title: The dual role of the RETINOBLASTOMA-RELATED protein in the DNA damage response is spatio-temporally coordinated by the interaction with LXCXE-containing proteins.

abstract: Living organisms face threats to genome integrity caused by environmental challenges or metabolic errors in proliferating cells. To avoid the spread of mutations, cell division is temporarily arrested while repair mechanisms deal with DNA lesions. Afterwards, cells either resume division or respond to unsuccessful repair by withdrawing from the cell cycle and undergoing cell differentiation or cell death. How the success rate of DNA repair connects to the execution of cell death remains incompletely known, particularly in plants. Here we provide evidence that the Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein, shown to play structural and transcriptional functions in the DNA damage response (DDR), coordinates these processes in time by successive interactions through its B-pocket sub-domain. Upon DNA damage induction, RBR forms nuclear foci; but the N849F substitution in the B-pocket, which specifically disrupts binding to LXCXE motif-containing proteins, abolishes RBR focus formation and leads to growth arrest. After RBR focus formation, the stress-responsive gene NAC044 arrests cell division. As RBR is released from nuclear foci, it can be bound by the conserved LXCXE motif in NAC044. RBR-mediated cell survival is inhibited by the interaction with NAC044. Disruption of NAC044-RBR interaction impairs the cell death response but is less important for NAC044 mediated growth arrest. Noteworthy, unlike many RBR interactors, NAC044 binds to RBR independent of RBR phosphorylation. Our findings indicate that the availability of the RBR B-pocket to interact with LXCXE-containing proteins couples RBR DNA repair functions and RBR transcriptional functions of in the cell death program.


title: Photosynthetic light harvesting and thylakoid organization in a CRISPR/Cas9 Arabidopsis thaliana LHCB1 knockout mutant.

abstract:
Light absorbed by chlorophylls of photosystem II and I drives oxygenic photosynthesis. Light-harvesting complexes increase the absorption cross-section of these photosystems. Furthermore, these complexes play a central role in photoprotection by dissipating the excess of absorbed light energy in an inducible and regulated fashion. In higher plants, the main light-harvesting complex is the trimeric LHCII.

In this work, we used CRISPR/Cas9 to knockout the five genes encoding LHCB1, which is the major component of the trimeric LHCII. In absence of LHCB1 the accumulation of the other LHCII isoforms was only slightly increased, thereby resulting in chlorophyll loss leading to a pale green phenotype and growth delay. Photosystem II absorption cross-section was smaller while photosystem I absorption cross-section was unaffected. This altered the chlorophyll repartition between the two photosystems, favoring photosystem I excitation. The equilibrium of the photosynthetic electron transport was partially maintained by a lower photosystem I over photosystem II reaction center ratio and by the dephosphorylation of LHCII and photosystem II. Loss of LHCB1 altered the thylakoid structure, with less membrane layers per grana stack and reduced grana width. Stable LHCB1 knock out lines allow characterizing the role of this protein in light harvesting and acclimation and pave the way for future in vivo mutational analyses of LHCII.

category: plant biology


title: Sexual and individual signatures are encoded in the temporal rate of Cape gannet display calls

abstract: 1.Abstract/SummaryVocalisations play a vital role in animal communication, as they are involved in many biological functions. Seabirds often breed in large and dense colonies, making successful recognition between mates or between parents -and offspring crucial for reproductive success. Most seabird species, including Cape gannets (Morus capensis), are monomorphic and likely rely on acoustic signals for mate selection and mate recognition. This study aimed to better understand the use of vocalisations for sex and individual recognition in Cape gannets by describing the acoustic structure of their display calls at the nest. Vocalisations of nesting Cape gannets were recorded and acoustic measurements were extracted in both temporal and frequency domains. Values of the fundamental frequency and the average of Inter-Onset-Interval appeared to be the most important acoustic variables for sex determination. Both temporal and frequency parameters showed a potential for individual identity coding, with the average units Inter-Onset-Interval being the most important variable for individual identification for both sexes. This study provides the first evidence of sex-specific and individual vocal signatures in adult breeding Cape gannets. From an applied perspective, identified sex specific differences could potentially be used as a non-invasive method for field-based sex-determination in research and monitoring projects on Cape gannets.

category: zoology


title: Learning enhances encoding of time and temporal surprise in primary sensory cortex

abstract: Primary sensory cortex has long been believed to play a straightforward role in the initial processing of sensory information. Yet, the superficial layers of cortex overall are sparsely active, even during sensory stimulation; moreover, cortical activity is influenced by other modalities, task context, reward, and behavioral state. Our study demonstrates that reinforcement learning dramatically alters representations among longitudinally imaged neurons in superficial layers of mouse primary somatosensory cortex. Learning an object detection task recruits previously unresponsive neurons, enlarging the neuronal population sensitive to touch and behavioral choice. In contrast, cortical responses decrease upon repeated exposure to unrewarded stimuli. Moreover, training improved population encoding of the passage of time, and unexpected deviations in trial timing elicited even stronger responses than touch did. In conclusion, the superficial layers of sensory cortex exhibit a high degree of learning-dependent plasticity and are strongly modulated by non-sensory but behaviorally-relevant features, such as timing and surprise.

category: neuroscience


title: Frequency transfer of the ventriloquism aftereffect

abstract:
The ventriloquism aftereffect (VAE) describes the persistent shift of perceived sound location after having been adapted to a ventriloquism condition, in which the sound was repeatedly paired with a displaced visual stimulus. In the latter case, participants consistently mislocalize the sound in the direction of the visual stimulus (ventriloquism effect, VE). Previous studies provide conflicting reports regarding the strength of the VAE, ranging from 0 to nearly 100%. Moreover, there is controversy about its generalization to different sounds than the one inducing the VE, ranging from no transfer at all, to full transfer across different sound spectra. Here, we imposed the VE for three different sounds: a low-frequency and a high-frequency narrow-band noise, and a broadband Gaussian white noise (GWN). In the adaptation phase, listeners generated fast goal-directed head movements to localize the sound, presented across a 70 deg range in the horizontal plane, while ignoring a visual distracter that was consistently displaced 10 deg to the right of the sound. In the post-adaptation phase, participants localized narrow-band sounds with center frequencies from 0.5 to 8 kHz, as well as GWN, without the visual distracter. Our results show that the VAE amounted to approximately 40% of the VE and generalized well across the entire frequency domain. We also found that the strength of the VAE correlated with the pre-adaptation sound-localization performance. We compare our results with previous reports and discuss different hypotheses regarding optimal audio-visual cue integration.

Significance statementWhere we believe sounds to emanate from is heavily influenced by what we see. We experience this illusion - the ventriloquism effect - regularly. For example, when watching movies, we perceive the voices of actors to originate from the actors lips rather than from the speakers. Long exposure to a spatial mismatch between what we hear and what we see, leads to a recalibration of our localization of sounds - the ventriloquism aftereffect. Unclear was whether exposure to an audiovisual mismatch for one sound type would also lead to adaptive changes for other sounds. The present study shows that the ventriloquism aftereffect generalizes, implying that it does not act at an early auditory stage, but at a late multisensory stage.


title: Influential factors of saliva microbiota composition

abstract:
BackgroundThe oral microbiota is emerging as an influential factor of host physiology and disease state. Factors influencing oral microbiota composition have not been well characterised. In particular, there is a lack of population-based studies. We undertook a large hypothesis-free study of the saliva microbiota, considering potential influential factors of host health (frailty; diet; periodontal disease), demographics (age; sex; BMI) and sample processing (storage time), in a sample (n=679) of the TwinsUK cohort of adult twins.

ResultsAlpha and beta diversity of the saliva microbiota was associated most strongly with frailty (alpha diversity: Q = 0.003, Observed; Q=0.002, Shannon; Q=0.003, Simpson; Beta diversity: Q = 0.002, Bray Curtis dissimilarity) and age (alpha diversity: Q=0.006, Shannon; Q=0.003, Simpson; beta diversity: Q=0.002, Bray Curtis dissimilarity; Q= 0.032, Weighted UniFrac) in multivariate models including age, frailty, sex, BMI, frailty and diet, and adjustment for multiple testing. Those with a more advanced age were more likely to be dissimilar in the saliva microbiota composition than younger participants (P = 5.125e-06, ANOVA). In subsample analyses, including consideration of periodontal disease (total n=138, periodontal disease n=66), the association with frailty remained for alpha diversity (Q=0.002, Observed ASVs; Q= 0.04 Shannon Index), but not beta diversity, whilst age was not demonstrated to associate with alpha or beta diversity in this subsample, potentially due to insufficient statistical power. Length of time that samples were stored prior to sequencing was associated with beta diversity (Q = 0.002, Bray Curtis dissimilarity). Six bacterial taxa were associated with age after adjustment for frailty and diet.

