pharmacodynamics by prolonging the residence time of the drug on the target of interest. This increase in target occupancy is limited only by the rate of target turnover. However, a limitation in such studies is to translate the in-vitro inhibition assessment to the appropriate in-cellulo target engagement parameter by covalent probes. Estimation of such parameters is often impeded by the low-throughput nature of current label-free approaches. In this study, an ultra-performance liquid chromatography-multiple reaction monitoring (UPLC-MRM) mass spectrometry platform was utilised to develop a targeted proteomics workflow that can evaluate cellular on-target engagement of covalent molecules in an increased throughput manner. This workflow enabled a throughput increase of 5-10 fold when compared to traditional nanoLC-based proteomics studies. To demonstrate the applicability of the method, KRASG12C was used as a model system to interrogate the interaction of an irreversible covalent small-molecule, compound 25, both in-vitro and in-cellulo. Initial biochemical studies confirmed that the small-molecule forms an adduct with the targeted cysteine on the protein, as assessed at the level of both intact protein and on the target peptide. In-cellulo studies were carried out to quantify target engagement and selectivity assessment in heterozygous NCI-H358 cell line with both WT type and KRASG12C alleles. The workflow enabled evaluation of in-cellulo target engagement kinetics providing mechanistic insights into the irreversible mode of inhibition. In summary, the method has the potential for target agnostic application in the assessment of on-target engagement of covalent probes compatible with the high-throughput requirements of early drug discovery.

category: biochemistry


title: CriSNPr: a single interface for the curated and de-novo design of gRNAs for CRISPR diagnostics using diverse Cas systems

abstract: Nucleic acid detection and variant calling through CRISPR-based diagnostics (CRISPRDx) has facilitated clinical decision-making, particularly during the COVID-19 pandemic. This has been further accelerated through the discovery of newer and engineered CRISPR effectors, expanding the portfolio of such diagnostic applications to a wide variety of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline requires customized detection schemes originating from fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is particularly relevant for variant detection, an attractive low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver CriSNPr (CRISPR based SNP recognition), which provides the user the opportunity to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. In addition, we provide a database of curated pre-designed gRNAs and target/off-target for all human and SARS-CoV-2 variants reported so far. CriSNPr has been validated on multiple Cas proteins and highlights its broad and immediate scope of utilization across multiple detection platforms. CriSNPr is available at URL http://crisnpr.igib.res.in/.


title: Benchmarking Peptide Structure Prediction with AlphaFold2

abstract: AlphaFold2 (AF2) is a computational tool developed for the determination of protein structures with high accuracy. AF2 has been used for the modeling of many soluble and membrane proteins, but its performance in modeling peptide structures has not been systematically investigated so far. We benchmarked the accuracy of AF2 in predicting peptide structures between 16 - 60 amino acids using experimentally-determined peptide structures as reference. Our results show that while AF2 can predict the structures of certain peptide scaffolds with RMSD values below 3 [A], it is less successful in predicting the structures of peptides that have kinks, turns, or have extended flexible regions. Further, AF2 had several shortcomings in predicting rotamer recoveries, disulfide bonds, and the lowest RMSD structures based on pLDDT values. In summary, AF2 can be a powerful tool to determine peptide structures, but additional steps may be necessary to analyze and validate the results.

category: biophysics


title: Mitochondrial mRNA localization is governed by translation kinetics and spatial transport

abstract: For many nuclear-encoded mitochondrial genes, mRNA localizes to the mitochondrial surface co-translationally, aided by the association of a mitochondrial targeting sequence (MTS) on the nascent peptide with the mitochondrial import complex. For a subset of these co-translationally localized mRNAs, their localization is dependent on the metabolic state of the cell, while others are constitutively localized. To explore the differences between these two mRNA types we developed a stochastic, quantitative model for MTS-mediated mRNA localization to mitochondria in yeast cells. This model includes translation, applying gene-specific kinetics derived from experimental data; and diffusion in the cytosol. Even though both mRNA types are co-translationally localized we found that the steady state number, or density, of ribosomes along an mRNA was insufficient to differentiate the two mRNA types. Instead, conditionally-localized mRNAs have faster translation kinetics which modulate localization in combination with changes to diffusive search kinetics across metabolic states. Our model also suggests that the MTS requires a maturation time to become competent to bind mitochondria. Our work indicates that yeast cells can regulate mRNA localization to mitochondria by controlling mitochondrial volume fraction (influencing diffusive search times) and gene translation kinetics (adjusting mRNA binding competence) without the need for mRNA-specifc binding proteins. These results shed light on both global and gene-specific mechanisms that enable cells to alter mRNA localization in response to changing metabolic conditions.


title: Vivaxin genes encode highly immunogenic non-variant antigens unique to the Trypanosoma vivax cell-surface

abstract:
Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal disease of livestock across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to many of these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum showed that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-{beta}8) that is among the most immunogenic and highly expressed but displays minimal polymorphism. VIV{beta}8 was localized across the cell body and flagellar membrane, suggesting that vivaxin is substantial family of novel surface proteins. Although vaccination of mice with VIV{beta}8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals, ultimately, it does not prevent disease. However, our phylogenetic analysis shows vivaxin includes other antigens shown to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence the host-parasite interaction.

Author summaryAnimal African trypanosomiasis (AAT) is an important livestock disease throughout sub-Saharan Africa and beyond. AAT is caused by Trypanosoma vivax, among other species, a unicellular parasite that is spread by biting tsetse flies and multiplies in the bloodstream and other tissues, leading to often fatal neurological conditions if untreated. Although concerted drug treatment and vector eradication programmes have succeeded in controlling Human African trypanosomiasis, AAT continues to adversely affect animal health and impede efficient food production and economic development in many less-developed countries. In this study, we attempted to identify parasite surface proteins that stimulated the strongest immune responses in naturally infected animals, as the basis for a vaccine. We describe the discovery of a new, species-specific protein family in T. vivax, which we call vivaxin. We show that one vivaxin protein (VIV{beta}8) is surface expressed and retards parasite proliferation when used to immunize mice, but does not prevent infection. However, we also reveal that vivaxin includes another protein previously shown to induce protective immunity (IFX/VIV{beta}1). Besides its great potential for novel approaches to AAT control, vivaxin is revealed as a significant component of the T. vivax cell surface and may have important, species-specific roles in host interactions.


title: HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification

abstract: HIV-1 DNA integration into the host chromosomes is carried out by the preintegration complex (PIC). The PIC contains the viral DNA, virally encoded integrase enzyme and other critical viral/host factors. The PIC-associated viral DNA is preferentially integrated into gene bodies of actively transcribing genes. Here, we identify a biochemical mechanism underlying the preference of PIC-mediated viral DNA integration (PIC-VDI). Specifically, we observed that the PIC-VDI into human chromatin is preferred over the genomic DNA. Surprisingly, nucleosome core particles without any histone modifications were not preferred for PIC-VDI when compared to the analogous naked DNA. However, PIC-VDI was markedly enhanced with nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to HIV-1 DNA integration preference. Interestingly, we observed that nucleosomes with flanking linker DNA promoted PIC-VDI in the presence of LEDGF/p75. We also discovered that nucleosomes with linker DNA and H3K36me3 served as the optimal substrate for PIC-VDI. Mapping of the integration sites within these substrates identified preference of specific regions of the nucleosome core DNA for integration. Finally, we provide biochemical and genetic evidence that histone H1 protein, that condenses the chromatin, negatively regulates HIV-1 DNA integration, consistent with the integration preference for open chromatin structure. Collectively, these results identify the role of specific chromatin marks that drive HIV-1 integration preference and define the optimal substrate requirement for efficient DNA integration by the PIC.


title: Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time

abstract:
The dynamic interplay between proteins and their ligands is central to molecular biology, pharmacology, and drug development but is difficult to resolve experimentally. Using time-resolved serial crystallography at a synchrotron and X-ray laser, we studied the release of the photochemical affinity switch azo-Combretastatin A4 from the anti-cancer target tubulin. Thirteen logarithmically spaced temporal snapshots at near-atomic resolution are complemented by time-resolved spectroscopy and molecular dynamics simulations. They show how the photoinduced cis to trans isomerization of the azobenzene bond stretches the ligand in the picosecond to nanosecond range, followed by stepwise opening of a gating loop within microseconds, and completion of the unbinding reaction within milliseconds. Ligand unbinding is accompanied by collapse of the binding pocket and global tubulin-backbone rearrangements. Our results have implications for the molecular basis of photopharmacology, the mechanism of action of anti-tubulin drugs and provide a general experimental framework to study protein-ligand interaction dynamics.