ConclusionsFrailty and age emerged as the most influential factors of saliva microbiota composition. Whilst frailty and age are correlates, the associations were independent of each other, suggesting that both biological and chronological ageing are key drivers of saliva microbiota composition.

category: microbiology


title: Primary productivity in subsidized green-brown food webs

abstract: 1Ecosystems worldwide receive large amounts of nutrients from both natural processes and human activities. While direct subsidy effects on primary productivity are relatively well known (the green food web), the indirect effects of subsidies on producers as mediated by the brown food web and predators have been neglected. With a dynamical green-brown food web model, parameterized using empirical estimates from the literature, we illustrate the effect of nutrient subsidies on net primary productivity (i.e., after removing loss to herbivory) in two generic ecosystems, terrestrial and aquatic. We find that nutrient subsidies increase net primary productivity because more nutrients are available, but this effect saturates with higher subsidies. Changing the quality of subsidies from inorganic to organic tends to increase net primary productivity in terrestrial ecosystems, but less often so in aquatic ecosystems. This occurs when organic nutrient inputs promote detritivores in the brown food web, and hence predators that in turn control herbivores, thus promoting primary productivity. This previously largely overlooked effect is further enhanced by ecosystem properties such as fast decomposition and low rates of nutrient additions, and demonstrates the importance of nutrient subsidy quality on ecosystem functioning.

category: ecology


title: High-throughput UHPLC-MS to screen metabolites in feces for gut metabolic health

abstract:
(1) BackgroundFeces are the product of our diets and have been linked to diseases of the gut, including Chrons disease and metabolic diseases such as diabetes. For screening metabolites in heterogeneous samples such as feces, it is necessary to use fast and reproducible analytical methods that maximize metabolite detection.

(2) MethodsAs sample preparation is crucial to obtain high quality data in MS-based clinical metabolomics, we developed a novel, efficient and robust method for preparing fecal samples for analysis with a focus in reducing aliquoting and detecting both polar and non-polar metabolites. Fecal samples (n= 475) from patients with alcohol-related liver disease and healthy controls were prepared according to the proposed method and analyzed in an UHPLC-QQQ targeted platform in order to obtain a quantitative profile of compounds that impact liver-gut axis metabolism.

(3) ResultsMS analyses of the prepared fecal samples have shown reproducibility and coverage of n=28 metabolites, mostly comprising bile acids and amino acids. We report metabolite-wise relative standard deviation (RSD) in quality control samples, inter-day repeatability, LOD, LOQ and range of linearity. The average concen-trations for 135 healthy participants are reported here for clinical applications.

(4) Conclusionsour high-throughput method provides an efficient tool for investigating gut-liver axis metabolism in liver-related diseases using a noninvasive collected sample.

category: biochemistry


title: Sensory axons induce epithelial lipid microdomain remodeling and determine the distribution of junctions in the epidermis

abstract: Epithelial cell properties are determined by the polarized distribution of membrane lipids, the cytoskeleton, and adhesive junctions. Epithelia are often profusely innervated, but little work has addressed how contact with neurites affects the polarized organization of epithelial components. In previous work, we found that basal keratinocytes in the larval zebrafish epidermis wrap around axons to enclose them in ensheathment channels sealed by autotypic cell junctions. In this study, we used live imaging to characterize how sensory axons remodel cell membranes, the actin cytoskeleton, and adhesive junctions in basal keratinocytes. At the apical surface of basal keratinocytes, axons promoted the formation of lipid microdomains quantitatively enriched in reporters for PI(4,5)P2 and liquid-ordered (Lo) membranes. Lipid microdomains supported the formation of cadherin-enriched F-actin protrusions, which wrapped around axons, likely initiating the formation of ensheathment channels. Lo reporters, but not reporters of liquid-disordered (Ld) membranes, became progressively enriched at axon-associated membrane domains as autotypic junctions matured at ensheathment channels. In the absence of axons, cadherin-enriched lipid microdomains still formed on basal cell membranes, but were not organized into the contiguous domains normally associated with axons. Instead, these isolated domains formed ectopic heterotypic junctions with overlying periderm cells, a distinct epithelial cell type in the epidermis. Thus, axons inhibit the formation of epithelial heterotypic junctions by recruiting cadherin-rich lipid microdomains to form autotypic junctions at ensheathment channels. These findings demonstrate that sensory nerve endings dramatically remodel polarized epithelial components and regulate the adhesive properties of the epidermis.

category: cell biology


title: Genome sequences hot and cold: a database of organisms with defined optimal growth temperatures

abstract: Despite the rapidly increasing number of organisms with sequenced genomes, there is no existing resource that simultaneously contains information about genome sequences and the optimal growth conditions for a given species. In the absence of such a resource, we cannot immediately sort genomic sequences by growth conditions, making it difficult to study how organisms and biological molecules adapt to distinct environments. To address this problem, we have created a database called GSHC (Genome Sequences: Hot, Cold, and everything in between). This database, available at http://melnikovlab.com/gshc, brings together information about the genomic sequences and optimal growth temperatures for 25,324 species, including ~89% of the bacterial species with known genome sequences. Using this database, it is now possible to readily compare genomic sequences from thousands of species and correlate variations in genes and genomes with optimal growth temperatures, at the scale of the entire tree of life. The database interface allows users to retrieve protein sequences sorted by optimal growth temperature for their corresponding species, providing a tool to explore how organisms, genomes, and individual proteins and nucleic acids adapt to certain temperatures. We hope that this database will contribute to medicine and biotechnology by helping to create a better understanding of molecular adaptations to heat and cold, leading to new ways to preserve biological samples, engineer useful enzymes, and develop new biological materials and organisms with the desired tolerance to heat and cold.

category: evolutionary biology


title: Accelerating manufacturing to start large-scale supply of a new adenovirus-vectored vaccine within 100 days

abstract:
The Coalition for Epidemic Preparedness Innovations  100-day moonshot aspires to launch a new vaccine within 100 days of pathogen identification. Here, we describe work to optimize adenovirus vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing the output from the available manufacturing footprint.

We describe a rapid viral seed expansion workflow that allows vaccine release to clinical trials within 60 days of antigen sequence identification, followed by vaccine release from globally distributed sites within a further 40 days. We also describe a new perfusion-based upstream production process, designed to maximize output while retaining simplicity and suitability for existing manufacturing facilities. This improves upstream volumetric productivity of ChAdOx1 nCoV-19 by around four-fold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10000 doses from each liter of bioreactor capacity.

Transition to a new production process across a large manufacturing network is a major task. In the short term, the rapid seed generation workflow could be used with the existing production process. We also use techno-economic modelling to show that, if linear scale-up were achieved, a single cleanroom containing two 2000 L bioreactors running our new perfusion-based process could supply bulk drug substance for around 120 million doses each month, costing <0.20 EUR/dose. We estimate that a manufacturing network with 32000 L of bioreactor capacity could release around 1 billion doses of a new vaccine within 130 days of genomic sequencing of a new pathogen, in a hypothetical  surge campaign with suitable prior preparation and resources, including adequate fill-and-finish capacity.

This accelerated manufacturing process, along with other advantages such as thermal stability, supports the ongoing value of adenovirus-vectored vaccines as a rapidly adaptable and deployable platform for emergency response.

category: bioengineering


title: Waning immune responses against SARS-CoV-2 among vaccinees in Hong Kong

abstract:
BackgroundNearly 4 billion doses of the BioNTech-mRNA and Sinovac-inactivated vaccines have been administrated globally, yet different vaccine-induced immunity against SARS-CoV-2 variants of concern (VOCs) remain incompletely investigated.

MethodsWe compare the immunogenicity and durability of these two vaccines among fully vaccinated Hong Kong people.

FindingsStandard BioNTech and Sinovac vaccinations were tolerated and induced neutralizing antibody (NAb) (100% and 85.7%) and spike-specific CD4 T cell responses (96.7% and 82.1%), respectively. The geometric mean NAb IC50 and median frequencies of reactive CD4 subsets were consistently lower among Sinovac-vaccinees than BioNTech-vaccinees. Against VOCs, NAb response rate and geometric mean IC50 against B1.351 and B.1.617.2 were significantly lower for Sinovac (14.3%, 15 and 50%, 23.2) than BioNTech (79.4%, 107 and 94.1%, 131). Three months after vaccinations, NAbs to VOCs dropped near to detection limit, along with waning memory T cell responses, mainly among Sinovac-vaccinees.

InterpretationOur results indicate that Sinovac-vaccinees may face higher risk to pandemic VOCs breakthrough infection.

FundingThis study was supported by the Hong Kong Research Grants Council Collaborative Research Fund (C7156-20GF to Z.C and C1134-20GF); the National Program on Key Research Project of China (Grant 2020YFC0860600, 2020YFA0707500 and 2020YFA0707504); Shenzhen Science and Technology Program (JSGG20200225151410198 and JCYJ20210324131610027); HKU Development Fund and LKS Faculty of Medicine Matching Fund to AIDS Institute; Hong Kong Innovation and Technology Fund, Innovation and Technology Commission and generous donation from the Friends of Hope Education Fund. Z.C.s team was also partly supported by the Theme-Based Research Scheme (T11-706/18-N).

category: immunology


title: Broad and Long-lasting Immune Response against SARS-CoV-2 Omicron and Other Variants by PIKA-Adjuvanted Recombinant SARS-CoV-2 Spike (S) Protein Subunit Vaccine (YS-SC2-010)

abstract: Recently SARS-CoV-2 Omicron (B.1.1.529) variant was identified in South Africa with numerous mutations in spike protein, and numerous community infections have been reported and raised grave concern around the world. Some studies found that the neutralization effects of several licensed vaccines against Omicron were dramatically reduced, which significantly affected antibody mediated protection, especially for individuals whose immunization were completed after extended period. In this regard, we studied the persistence and neutralization activity toward mutant strains in animal serum immunized with PIKA-adjuvanted recombinant SARS-CoV-2 spike protein subunit vaccine (YS-SC2-010). Here we are reporting that animal serum collected at 596 days after immunization with YS-SC2-010 still retains high and persistent neutralizing activity against all the Variant of Concern (VOC) variants, including Omicron variant. Although it is a blessed event to achieve 20 months long neutralization against Omicron variant after immunization with YS-SC2-010, it was also founded that the neutralization effect of immune serum on Omicron decreased by 6.29 folds as compared to D614G, more significantly when compared with other mutant strains.


title: Interferon-induced transmembrane protein 3 (IFITM3) limits lethality of SARS-CoV-2 in mice

abstract: Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.


title: Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant

abstract:
The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

One Sentence SummaryOmicron, carrying [~]30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.


title: SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster

abstract:
The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants.

One Sentence SummaryThird dose of Pfizer/BioNTech COVID-19 vaccine significantly boosts neutralizing antibodies to the Omicron variant compared to a second dose, while neutralization of Omicron by convalescent sera, two-dose vaccine-elicited sera, or therapeutic antibodies is variable and often low.


title: SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-weeks interval between doses

abstract: Continuous emergence of SARS-CoV-2 variants of concern (VOC) is fueling the COVID-19 pandemic. Omicron (B.1.1.529), is rapidly spreading worldwide. The large number of mutations in its Spike raised concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses was shown to elicit antibodies that efficiently recognize Spikes from different VOCs. Here we evaluated the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously-infected individuals that received their BNT162b2 mRNA vaccine 16-weeks apart. Omicron Spike was recognized less efficiently than D614G, Alpha, Beta, Gamma and Delta Spikes. We compared to plasma activity from participants receiving a short (4-weeks) interval regimen. Plasma from individuals of the long interval cohort recognized and neutralized better the Omicron Spike compared to those that received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.


title: Protein homeostasis is maintained by proteasomes containing PSMB9 induced by EEF1A2 upon mitochondrial stress

abstract: Perturbed proteostasis and mitochondrial dysfunction are often associated with age-related diseases such as Alzheimers and Parkinsons diseases. However, the link between them remains incompletely understood. Mitochondrial dysfunction causes proteostasis imbalance, and cells respond to restore proteostasis by increasing proteasome activity and molecular chaperons in yeast and C. elegans. Here, we demonstrate the presence of similar responses in humans. Mitochondrial dysfunction upregulates a small heat shock protein HSPB1 and an immunoproteasome subunit PSMB9 leading to an increase in proteasome activity. HSPB1 and PSMB9 are required to prevent protein aggregation upon mitochondrial dysfunction. Moreover, PSMB9 expression is dependent on a translation elongation factor EEF1A2, and PSMB9-containing proteasomes are located near mitochondria, enabling fast local degradation of aberrant proteins. Our findings put a step forward in understanding the stress response triggered by mitochondrial dysfunction, and may be useful for therapeutic strategies to prevent or delay the onset of age-related diseases and attenuate their progression.


title: Rapid longitudinal SARS-CoV-2 intra-host emergence of novel haplotypes regardless of immune deficiencies

abstract:
On February 2020, the municipality of Vo, a small town near Padua (Italy), was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. The entire population was swab tested in two sequential surveys. Here we report the analysis of the viral genomes, which revealed that the unique ancestor haplotype introduced in Vo belongs to lineage B and, more specifically, to the subtype found at the end of January 2020 in two Chinese tourists visiting Rome and other Italian cities, carrying mutations G11083T and G26144T. The sequences, obtained for 87 samples, allowed us to investigate viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptoms severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype, carried by an index case. The lockdown extinguished both viral spread and the emergence of new variants, confirming the efficiency of this containment strategy. The information gathered from household was used to reconstructs possible transmission events.

AUTHOR SUMMARYIt is of great interest and importance to understand SARS-CoV-2 ability to mutate generating new viral strains, and to assess the impact of containment strategies on viral transmission. In this study we highlight the rapid intra-host haplotype evolution regardless of symptom severity and immune deficiencies that we observed during the first wave of the pandemic in the municipality of Vo in Italy. The confirmation that all the haplotypes found in this small community derive from a common ancestor haplotype, has allowed us to track the rapid emergence of new variants but lockdown and mass testing efficiently prevented their spread elsewhere.


title: Tutorial: Investigating SARS-CoV-2 evolution and phylogeny using MNHN-Tree-Tools

abstract: The Covid-19 pandemic has caused at more than 3 million deaths by Mai this year [1]. It had a significant impact on the daily life and the global economy [2]. The virus has since its first recorded outbreak in China [3] mutated into new strains [4]. The Nextstrain [5] project has so far been monitoring the evolution of the virus. At the same time we were developing in our lab the MNHN-Tree-Tools [6] toolkit, primarily for the investigation of DNA repeat sequences. We have further extended MNHN-Tree-Tools [6] to guide phylogenetics. As such the toolkit has evolved into a high performance code, allowing for a fast investigation of millions of sequences. Given the context of the pandemic it became evident that we will use our versatile tool to investigate the evolution of SARS-CoV-2 sequences. Our efforts have cumulated in this tutorial that we share with the scientific community.

category: genetics


title: Computational Mapping of the Human-SARS-CoV-2 Protein-RNA Interactome

abstract: Strong evidence suggests that human human RNA-binding proteins (RBPs) are critical factors for viral infection, yet there is no feasible experimental approach to map exact binding sites of RBPs across the SARS-CoV-2 genome systematically at a large scale. We investigated the role of RBPs in the context of SARS-CoV-2 by constructing the first in silico map of human RBP / viral RNA interactions at nucleotide-resolution using two deep learning methods (pysster and DeepRiPe) trained on data from CLIP-seq experiments. We evaluated conservation of RBP binding between 6 other human pathogenic coronaviruses and identified sites of conserved and differential binding in the UTRs of SARS-CoV-1, SARS-CoV-2 and MERS. We scored the impact of variants from 11 viral strains on protein-RNA interaction, identifying a set of gain-and loss of binding events. Lastly, we linked RBPs to functional data and OMICs from other studies, and identified MBNL1, FTO and FXR2 as potential clinical biomarkers. Our results contribute towards a deeper understanding of how viruses hijack host cellular pathways and are available through a comprehensive online resource (https://sc2rbpmap.helmholtz-muenchen.de).

category: bioinformatics


title: Conformational Flexibility and Local Frustration in the Functional States of the SARS-CoV-2 Spike B.1.1.7 and B.1.351 Variants: Mutation-Induced Allosteric Modulation Mechanism of Functional Dynamics and Protein Stability

abstract: The experimental and computational studies of the SARS-CoV-2 spike protein variants revealed an important role of the D614G mutation that is shared across variants of concern(VOCs), linking the effect of this mutation with the enhanced virus infectivity and transmissibility. The recent structural and biophysical studies characterized the closed and open states of the B.1.1.7 (B.1.1.7) and B.1.351 (Beta) spike variants allowing for a more detailed atomistic characterization of the conformational landscapes and functional changes. In this study, we employed coarse-grained simulations of the SARS-CoV-2 spike variant trimers together with the ensemble-based mutational frustration analysis to characterize the dynamics signatures of the conformational landscapes. By combining the local frustration analysis of the conformational ensembles with collective dynamics and residue-based mutational scanning of protein stability, we determine protein stability hotspots and identify potential energetic drivers favoring the receptor-accessible open spike states for the B.1.1.7 and B.1.351 spike variants. Through mutational scanning of protein stability changes we quantify mutational adaptability of the S-G614, S-B.1.1.7 and S-B.1.351 variants in different functional forms. Using this analysis, we found a significant conformational and mutational plasticity of the open states for all studied variants. The results of this study suggest that modulation of the energetic frustration at the inter-protomer interfaces can serve as a mechanism for allosteric couplings between mutational sites, the inter-protomer hinges of functional motions and motions of the receptor-binding domain required for binding of the host cell receptor. The proposed mechanism of mutation-induced energetic frustration may result in the greater adaptability and the emergence of multiple conformational substates in the open form. This study also suggested functional relationships between mutation-induced modulation of protein dynamics, local frustration and allosteric regulation of the SARS-CoV-2 spike protein.