One-Sentence SummaryTime-resolved crystallography reveals structural changes in tubulin upon release of a photoswitchable microtubule inhibitor.


title: The stationary phase-specific sRNA fimR2 is a multifunctional regulator of bacterial motility, biofilm formation and virulence

abstract:
Bacterial pathogens employ a plethora of virulence factors for host invasion, and their use is tightly regulated to maximize infection efficiency and manage resources in a nutrient-limited environment. Here we show that during Escherichia coli stationary phase the small non-coding RNA fimR2 regulates fimbrial and flagellar biosynthesis at the post-transcriptional level, leading to biofilm formation as the dominant mode of survival under conditions of nutrient depletion. fimR2 interacts with the translational regulator CsrA, antagonizing its functions and firmly tightening control over motility and biofilm formation. Generated through RNase E cleavage, fimR2 regulates stationary phase biology independently of the chaperones Hfq and ProQ. The Salmonella enterica version of fimR2 induces effector protein secretion by the type III secretion system and stimulates infection, thus linking the sRNA to virulence. This work reveals the importance of bacterial sRNAs in modulating various aspects of bacterial physiology including stationary phase and virulence.

HighlightsO_LIfimR2 expression causes biofilm formation and alters bacterial outer membrane architecture
C_LIO_LIfimR2 modulates CsrA activity and sequesters it from its targets
C_LIO_LIThe Salmonella fimR2 variant is functional in E. coli
C_LIO_LIfimR2 is generated through RNase E processing and enhances infectivity
C_LI


title: Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity

abstract: In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated, so as to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants as well as in the wild type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and also upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.


title: Excited-state dynamics of the all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics

abstract: The rhodopsin mimic is a chemically synthetized complex with the retinyl Schiff base (RSB) formed between protein and the retinal chromophore, which can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the transient absorption spectra (TAS) of three mutants of the cellular retinoic acid-binding protein II (CRABPII)-based rhodopsin mimic at PH = 3 were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra (TFS) measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB (15C-PRSB) of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the {beta}-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. Furthermore, from the three mutants, we find one protein with near-infrared fluorescence emission up to 688 nm, leading to further possible applications in sensing or bioimaging.


title: Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.

abstract: The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with impaired protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). Yet how Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Unexpectedly, substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK-linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. Our findings illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.


title: Molecular architecture of the human caveolin-1 complex

abstract:
Membrane sculpting proteins shape the morphology of cell membranes and facilitate remodeling in response to physiological and environmental cues. Complexes of the monotopic membrane protein caveolin function as essential curvature-generating components of caveolae, flask-shaped invaginations that sense and respond to plasma membrane tension. However, the structural basis for caveolins membrane remodeling activity is currently unknown. Here, we show, using cryo-electron microscopy, that the human caveolin-1 complex is composed of 11 protomers organized into a tightly packed disc with a flat membrane-embedded surface. The structural insights suggest a new mechanism for how membrane sculpting proteins interact with membranes and reveal how key regions of caveolin-1, including its scaffolding, oligomerization, and intramembrane domains, contribute to its function.

One-Sentence SummaryCryo-electron microscopy reveals that Caveolin-1 oligomerizes into a tightly packed disc with a flat membrane-binding surface.


title: A new insight into MYC action: control of RNA polymerase II methylation and transcription termination

abstract: A common catastrophic event in most human cancers is deregulation of MYC, a multifunctional transcription factor that controls gene expression in partnership with MAX and drives key biological mechanisms of the cell. Restraining its activity impairs cancer cell features and prevents tumor development, as shown by Omomyc - a 90 amino acid mini-protein interfering with MYC activity. MYC regulates many aspects of transcription by RNA polymerase II (RNAPII), such as activation, pause release, and elongation. That it may have a role in transcription termination as well is suggested by our finding of an interaction between MYC and the Protein Arginine Methyltransferase 5 (PRMT5), which catalyzes symmetrical dimethylation of RNAPII at the arginine residue R1810 (R1810me2s) allowing proper termination and splicing of transcripts. Here we show that MYC overexpression strongly increases R1810me2s, while the concomitant expression of Omomyc or a MYC-specific shRNA counteracts this capacity. Omomyc impairs as well Serine 2 phosphorylation in the RNAPII carboxyterminal domain, a modification that sustains transcript elongation and is enhanced by MYC. By displacing MYC on DNA, Omomyc reshapes RNAPII distribution along genes, leading to greater occupancy of promoter and termination sites. It is unclear how this may affect expression of the variety of genes that control metabolic, biosynthetic, and other pathways and are up or down regulated upon MYC inhibition. Genes belonging to a signature of direct MYC targets are instead strongly downregulated following MYC inhibition, with a weak correlation with RNAPII occupancy at promoters. Our data point to a MYC/ PRMT5/RNAPII axis that controls termination via RNAPII dimethylation (R1810me2s) and may contribute to fine-tune the expression of genes altered by MYC overexpression in cancer cells. It remains to be seen which role this may have in tumor development and maintenance.