category: biophysics


title: Directing Min protein patterns with advective bulk flow

abstract: We theoretically predict and experimentally show that the propagation direction of in vitro Min protein patterns can be controlled by a hydrodynamic flow of the bulk solution. We find downstream propagation of Min wave patterns relative to the bulk flow direction for low MinE:MinD concentration ratios, but upstream propagation for large MinE:MinD ratios, with multistability of both propagation directions in between. A theoretical model for the Min system reveals the mechanism underlying the upstream propagation and links it to the fast conformational switching of MinE in the bulk. For high MinE:MinD ratios, upstream propagation can be reproduced by a reduced model in which increased MinD bulk concentrations on the upstream side promote protein attachment and hence, propagation in that direction. For low MinE:D ratios, downstream propagation is described by the minimal model, as additionally confirmed by experiments with a non-switching MinE mutant. No advection takes place on the membrane surface where the protein patterns form, but advective bulk flow shifts the protein-concentration profiles in the bulk relative to the membrane-bound pattern. From a broader perspective, differential flows in a bulk volume relative to a surface are a relevant general feature in bulk-surface coupled systems. Our study shows how such a differential flow can control surface-pattern propagation and demonstrates how the global patterns response may depend on specific molecular features of the reaction kinetics.


title: Allosteric Determinants of the SARS-CoV-2 Spike Protein Binding with Nanobodies: Examining Mechanisms of Mutational Escape and Sensitivity of the Omicron Variant

abstract: Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this study, we performed a comprehensive computational analysis of the SARS-CoV-2 spike trimer complexes with Nb6, VHH E and bi-paratopic VHH VE nanobodies. We combined atomistic dynamics and collective motions analysis with binding free energy scanning, perturbation-response scanning and network centrality analysis to examine mechanisms of nanobody-induced allosteric modulation and cooperativity in the SARS-CoV-2 spike trimer complexes with these nanobodies. By quantifying energetic and allosteric determinants of the SARS-CoV-2 spike protein binding with nanobodies, we also examined nanobody-induced modulation of escaping mutations and the effect of the Omicron variant on nanobody binding. The mutational scanning analysis supported the notion that E484A mutation can have a significant detrimental effect on nanobody binding and result in Omicron-induced escape from nanobody neutralization. Our findings showed that SARS-CoV-2 spike protein may exploit plasticity of specific allosteric hotspots to generate escape mutants that alter response to binding without compromising activity. The network analysis supported these findings showing that VHH VE nanobody binding can induce long-range couplings between the cryptic binding epitope and ACE2-binding site through a broader ensemble of communication paths that is less dependent on specific mediating centers and therefore may be less sensitive to mutational perturbations of functional residues. The results suggest that binding affinity and long-range communications of the SARS-CoV-2 complexes with nanobodies can be determined by structurally stable regulatory centers and conformationally adaptable hotspots that are allosterically coupled and collectively control resilience to mutational escape.


title: Machine learning guided design of high affinity ACE2 decoys for SARS-CoV-2 neutralization

abstract: A potential therapeutic candidate for neutralizing SARS-CoV-2 infection is engineering high-affinity soluble ACE2 decoy proteins to compete for binding of the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant ACE2 variant, named ACE22.v.2.4, that exhibits nanomolar affinity binding to the RBD domain of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identified other ACE2 variants, namely double mutants, that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified several ACE2 double mutants that bind to RBD with tighter affinity as compared to the wild type, most notably, L79V;N90D that binds RBD with similar affinity to ACE22.v.2.4. The successful experimental validation of the double mutants demonstrated the use transfer and supervised learning approaches for engineering protein-protein interactions and identifying high affinity ACE2 peptides for targeting SARS-CoV-2.


title: Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

abstract: Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyPs many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we characterize the motion of chromosomal loci and free tracer particles in the cytoplasm. In the absence of polyP and upon starvation, we observe an increase in mobility both for chromosomal loci and for tracer particles. Tracer particles reveal that polyP also modulates the partitioning between a  more mobile and a  less mobile population: small particles in cells unable to make polyP are more likely to be  mobile and explore more of the cytoplasm, particularly during starvation. We speculate that this larger freedom of motion may be a consequence of nucleoid decompaction, which we also observe in starved cells deficient in polyP. Our observations suggest that polyP limits cytoplasmic mobility and accessibility during nitrogen starvation, which may help to explain the pleiotropic phenotypes observed in the absence of polyP.


title: An in vivo, neuron-specific approach for pairing translational and epigenetic signatures of early-life exercise

abstract: Aerobic exercise promotes physiological and molecular adaptations in neurons to influence brain function and behavior. The most well studied neurobiological consequences of exercise are those which underlie exercise-induced improvements in hippocampal memory, including the expression and regulation of the neurotrophic factor Bdnf. Whether aerobic exercise taking place during early-life periods of postnatal brain maturation has similar impacts on gene expression and its regulation remains to be investigated. Using unbiased next-generation sequencing we characterize gene expression programs and their regulation by specific, memory-associated histone modifications during juvenile-adolescent voluntary exercise (ELE). Traditional transcriptomic and epigenomic sequencing approaches have either used heterogeneous cell populations from whole tissue homogenates or flow cytometry for single cell isolation to distinguish cell types / subtypes. These methods fall short in providing cell-type specificity without compromising sequencing depth or procedure-induced changes to cellular phenotype. In this study, we use simultaneous isolation of translating mRNA and nuclear chromatin from a neuron-enriched cell population to more accurately pair ELE-induced changes in gene expression with epigenetic modifications. We employ a line of transgenic mice expressing the NuTRAP (Nuclear Tagging and Translating Ribosome Affinity Purification) cassette under the Emx1 promoter allowing for brain cell-type specificity. We then developed a technique that combines nuclear isolation using Isolation of Nuclei TAgged in Specific Cell Types (INTACT) with Translating Ribosomal Affinity Purification (TRAP) methods to determine cell type-specific epigenetic modifications influencing gene expression programs from a population of Emx1 expressing hippocampal neurons. Data from RNA-seq and CUT&RUN-seq were coupled to evaluate histone modifications influencing the expression of translating mRNA in neurons after early-life exercise (ELE). We also performed separate INTACT and TRAP isolations for validation of our protocol and demonstrate similar molecular functions and biological processes implicated by gene ontology (GO) analysis. Finally, as prior studies use tissue from opposite brain hemispheres to pair transcriptomic and epigenomic data from the same rodent, we take a bioinformatics approach to compare hemispheric differences in gene expression programs and histone modifications altered by by ELE. Our data reveal transcriptional and epigenetic signatures of ELE exposure and identify novel candidate gene-histone modification interactions for further investigation. Importantly, our novel approach of combined INTACT/TRAP methods from the same cell suspension allows for simultaneous transcriptomic and epigenomic sequencing in a cell-type specific manner.


title: Quantifying the inverted U: A meta-analysis of prefrontal dopamine, D1-receptors, and working memory

abstract: Dopamine in the prefrontal cortex can be disrupted in human disorders that affect cognitive function such as Parkinsons disease (PD), attention-deficit hyperactivity disorder (ADHD), and schizophrenia. Dopamine has a powerful effect on prefrontal circuits via the D1-type dopamine receptor (D1DR). It has been proposed that prefrontal dopamine has "inverted U-shaped" dynamics, with optimal dopamine and D1DR signaling required for optimal cognitive function. However, the quantitative relationship between prefrontal dopamine and cognitive function is not clear. Here, we conducted a meta-analysis of published manipulations of prefrontal dopamine and the effects on working memory, a high-level executive function in humans, primates, and rodents that involves maintaining and manipulating information over seconds to minutes. We reviewed 646 papers and found that 75 studies met criteria for inclusion. Our quantification of effect sizes for dopamine, D1DRs, and behavior revealed a negative quadratic slope. This is consistent with the proposed inverted U-shape of prefrontal dopamine and D1DRs and working memory performance, explaining 10% of the variance. Of note, the inverted quadratic fit was much stronger for prefrontal D1DRs alone, explaining 26% of the variance, compared to prefrontal dopamine alone, explaining 10% of the variance. Taken together, these data, derived from a variety of manipulations and systems, demonstrate that optimal prefrontal dopamine signalling is linked with higher cognitive function. Our results provide insight into the fundamental dynamics of prefrontal dopamine, which could be useful for pharmacological interventions targeting prefrontal dopaminergic circuits, and into the pathophysiology of human brain disease.