title: Leukemia-intrinsic determinants of CAR-T response revealed by in vivo genome-wide CRISPR screening.

abstract: CAR-T cells are a promising new treatment for B cell malignancies. However, the majority of patients inevitably go on to experience disease relapse through largely unknown means. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screening in an immunocompetent murine model of B cell acute lymphoblastic leukemia (B-ALL). We identified IFN{gamma}/JAK/STAT signaling and components of the antigen processing and presentation pathway as key mediators of resistance to anti-CD19 CAR-T therapy in vivo, but not in vitro. Transcriptional characterization of this model demonstrated an upregulation of these pathways in CAR-T treated relapsed tumors, and examination of data from CAR-T treated B-ALL patients revealed an association between poor outcomes and increased expression of JAK/STAT/MHC-I in leukemia cells. Together, our data identify an unexpected mechanism of resistance to immunotherapy, in which tumor cell interaction with IFN{gamma}-secreting CAR-T cells in vivo activates the expression of an MHC-I inhibitory T cell program.


title: Sex differences in the immediate extinction deficit and renewal of extinguished fear in rats

abstract: Extinction learning is central to exposure-based behavioral therapies for reducing fear and anxiety in humans. However, patients with fear and anxiety disorders are often resistant to extinction. Moreover, trauma and stress-related disorders are highly prone to relapse and are twice as likely to occur in females compared to males, suggesting that females may be more susceptible to extinction deficits and fear relapse phenomena. In this report, we tested this hypothesis by examining sex differences in a stress-induced extinction learning impairment, the immediate extinction deficit (IED), and renewal, a common form of fear relapse. In contrast to our hypothesis, there were no sex differences in the magnitude of the immediate extinction deficit in two different rat strains (Long-Evans and Wistar). However, we did observe a sex difference in the renewal of fear when the extinguished conditioned stimulus was presented outside the extinction context. Male Wistar rats exhibited significantly greater renewal than female rats, a sex difference that has previously been reported after appetitive extinction. Collectively, these data reveal that stress-induced extinction impairments are similar in male and female rats, though the context-dependence of extinction is more pronounced in males.

category: animal behavior and cognition


title: Substrate Temperature and Seed Scarification on Germination Parameters of Butterfly Pea (Clitoria ternatea)

abstract: Recent interest in the cultivation and use of butterfly pea (Clitoria ternatea) has increased demand for its commercial production. Successful germination of seed is a critical first step to its production; however, few studies have documented factors influential to germination success. To help support commercial production efforts, the influence of substrate temperature and seed scarification technique on germination parameters were evaluated. Two scarification (nicked and nicked and soaked) and three substrate temperature treatments (21, 24, and 27 {degrees}C) were imposed on C. ternatea seed. Following treatments, six germination parameters were recorded. Data showed seeds maintained at a substrate temperature of 21 {degrees}C reached maximal germination (54.8%) at the longest mean germination time (2.61 d) and slowest mean germination rate (0.39 d-1). Mechanical scarification by nicking the seed coat with a razor blade prior to soaking the seed for 24 hours reached maximal germination (80.95%) at the lowest mean germination time (2.06 d) and fastest mean germination rate (0.51 d-1). Optimal germination was attained when seed was scarified and soaked and then germinated in rockwool substrate maintained at a temperature (To) of 21 {degrees}C. Results provide commercial growers with production technique information helpful to fast and efficient germination of C. ternatea seed.


title: Sex differences in insular gyri responses to the cold pressor challenge

abstract:
IntroductionSex differences in autonomic control may contribute to physiological sex differences in cardiovascular disease, thermoregulation, and the experience of pain. We previously showed sex differences in functional mapping of autonomic responses across the insula, a central autonomic control region. Anterior insula response patterns to sympathetic activation (Valsalva) and parasympathetic withdrawal (handgrip) differed between men and women. We here assessed sex differences in autonomic insula gyrus responses to a cold pressor challenge, which involves temperature and pain regulation.