title: Neurons burdened by DNA double strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration.

abstract:
DNA double strand breaks (DSBs) are linked to aging, neurodegeneration, and senescence1,2. However, the role played by neurons burdened with DSBs in disease-associated neuroinflammation is not well understood. Here, we isolate neurons harboring DSBs from the CK-p25 mouse model of neurodegeneration through fluorescence-activated nuclei sorting (FANS), and characterize their transcriptomes using single-nucleus, bulk, and spatial sequencing techniques. We find that neurons harboring DSBs enter a late-stage DNA damage response marked by the activation of senescent and antiviral-like immune pathways. We identify the NFkB transcription factor as a master regulator of immune gene expression in DSB-bearing neurons, and find that the expression of cytokines like Cxcl10 and Ccl2 develop in DSB-bearing neurons before glial cell types. Alzheimers Disease pathology is significantly associated with immune activation in excitatory neurons, and direct purification of DSB-bearing neurons from Alzheimers Disease brain tissue further validates immune gene upregulation. Spatial transcriptomics reveal that regions of brain tissue dense with DSB-bearing neurons also harbor signatures of inflammatory microglia, which is ameliorated by NFkB knock down in neurons. Inhibition of NFkB or depletion of Ccl2 and Cxcl10 in DSB-bearing neurons also reduces microglial activation in organotypic brain slice culture. In conclusion, we find that in the context of age-associated neurodegenerative disease, DSBs activate immune pathways in neurons, which in turn adopt a senescence associated secretory phenotype to elicit microglia activation. These findings highlight a novel role for neurons in the mechanism of age-associated neuroinflammation.

SummaryIt is unclear how age-associated DNA double strand break (DSB) accumulation in neurons influences the progression of cellular senescence and neurodegenerative disease. Here, we leverage mouse models of neurodegeneration, single-nucleus, bulk, and spatial transcriptomics from Alzheimers disease patients, mouse models, and primary neuron cultures to dissect the immune signaling pathways initiated by DSB-bearing neurons that trigger neuroinflammation.


title: Copy number variation of the putative speciation genes in the European house mouse hybrid zone

abstract: AO_SCPLOWBSTRACTC_SCPLOWHybrid zones have long been described as "windows on the evolutionary process", and studying them has become even more important since the advance in the genome analysis tools. The hybrid zone between two subspecies of the house mouse (Mus musculus musculus and Mus m. domesticus) is a unique model speciation system to study fine scale interactions of recently diverged genomes. Here, we explore the role of gene Copy Number Variation in shaping the barrier to introgression in the hybrid zone within a previously established transect in Central Europe. The CNV of seven pre-selected candidate genes was determined via droplet-digital PCR and analyzed in the context of ~500k SNPs, with the ancestral population (i.e. musculus or domesticus) of every SNP allele previously inferred in the admixed individuals (Baird et al., in prep.). The copy numbers of five genes were clearly associated with the prevalence of either musculus or domesticus genomes across the hybrid zone. In three cases, the highest and/or outlying levels of association were observed at or very close to the annotated positions of the respective gene amplicons, demonstrating the power of our approach in confirming the reference locations of copy number variants. Notably, several other reference locations were recognized as positive outliers in the association with particular CNV genes, possibly representing the extra gene copies and/or their epistatic interaction sites.


title: TACAN is a novel regulator of PKD2

abstract: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in membrane receptor PKD1 or cation channel PKD2. TACAN (also named TMEM120A), recently reported as an ion channel in neuron cells for mechano and pain sensing, is also distributed in diverse non-neuronal tissues such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes we found that TACAN inhibited the channel activity of PKD2 gain-of-function mutant F604P. The first and last transmembrane domains of TACAN were found to interact with the PKD2 C-and N-terminal portions, respectively. We showed that the TACAN N-terminus acted as a blocking peptide and that TACAN inhibits the PKD2 function through the PKD2/TACAN binding. By patch clamping in mammalian cells, we found that TACAN inhibits both the single channel conductance and open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we also found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish, in support of the in vitro inhibitory effects of TACAN. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechano sensitivity of the PKD2/TACAN channel complex.

category: physiology


title: Regulation of the PKD2 channel function by TACAN


title: Astroglial CD38 regulates social memory and synapse formation through SPARCL1 in the medial prefrontal cortex

abstract: Social behavior is essential for the health, survival and reproduction of animals, yet the role of astrocytes in social behavior is largely unknown. CD38 is critical for social behaviors by regulating oxytocin release from hypothalamic neurons. On the other hand, CD38 is most abundantly expressed in astrocytes especially in the postnatal cortex, and is important for astroglial development. Here, we demonstrate that astroglial CD38 plays a pivotal role in the social behavior. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, specifically impaired social memory without any other behavioral abnormalities. Morphological analysis revealed reductions in spine numbers, mature spines and excitatory synapse numbers in the pyramidal neurons of the medial prefrontal cortex (mPFC) due to deletion of astroglial CD38 in the postnatal brain. Astrocyte-conditioned medium (ACM) of CD38 KO astrocytes reduced synaptogenesis of cortical neurons by reducing extracellular SPARCL1, a synaptogenic protein. Finally, the release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. Our data indicate that astroglial CD38 developmentally regulates social memory and neural circuit formation in the developing brain by promoting synaptogenesis through SPARCL1.


title: An extended toolkit for production and use of RVdG-CVS-N2c rabies viral vectors uncovers hidden hippocampal connections

abstract: To understand the function of neuronal circuits it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low-throughput, low-selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotrophic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of "starter" AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo.


title: Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling


title: Disruption of synaptic transmission in the Bed Nucleus of the Stria Terminalis reduces seizure-induced death in DBA/1 mice and alters brainstem E/I balance

abstract:
Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in refractory epilepsy patients. Accumulating evidence from recent human studies and animal models suggests that seizure-related respiratory arrest may be important for initiating cardiorespiratory arrest and death. Prior evidence suggests that apnea onset can coincide with seizure spread to the amygdala and that stimulation of the amygdala can reliably induce apneas in epilepsy patients, potentially implicating amygdalar regions in seizure-related respiratory arrest and subsequent postictal hypoventilation and cardiorespiratory death. This study aimed to determine if an extended amygdalar structure, the dorsal bed nucleus of the stria terminalis (dBNST), is involved in seizure-induced respiratory arrest (S-IRA) and death using DBA/1 mice, a mouse strain which has audiogenic seizures and a high incidence of postictal respiratory arrest and death. The presence of S-IRA significantly increased c-Fos expression in the dBNST of DBA/1 mice. Furthermore, disruption of synaptic output from the dBNST via viral-induced tetanus neurotoxin significantly improved survival following S-IRA in DBA/1 mice without affecting baseline breathing or hypercapnic and hypoxic ventilatory response. This disruption in the dBNST resulted in changes to the balance of excitatory/inhibitory synaptic events in the downstream brainstem regions of the lateral parabrachial nucleus (PBN) and the periaqueductal gray (PAG). These findings suggest that the dBNST is a potential subcortical forebrain site necessary for the mediation of seizure-induced respiratory arrest, potentially through its outputs to brainstem respiratory regions.

SUMMARY STATEMENTThis study used a viral expression technique to disrupt synaptic output in the bed nucleus of the stria terminalis (BNST) of DBA/1 audiogenic seizure mice. Inactivating the BNST significantly improved survival following seizures and altered brainstem excitation/inhibition balance.


title: Age related susceptibility to grey matter demyelination and neurodegeneration is associated with meningeal neutrophil accumulation in an animal model of Multiple Sclerosis

abstract: People living with multiple sclerosis (MS) experience episodic central nervous system (CNS) white matter lesions instigated by autoreactive T cells. With age, MS patients show evidence of grey matter demyelination and experience devastating non-remitting symptomology. What drives progression is unclear and has been hampered by the lack of suitable animal models. Here we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induces a non-remitting clinical phenotype that is associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young SJL/J mice. While the quantity and quality of T cells did not differ in the brains of old vs young EAE mice, an increase in neutrophils and a decrease in B cells was observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and grey matter pathology are dictated by age and associated with other immune cells such as neutrophils.


title: Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses

abstract:
Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. Here, we find that deficiency of EP2 rather than EP4 augments IL-33-induced lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 but not EP2 markedly restricts IL-33- and Alternaria alternata-induced lung ILC2 responses and eosinophilic inflammation. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice lacking endogenous PG synthesis but not in PG-competent mice. Together, we have defined a mechanism for optimal suppression of lung ILC2 responses by endogenous PGE2-EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2-EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating ILC2-initiated lung inflammation in asthma and NERD.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/474031v1_ufig1.gif" ALT="Figure 1">
View larger version (45K):
org.highwire.dtl.DTLVardef@17dd2caorg.highwire.dtl.DTLVardef@199ce97org.highwire.dtl.DTLVardef@ca1f67org.highwire.dtl.DTLVardef@1d3a49f_HPS_FORMAT_FIGEXP  M_FIG C_FIG Schematic of potential roles for activation of EP2 and EP4 by endogenous versus exogenous ligands in regulation of lung ILC2 immune responses. Endogenous PGE2 in the lung preferentially activates EP2 rather than EP4 to inhibit ILC2 responses and eosinophilic inflammation, and ablation of EP2 enhances lung ILC2 responses. Conversely, lung ILC2 responses are not altered by EP4 deficiency. However, they are markedly inhibited by EP4 agonism but not EP2 agonism. Mechanistically, PGE2-EP2/EP4 signaling activates the cAMP pathway which inhibits ILC2 energy metabolism, possibly through interruption of NF-{kappa}B (reported in Nagashima H, et al. Immunity 2019;51:682-695) and STAT5 signaling, leading to decline of ILC2 survival, proliferation and type 2 cytokine production.


title: The cardiac lncRNA Chantico directly regulates Cxcl1 chemokine transcription

abstract: In local gene regulation, long noncoding RNA (lncRNA) loci can function at three distinct, but non-mutually exclusive levels: the RNA molecule itself, the process of its transcription, and/or the underlying DNA element. Yet for cis-acting lncRNA, these distinctions are particularly challenging with present tools. To address this problem, we developed Omegazymes, catalytic nucleic acid enzymes to specifically target lncRNA for degradation without triggering premature termination of their transcription. We use Omegazymes to selectively target one of a highly refined set of lncRNAs in the mouse cardiomyocytes, demonstrating that the Chantico lncRNA molecule directly potentiates the transcription of Cxcl1, a neighboring chemokine gene. We find that the Chantico locus also acts at the DNA-level, as the binding of an essential cardiac transcription factor to the Chantico promoter is necessary for Cxcl1 transcription in mature cardiomyocytes. This Chantico regulation of Cxcl1 impacts cardiomyocyte signaling to immune cells, potentially regulating tissue-residence and inflammation.

category: molecular biology


title: A Population Genomic Assessment of Three Decades of Evolution in a Natural Drosophila Population

abstract: Population genetics seeks to illuminate the forces shaping genetic variation, often based on a single snapshot of genomic variation. However, utilizing multiple sampling times to study changes in allele frequencies can help clarify the relative roles of neutral and non-neutral forces on short time scales. This study compares whole-genome sequence variation of recently collected natural population samples of Drosophila melanogaster against a collection made approximately 35 years prior from the same locality - encompassing roughly 500 generations of evolution. The allele frequency changes between these time points would suggest a relatively small local effective population size on the order of 10,000, significantly smaller than the global effective population size of the species. Some loci display stronger allele frequency changes than would be expected anywhere in the genome under neutrality - most notably the tandem paralogs Cyp6a17 and Cyp6a23, which are impacted by structural variation associated with resistance to pyrethroid insecticides. We find a genome-wide excess of outliers for high genetic differentiation between old and new samples, but a larger number of adaptation targets may have affected SNP-level differentiation versus window differentiation. We also find evidence for strengthening latitudinal allele frequency clines: northern-associated alleles have increased in frequency by an average of nearly 2.5% at SNPs previously identified as clinal outliers, but no such pattern is observed at random SNPs. This project underscores the scientific potential of using multiple sampling time points to investigate how evolution operates in natural populations, by quantifying how genetic variation has changed over ecologically relevant timescales.


title: A novel high-throughput assay using mixed genomic DNA for fast screening germline pathogenic variants in breast cancer susceptibility genes

abstract: The demand for genetic testing for breast cancer susceptibility genes is increasing for both breast cancer patients and healthy individuals. Here we established a novel high-throughput assay to detect germline pathogenic variants in breast cancer susceptibility genes. In general, up 10 to 50 individual genomic DNA samples were mixed together to create a mixed DNA sample and the mixed DNA sample was subjected to a next-generation multigene panel. Germline pathogenic variants in breast cancer susceptibility genes could be found in the mixed DNA sample; next, site-specific Sanger sequencing was performed to identify individuals who carried he pathogenic variant in the mixed samples. We found that the recall and precision rates were 89.9% and 92.9% when twenty individual genomic samples were mixed. Therefore, our new assay can increase an approximately 20-fold of efficacy to identify the pathogenic variants in breast cancer susceptibility genes in individuals when compared with current assay.


title: Experimental Evolution Reveals Redox State Modulates Mycobacterial Pathogenicity

abstract: Understanding how Mycobacterium tuberculosis has evolved into a professional pathogen is helpful in studying its pathogenesis and for designing vaccines. We investigated how the evolutionary adaptation of M. smegmatis mc251 to an important clinical stressor H2O2 allows bacteria undergo coordinated genetic mutations, resulting in increased pathogenicity. Whole-genome sequencing identified a mutation site in the fur gene, which caused increased expression of katG. Using a Wayne dormancy model, mc251 showed a growth advantage over its parental strain mc2155 in recovering from dormancy under anaerobic conditions. Meanwhile, the high level of KatG in mc251 was accompanied by a low level of ATP, which meant that mc251 is at a low respiratory level. Additionally, the redox-related protein Rv1996 showed different phenotypes in different specific redox states in M. smegmatis mc2155, mc251, M. bovis BCG and M. tuberculosis mc27000. In conclusion, our study shows that the same gene presents different phenotypes under different physiological conditions. This may partly explain why M. smegmatis and M. tuberculosis have similar virulence factors and signaling transduction systems such as two-component systems and sigma factors, but due to the different redox states in the corresponding bacteria, M. smegmatis is a nonpathogen, while M. tuberculosis is a pathogen. As mc251 overcomes its shortcomings of rapid removal, it can be potentially developed as a vaccine vector.


title: Novel allosteric mechanism of dual p53/MDM2 and p53/MDM4 inhibition by a small molecule

abstract:
Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising strategy. However, high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance.

Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which blocks both p53/MDM2 and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA and protoporphyrin IX (PpIX). Ion-mobility mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions.

Contribution to the fieldGiven the immense importance of the p53 tumor suppressor for cancer, efforts have been made to identify p53 activators, which sterically inhibit MDM2. Because high-affinity MDM2 inhibitors are facing problems with considerable side effects, other approaches are needed to reactivate p53 for improved cancer therapy. The allosteric mechanism of action of p53 activator RITA, which we discovered, and its dependence on the oncogenic switch, is an unexpected turn in the p53 story. Our findings provide a basis for the development of p53 activators with a similar mode of functioning, either through the classical drug discovery route or through the drug repurposing approach. Allosteric modulators might have great potential as single agents, or in combination with the standard of care. Further, p53 modulators could serve as invaluable tools to better understand its biology.


title: Two distinct binding modes provide the RNA binding protein RbFox with extraordinary sequence specificity

abstract: The specificity of RNA-binding proteins for their target sequences varies considerably. Yet, it is not understood how certain proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two different binding modes. The first exclusively binds the cognate and a closely related RNA variant with high affinity. The second mode accommodates all other RNAs with greatly reduced affinity, thereby imposing large thermodynamic penalties on even near-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA binding site. Distinct binding modes by a single RNA binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation for RNA-protein interactions.


title: Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices

abstract: During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~45 residues away from the peptidyl transferase center. However, we recently uncovered instances where the first contact is delayed by up to ~10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the BtuC protein: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion.


title: Microfluidic device integrating a network of hyper-elastic valves for automated glucose stimulation and insulin secretion collection from a single pancreatic islet

abstract: Advances in microphysiological systems have prompted the need for robust and reliable cell culture devices. While microfluidic technology has made significant progress, devices often lack user-friendliness and are not designed to be industrialized on a large scale. Pancreatic islets are often being studied using microfluidic platforms in which the monitoring of fluxes is generally very limited, especially because the integration of valves to direct the flow is difficult to achieve. Considering these constraints, we present a thermoplastic manufactured microfluidic chip with an automated control of fluxes for the stimulation and secretion collection of pancreatic islet. The islet was directed toward precise locations through passive hydrodynamic trapping and both dynamic glucose stimulation and insulin harvesting were done automatically via a network of large deformation valves, directing the reagents and the pancreatic islet toward different pathways. This device we developed enables monitoring of insulin secretion from a single islet and can be adapted for the study of a wide variety of biological tissues and secretomes.


title: Reciprocal interactions between the gut microbiome and mammary tissue mast cells promote metastatic dissemination of HR+ breast tumors

abstract:
Establishing commensal dysbiosis, defined as an inflammatory gut microbiome with low biodiversity, prior to breast tumor initiation, enhances early dissemination of hormone-receptor positive (HR+) mammary tumor cells. Here, we sought to define mammary tissue mediators of dysbiosis-induced tumor dissemination. We found that commensal dysbiosis increased both the frequency and profibrogenicity of mast cells in the mammary tissue, a phenotypic change that persisted after tumor implantation. Fibroblast activation and tissue remodeling associate with enhanced breast tumor metastasis. We employed pharmacological and adoptive transfer approaches to demonstrate that mammary tissue mast cells from dysbiotic animals enhances dissemination of HR+ tumor cells. Collagen levels in mammary tissues from HR+ breast cancer patients correlated with mast cell abundance, suggesting clinical relevance of mast cell-mediated fibroblast activation. Together, these data demonstrate that a gut-mast cell axis exists that induces fibroblast activation and orchestrates early dissemination of HR+ breast tumors.