MethodsFunctional MRI (fMRI) involved a 1-minute right-foot cold pressor challenge in 22 women (age; mean{+/-}std: 50{+/-}4 yrs), and 39 men (45{+/-}3 yrs). Regions of interest (ROI) comprised left and right anterior short gyrus (ASG), middle short gyrus (MSG), posterior short gyrus (PSG), anterior long gyrus (ALG), and posterior long gyrus (PLG). Two-second time intervals of fMRI signal responses per ROI and concurrently recorded heart rate and blood oxygen saturation (SaO2) were tested for within- and between-group effects over time (repeated measures ANOVA P[≤]0.05). We tested sex differences in 1) each ROI; 2) lateralization effects; and 3) posterior-to-anterior gradients in responses.

ResultsNo sex differences emerged in heart rate or SaO2. Women showed larger signal changes in several gyri than men after cold pressor onset and offset. Most consistently, women showed a greater right-over-left hemisphere dominance in cold pressor response compared to men in the MSG, PSG and ALG. Greater right-over-left hemisphere anterior-to-posterior dominance was more pronounced in men.

ConclusionsThe findings confirm anterior and right-hemispheric dominance of insular responses during sympathetic activation that includes pain and cold. Although distinct transient male-female differences in fMRI responses appeared during the cold pressor, unlike the Valsalva and handgrip, no sex differences in ASG lateralization emerged, suggesting that the previously found sex differences may not solely relate to regulating blood pressure responses.


title: MafB, WDR77, and ss-catenin interact with each other and have similar genome association profiles

abstract: MafB (a bZIP transcription factor), {beta}-catenin (the ultimate target of the Wnt signal transduction pathway that acts as a transcriptional co-activator of LEF/TCF proteins), and WDR77 (a transcriptional co-activator of multiple hormone receptors) are important for breast cellular transformation. Unexpectedly, these proteins interact directly with each other, and they have similar genomic binding profiles. Furthermore, while some of these common target sites coincide with those bound by LEF/TCF, the majority are located just downstream of transcription initiation sites at a position near paused RNA polymerase (Pol II) and the +1 nucleosome. Occupancy levels of these factors at these promoter-proximal sites are strongly correlated with the level of paused Pol II and transcriptional activity.


title: How drought affects range and variability of environmental conditions in subtropical South African estuaries

abstract: Globally estuaries are under increasing pressure from human development impacts as well as the growing effects of climate change that is already, and increasingly so, causing major changes in events such as droughts. Although droughts are regular occurrences and are projected to become progressively more significant in many parts of the world, their potential impacts on estuaries requires better understanding and quantification. This study aims to quantify environmental and phytoplankton parameter changes in two contrasting subtropical estuary types in South Africa; the temporarily closed uMdloti Estuary and the predominantly open to the sea uMlalazi Estuary during a hydrological drought event and subsequent non-drought period. Drought caused lower salinities, almost exclusively freshwater, throughout the temporarily closed estuarine type uMdloti. The marine-estuarine salinity environment of the predominantly open uMlalazi during drought expanded to include lower salinities in the non-drought period. Dissolved oxygen levels were more variable during drought conditions including periods of hypoxia, but more stable at elevated levels in both estuaries during the non- drought period. Temperature measurements included higher values during drought, especially in the dry season. Chl-a concentrations were overall higher and more variable during drought in uMdloti, with periods of bloom formation as well as decay. Drought-induced conditions can span several generations for estuarine and estuarine associated organisms. The results from the study provide quantitative support for the hypothesis that extended periods of drought conditions lead to a decrease in niche availability to estuarine organisms in a range of estuary types.