SignificanceOur study defines the mechanism by which an inflammatory gut microbiome facilitates HR+ breast tumor cell dissemination. We establish that gut commensal dysbiosis triggers mammary tissue mast cells to facilitate early metastatic dissemination. These findings highlight a novel gut microbiome-innate immune cell axis involved in negative breast cancer outcomes.

category: cancer biology


title: Neural stemness unifies cell tumorigenicity and pluripotent differentiation potential

abstract: Tumorigenicity and pluripotent differentiation potential are kernel cell properties for tumorgenesis and embryogenesis. A growing number of studies have demonstrated that neural stemness is the source of the two cell properties, because neural stem cells and cancer cells share cell features and regulatory networks and neural stemness has an evolutionary advantage. However, it needs to validate whether neural stemness is a cell property that would unify tumorigenicity and pluripotent differentiation potential. SETDB1/Setdb1 is an epigenetic factor that is upregulated in cancer cells and promotes cancers, and correspondingly, is enriched in embryonic neural cells during vertebrate embryogenesis. We show that knockdown of SETDB1/Setdb1 led to neuronal differentiation in neural stem and cancer cells, concomitant with reduced tumorigenicity and pluripotent differentiation potential in these cells; whereas overexpression caused an opposite effect. On one hand, SETDB1 maintains a regulatory network comprised of proteins involved in developmental programs and basic cellular functional machineries, including epigenetic modifications (EZH2), ribosome biogenesis (RPS3), translation initiation (EIF4G), spliceosome assembly (SF3B1), etc., all of which play active roles in cancers. On the other, it represses transcription of genes promoting differentiation and cell cycle and growth arrest. Moreover, neural stemness, tumorigenicity and pluripotent differentiation potential were simultaneously enhanced during serial transplantation of cancer cells. Expression of proteins involved in developmental programs and basic cellular functional machineries, including SETDB1 and other proteins above, was gradually increased. In agreement with increased expression of spliceosome proteins, alternative splicing events also increased in tumor cells derived from later transplantations, suggesting that different machineries should work concertedly to match the status of high proliferation and pluripotent differentiation potential. The study presents the evidence that neural stemness unifies tumorigenicity and differentiation potential. Tumorigenesis represents a process of gradual loss of original cell identity and gain of neural stemness in somatic cells, which might be a distorted replay of neural induction during normal embryogenesis.


title: ROKET: Associating Somatic Mutation with Clinical Outcomes through Kernel Regression and Optimal Transport

abstract: Somatic mutations in cancer patients are inherently sparse and potentially high dimensional. Cancer patients may share the same set of deregulated biological processes perturbed by different sets of somatically mutated genes. Therefore, when assessing the associations between somatic mutations and clinical outcomes, gene-by-gene analyses is often under-powered because it does not capture the complex disease mechanisms shared across cancer patients. Rather than testing genes one by one, an intuitive approach is to aggregate somatic mutation data of multiple genes to assess the joint association. The challenge is how to aggregate such information. Building on the optimal transport method, we propose a principled approach to estimate the similarity of somatic mutation profiles of multiple genes between tumor samples, while accounting for gene-gene similarity defined by gene annotations or empirical mutational patterns. Using such similarities, we can assess the associations between somatic mutations and clinical outcomes by kernel regression. We have applied our method to analyze somatic mutation data of 17 cancer types and identified at least three cancer types harboring associations between somatic mutations and overall survival, progression-free interval or cytolytic activity.

category: genomics


title: Control effectiveness of APL formulation against dengue- and Zika-transmitting Aedes mosquitoes in Gia Lai province, Vietnam

abstract:
BackgroundDengue fever and Zika are two of the Aedes-borne diseases. Despite being widely used, synthetic mosquitocides become abortive for the mosquito control due to growing resistance and environmental pollution. In Gia Lai province (dengue-endemic area), a huge amount of cashew nut shell waste with roughly 100,000 tons/year has been disposed of into the environment, potentiating a high risk of pollution.

Methodology/Principal findingsTo utilize it, anacardic acid was extracted and combined it with ethanol extract of the local lime peel, which contains limonene, to generate APL formulation. APL robustly exhibited inhibition of egg hatching, larvicidal effect, and repellent effect against female mosquitoes from oviposition sites in the laboratory and field. The results showed that, at a dose of 12.5 ppm, the APL formulation after 24 hours of treatment demonstrated oviposition deterrence against Ae. aegypti (43.6%) and Ae. albopictus (59.6%); inhibited egg hatching of Ae. aegypti (49.6%) and Ae. albopictus (59.6%); caused larval lethality in Ae. aegypti (LC50 = 9.5 ppm, LC90 = 21 ppm) and Ae. albopictus (LC50 = 7.6 ppm, LC90 = 18 ppm). Under natural field conditions, it showed a 100% reduction in larval density after 48 and 72 hours of the APL treatment at a tested concentration of 120 mg a.i./m2 and maintained a mortality rate of 100% in the next 14 days.

Conclusions/SignificanceThe APL formulation is promisingly to become an environmentally friendly and highly effective biological product for future management programs of dengue and Zika-transmitting vectors. Here offer prospects in controlling critical illnesses transmitted by several mosquito species in dengue-endemic areas.

Author summaryThe use of synthetic insecticide to control the dengue and Zika vector population has contributed to drug resistance and caused negative impacts on the environment. The plant-based insecticide should be beneficial for mosquito management in the current situation. Gia Lai province in Vietnam is a dengue-endemic area. A large amount of cashew nut shell waste gets discarded in the area every year, which imposes an increased risk of pollution. The authors took advantage of this by extracting anacardic acid to combine with ethanol extract of local lime peel (containing limonene) to produce APL formulation. This formulation demonstrated potential activities and efficiency in controlling mosquitoes transmitting disease. In the laboratory condition, at a low dose of 12.5 ppm, APL showed activities in inhibiting egg hatching, larviciding, and repelling female Aedes aegypti and Aedes albopictus. In the field condition, APL at a dose of 120 mg a.i./m2 thoroughly reduced the dengue larval density after two days of contact, and this effect lasted to the next 14 days. APL is a promising and environmentally friendly larvicidal product that is highly effective in controlling dengue and Zika vectors and can play as an alternative measure for vector-borne dengue in the locality.


title: Sorghum Association Panel Whole-Genome Sequencing Establishes Pivotal Resource for Dissecting Genomic Diversity

abstract:
Association mapping panels represent foundational resources for understanding the genetic basis of phenotypic diversity and serve to advance plant breeding by exploring genetic variation across diverse accessions with distinct histories of evolutionary divergence and local adaptation. We report the whole-genome sequencing (WGS) of 400 sorghum [Sorghum bicolor (L.) Moench] accessions from the Sorghum Association Panel (SAP) at an average coverage of 38X (25X-72X), enabling the development of a high-density genomic-marker set of 43,983,694 variants including SNPs (~ 38 million), indels (~ 5 million), and CNVs (170,000). We observe slightly more deletions among indels and a much higher prevalence of deletions among copy number variants compared to insertions. This new marker set enabled the identification of several putatively novel genomic associations for plant height and tannin content, which were not identified when using previous lower-density marker sets. WGS identified and scored variants in 5 kb bins where available genotyping-by-sequencing (GBS) data captured no variants, with half of all bins in the genome falling into this category. The predictive ability of genomic best unbiased linear predictor (GBLUP) models was increased by an average of 30% by using WGS markers rather than GBS markers. We identified 18 selection peaks across subpopulations that formed due to evolutionary divergence during domestication, and we found six Fst peaks resulting from comparisons between converted lines and breeding lines within the SAP that were distinct from the peaks associated with historic selection. This population has been and continues to serve as a significant public resource for sorghum research and demonstrates the value of improving upon existing genomic resources.

Author summary

category: plant biology


title: Growth-limiting drought stress induces time-of-day dependent transcriptome and physiological responses in hybrid poplar

abstract:
Drought stress negatively impacts the health of long-lived trees. Understanding the genetic mechanisms that underpin response to drought stress is requisite for selecting or enhancing climate change resilience. We aimed to determine how hybrid poplars respond to prolonged and uniform exposure to drought; how responses to moderate and more severe growth-limiting drought stresses differed; and, how drought responses change throughout the day. We established hybrid poplar trees (Populus x  Okanese) from unrooted stem cutting with abundant soil moisture for six weeks. We then withheld water to establish well-watered, moderate, and severe growth-limiting drought conditions. These conditions were maintained for three weeks during which growth was monitored. We then measured photosynthetic rates and transcriptomes of leaves that had developed during the drought treatments at two times of day. The moderate and severe drought treatments elicited distinct changes in growth and development, photosynthetic rates, and global transcriptome profiles. Notably, the time of day of sampling produced the strongest signal in the transcriptome data. The moderate drought treatment elicited global transcriptome changes that were intermediate to the severe and well-watered treatments in the early evening, but did not elicit a strong drought response in the morning, emphasizing the complex nature of drought regulation in long-lived trees.

HighlightPoplar drought transcriptome is defined by the time of day of sampling and by the extent of water deficit.


title: AFP2 inhibits ABA responses during germination without ABI5 degradation but DWAs reduce desiccation tolerance

abstract:
Overexpression of ABI5/ABF interacting proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. This study tests the hypothesis that the AFPs promote germination by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING (KEG), were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although the AFPs and E3 ligases showed weak direct interactions, loss of function for the E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 controls ABA sensitivity during germination independently of ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance that we investigated by quantitative proteomics. Our analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of the different genotypes allowed us to separate and refine the changes in protein accumulation patterns correlating with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

Summary SentenceExtreme ABA resistance conferred by overexpression of AFP2 is not mediated by interactions with E3 ligases, but the dwa background maintains desiccation tolerance despite ABA resistance.


title: A spatiotemporal steroidogenic regulatory network in human fetal adrenal glands and gonads

abstract: Human fetal adrenal glands produce substantial amounts of dehydroepiandrosterone (DHEA), which is one of the most important precursors of sex hormones. However, the underlying biological mechanism remains largely unknown. Herein, we sequenced human fetal adrenal glands and gonads from 7 to 14 GW via the 10x Genomics single-cell transcriptome techniques and reconstructed their location information by Spatial Transcriptome, conducted COOL-seq for the MC2R+ inner zone steroidogenic cells during the time window of sex differentiation (8-12GW). We found that relative to gonads, adrenal glands begin to synthesize steroids early. The coordination among steroidogenic cells and multiple nonsteroidogenic cells promotes adrenal cortex construction and steroid synthesis. Notably, during the time window of sex differentiation (8-12 GW), key enzyme gene expression shifts to accelerate DHEA synthesis in males and cortisol synthesis in females. Furthermore, high SST+ expressions in the adrenal gland and testis amplify androgen synthesis in males. Our research highlights the robustness of the action of fetal adrenal glands on gonads to modify the process of sexual differentiation.

category: systems biology


title: Hypersensitivity to uncertainty is key feature of subjective cognitive impairment

abstract: With an increasingly ageing global population, more people are presenting with concerns about their cognitive function, but not all have an underlying neurodegenerative diagnosis. Subjective cognitive impairment (SCI) is a common condition describing self-reported deficits in cognition without objective evidence of cognitive impairment. Many individuals with SCI suffer from depression and anxiety, which have been hypothesised to account for their cognitive complaints. Despite this association between SCI and affective features, the cognitive and brain mechanisms underlying SCI are poorly understood. Here, we show that people with SCI are hypersensitive to uncertainty and that this might be a key mechanism accounting for their affective burden. Twenty-seven individuals with SCI performed an information sampling task, where they could actively gather information prior to decisions. Across different conditions, SCI participants sampled faster and obtained more information than matched controls to resolve uncertainty. Remarkably, despite their  urgent sampling behaviour, SCI participants were able to maintain their efficiency. Hypersensitivity to uncertainty indexed by this sampling behaviour correlated with the severity of affective burden including depression and anxiety. Analysis of MRI resting functional connectivity revealed that both uncertainty hypersensitivity and affective burden were associated with stronger insular-hippocampal connectivity. These results suggest that altered uncertainty processing is a key mechanism underlying the psycho-cognitive manifestations in SCI and implicate a specific brain network target for future treatment.

category: neuroscience


title: Combination of Mycobacterium tuberculosis RS ratio and CFU improves the ability of murine efficacy experiments to distinguish between drug treatments

abstract: Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on colony forming units of M. tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process: ongoing ribosomal RNA synthesis. Here we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with colony forming units showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

category: microbiology


title: Bionized nanoferrite particles alter the course of experimental Cryptococcus neoformans pneumonia

abstract: Cryptococcosis is a devastating fungal disease associated with high morbidity and mortality even when treated with antifungal drugs. Bionized nanoferrite (BNF) nanoparticles are powerful immunomodulators, but their efficacy for infectious diseases has not been investigated. Administration of BNF nanoparticles to mice with experimental cryptococcal pneumonia altered the outcome of infection in a dose response manner as measured by colony forming units and survival. The protective effects were higher at lower doses, with reductions in IL-2, IL-4 and TNF-, consistent with immune modulation whereby reductions in inflammation translate into reduced host damage, clearance of infection and longer survival.


title: Regulation of KHNYN antiviral activity by the extended di-KH domain and nucleo-cytoplasmic trafficking

abstract:
The zinc finger antiviral protein (ZAP) restricts a broad range of viruses by binding CpG dinucleotides in viral RNA to target it for degradation and inhibit its translation. KHNYN was recently identified as an antiviral protein required for ZAP to inhibit retroviral replication, though little is known about its functional determinants. KHNYN contains an N-terminal extended di-KH-like domain, a PIN endoribonuclease domain and a C-terminal CUBAN domain that binds NEDD8 and ubiquitin. We show that deletion of the extended di-KH domain reduces its antiviral activity. However, despite its similarity to RNA binding KH domains, the extended di-KH domain in KHNYN does not appear to bind RNA. Mutation of residues in the CUBAN domain that bind NEDD8 increase KHNYN abundance but do not alter its antiviral activity, suggesting that this interaction regulates KHNYN homeostatic turnover. In contrast, a CRM1-dependent nuclear export signal (NES) at the C-terminus of the CUBAN domain is required for antiviral activity. Deletion of this signal retains KHNYN in the nucleus and inhibits its interaction with ZAP. Interestingly, this NES appeared in the KHNYN lineage at a similar time as when ZAP evolved in tetrapods, indicating that these proteins may have co-evolved to restrict viral replication.

AUTHOR SUMMARYAntiviral proteins restrict viral replication in many different ways, including inhibiting viral gene expression. ZAP is an antiviral RNA binding protein that must interact with other cellular proteins to inhibit viral protein synthesis. KHNYN is a ZAP cofactor that is required for it to inhibit retroviral replication. Because little is known about how KHNYN functions in this role, we have analyzed how two of its domains regulate its antiviral activity. We first show that the extended di-KH domain in KHNYN is required for its antiviral activity. While it is related to di-KH domains in RNA binding proteins, it appears to have lost its ability to bind RNA and KHNYN likely acts in the restriction pathway after ZAP binds a target viral RNA. Second, we show that the KHNYN CUBAN domain regulates both its protein abundance and trafficking within the cell. The CUBAN domain contains a nuclear export signal and, when this signal is mutated, KHNYN is sequestered in the nucleus, has substantially reduced antiviral activity and does not interact with ZAP. Overall, we show that the extended di-KH and CUBAN domains in KHNYN are required for it to act as a cofactor for ZAP to restrict viral replication.


title: XAB2 Dynamics during DNA Damage-Dependent Transcription Inhibition

abstract: Xeroderma Pigmentosum group A (XPA)-binding protein 2 (XAB2) is a multi-functional protein that plays a critical role in distinct cellular processes including transcription, splicing, DNA repair and mRNA export. In this study, we detailed XAB2 involvement during Nucleotide Excision Repair (NER), a repair pathway that guarantees genome integrity against UV light-induced DNA damage and that specifically removes transcription-blocking damage in a dedicated process known as Transcription-Coupled repair (TC-NER). Here, we demonstrated that XAB2 is involved specifically and exclusively in TC-NER reaction and solely for RNA Polymerase 2 transcribed genes. Surprisingly, contrary to all the other NER proteins studied so far, XAB2 does not accumulate on the local UV-C damage but on the contrary is remobilized after damage induction. This fast change in mobility is restored when DNA repair reactions are completed. By scrutinizing from which cellular complex/partner/structure XAB2 is released, we have identified that XAB2 is detached after DNA damage induction from the DNA:RNA hybrids, commonly known as R-loops, and from the CSA and XPG protein and this release is thought to contribute to the DNA damage recognition step during TC-NER. Importantly, we have disclosed a role for XAB2 in retaining RNAP2 on its substrate.