title: Minus one frameshifted puromycin N-acetyltransferase is targeted to the nucleolus: sporadic but reproducible chimera formation by a transfected Lc3b construct

abstract: Map1Lc3b is a protein that has pivotal functions in cellular autophagy. At least three groups in the past decade have reported its presence in the nucleoli of cells, but its functions in that organelle remain unknown. We isolated a few clonal populations of cells stably expressing V5-tagged mouse Lc3b highly enriched in the nucleoli, but the frequency of occurrence of such clones was strikingly low. The phenomenon was readily reproducible, though the protein in the nucleolus puzzlingly had varying molecular masses in different clones but consistently displayed a very strong interaction with the mitochondrial protein C1qbp, which has well-documented functions in the nucleolus. We investigated further and discovered that, in at least one of the clones, Lc3b had formed a chimera with the puromycin resistance gene in the plasmid, plausibly by illegitimate recombination during or after integration of the construct into the cellular genomic DNA. The -1 shifted reading frame of puromycin N-acetyltransferase (pac) can encode a protein that is equally long as the one encoded by the complete pac ORF, but is targeted to the nucleoli due to a drastic shift in the isoelectric point (pI). Notably, this set of events again brings into focus the low threshold often reported for recombination events to occur in eukaryotic cells, the multiple factors influencing them, and calls for increased vigilance in experiments involving DNA transfection and gene targeting.


title: CRISPR-mediated correction of skeletal muscle Ca2+ handling in a novel DMD patient-derived pluripotent stem cell model

abstract: Mutations in the dystrophin gene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+ handling is a pathological feature of DMD, mechanisms underlying defective Ca2+ homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+ handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+ transients and mathematical modeling of Ca2+ dynamics reveal significantly reduced cytosolic Ca2+ clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+ flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wild type PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+ homeostasis in DMD and suggests that Ca2+ signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevant in vitro model enabling rapid pre-clinical testing of potential therapies for DMD.


title: Vps60 initiates formation of alternative membrane-bound ESCRT-III filaments

abstract: Endosomal sorting complex required for transport-III (ESCRT-III)-driven membrane remodeling participates in many crucial cellular functions, from cell division to endosome maturation, and occurs on essentially all cellular organelles. In eukaryotes, ESCRT-III displays a remarkable molecular diversity in its subunits which may have been acquired through evolution to perform novel cellular functions. Here, we describe and characterize a novel ESCRT-III polymer initiated by the subunit Vps60. Membrane-bound Vps60 polymers recruit ESCRT-III subunits Vps2, Vps24, Did2 and Ist1, and undergo polymer turnover powered by the ATPase Vps4. Snf7- and Vps60 filaments can coexist on membranes without interacting. Their nucleation, polymerization and recruitment of downstream subunits remains unaffected by the presence of the respective other polymer. Taken together, our results suggest Vps60 and Snf7 form distinct ESCRT-III polymers, which overall, supports the notion of evolutionary diversification of ESCRT-III assemblies to perform specific cellular functions.


title: Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events trigger two-step maturation

abstract: Macroautophagy is a homeostatic process required to clear cellular waste including aggregated proteins and dysfunctional organelles. Neuronal autophagosomes form constitutively in the distal tip of the axon and are actively transported toward the soma, with cargo degradation initiated en route. Cargo turnover requires autophagosomes to fuse with lysosomes to acquire degradative enzymes; however, the timing and number of these fusion events in the axon have proven difficult to detect using microscopy alone. Here we use a quantitative model, parameterized and validated using data from live and fixed imaging of primary hippocampal neurons, to explore the autophagosome maturation process on a cellular scale. We demonstrate that retrograde autophagosome motility is independent from lysosomal fusion, and that most autophagosomes fuse with only a few lysosomes by the time they reach the soma. Furthermore, our imaging and model results highlight the two-step maturation of the autophagosome: fusion with a lysosome or late endosome is followed by the slow degradation of the autophagosomal inner membrane before actual cargo degradation can occur. Together, rigorous quantitative measurements and mathematical modeling elucidate the dynamics of autophagosome-lysosome interaction and autophagosomal maturation in the axon.


title: A centimeter-long bacterium with DNA compartmentalized in membrane-bound organelles

abstract:
Cells of most bacterial species are around 2 {micro}m in length, with some of the largest specimens reaching 750 {micro}m. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Ca. Thiomargarita magnifica, a bacterium with an average cell length greater than 9,000 {micro}m that is visible to the naked eye. We found that these cells grow orders of magnitude over theoretical limits for bacterial cell size through unique biology, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes in daughter cells. These features, along with compartmentalization of genomic material and protein synthesis in membrane-bound organelles, indicate gain of complexity in the Thiomargarita lineage, and challenge traditional concepts of bacterial cells.

One Sentence SummaryCa. T. magnifica are compartmentalized centimeter-long bacteria


title: The probability of chromatin to be at the nuclear lamina has no systematic effect on its transcription level.

abstract:
Multiple studies have shown a correlation between gene expression and positioning of genes at nuclear envelope (NE) lined by nuclear lamina. Here, we ask whether there is a causal, systematic connection between the expression level of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probability of TADs to be found at the NE. To investigate the connection, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with the genome-wide gene expression data for both the control and lamins depleted (LD) nuclei. We analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be near the NE. Our findings suggest that, within statistical error margin, positioning of Drosophila TADs at the nuclear envelope does not, by itself, systematically affect the average gene expression in these TADs, while the expected negative correlation is confirmed. Verifiable hypotheses of the underlying mechanism for the presence of correlation without causality are discussed.

We introduce a TAD length normalized metric for the average transcription activity of genes in a TAD: number of Reads mapped to all genes in a TAD Per Kilobase of TAD length per Million reads mapped to all TADs (RPKMT).


title: The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator

abstract: How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins (ASPPs) are protein phosphatase 1 (PP1) binding partners associated with cardiocutaneous diseases. ASPPs localize PP1 to cell-cell junctions, but how ASPPs localize and whether they regulate PP1 activity in vivo is unclear. Through a C. elegans genetic screen, we find that loss of the ASPP homolog, APE-1, suppresses a pathology called  jowls, providing us with an in vivo assay for APE-1 activity. Using structure-function analysis, we discover that APE-1s N-terminal half directs the APE-1-PP1 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1s ankyrin repeats that suppress jowls yet do not preclude PP1 binding, implying ASPPs do more than simply localize PP1. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.


title: Kinesin-1 promotes centrosome clustering and nuclear migration in the Drosophila oocyte

abstract:
Accurate positioning of the nucleus is essential in many cellular contexts. Microtubules and their associated motors are important players in this process. Although nuclear migration in Drosophila oocytes is controlled by microtubule-generated forces, a role for microtubule-associated molecular motors in nuclear positioning has yet to be reported. In this study, we first characterize novel landmarks that allow a precise description of the pre-migratory stages. Using these newly defined stages, we report that, prior to migration, the nucleus moves from the anterior side of the oocyte toward the center and concomitantly the centrosomes cluster at the posterior of the nucleus. In absence of Kinesin-1, centrosome clustering is impaired and the nucleus fails to position and migrate properly. In addition, we show that maintaining a high level of Polo-kinase at centrosomes prevents centrosome clustering and impairs nuclear positioning, suggesting that Kinesin-1 associated defects result from a failure to reduce centrosome activity. Consistently, depleting centrosomes rescues the nuclear migration defects induced by Kinesin-1 inactivation. In summary, our results suggest that Kinesin-1 controls nuclear migration in the oocyte by modulating centrosome activity.

Summary statementIn this study, we identified a crucial role of Kinesin-1 in centrosome clustering required for nuclear positioning and migration in the Drosophila oocyte.

category: developmental biology


title: Clonal behaviour of myogenic precursor cells throughout the vertebrate lifespan.

abstract:
To address questions of stem cell diversity during skeletal myogenesis, a Brainbow-like genetic cell lineage tracing method, dubbed Musclebow, was derived by enhancer trapping in zebrafish. It is shown that at least 15 muscle precursor cells (mpcs) seed each somite, where they proliferate but contribute little to muscle growth prior to hatching. Thereafter, dermomyotome-derived mpc clones rapidly expand while some progeny undergo terminal differentiation, leading to stochastic clonal drift. No evidence of cell lineage-based clonal fate diversity was obtained. Neither fibre nor mpc death was observed in uninjured animals. Individual marked muscle fibres persist across much of the lifespan indicating low rates of nuclear turnover. In adulthood, early-marked mpc clones label stable blocks of tissue comprising a significant fraction of either epaxial or hypaxial somite. Fusion of cells from separate early-marked clones occurs in regions of clone overlap. Wounds are regenerated from many/most local mpcs; no evidence for specialised stem mpcs was obtained. In conclusion, our data indicate that most mpcs in muscle tissue contribute to local growth and repair and suggest that cellular turnover is low in the absence of trauma.

Summary StatementMusclebow clonal cell lineage analysis is introduced to reveal the cellular dynamics of skeletal muscle formation, repair and maintenance throughout the life of zebrafish.