title: Differences in environmental stability among SARS-CoV-2 variants of concern: Omicron has higher stability

abstract: SARS-CoV-2 variants of concern (VOCs) could cause significant human and economic damage owing to increased infectivity and transmissibility, and understanding their characteristics is crucial for infection control. Here, we analyzed differences in viral stability and disinfection efficacy between the Wuhan strain and all VOCs. On plastic and skin surfaces, Alpha, Beta, Delta, and Omicron variants exhibited more than two-fold longer survival than the Wuhan strain, and the Omicron variant had the longest survival time. Specifically, survival times of the Wuhan strain, Alpha variant, Beta variant, Gamma variant, Delta variant, and Omicron variant on skin surfaces were 8.6 h (95% CI, 6.5-10.9 h), 19.6 h (95% CI, 14.8-25.3 h), 19.1 h (95% CI, 13.9- 25.3 h), 11.0 h (95% CI, 8.1-14.7 h), 16.8 h (95% CI, 13.1-21.1 h), and 21.1 h (95% CI, 15.8- 27.6 h), respectively. In vitro, disinfectant effectiveness evaluations showed that Alpha, Beta, Delta, and Omicron were slightly more resistant to ethanol than the Wuhan strain. However, ex vivo evaluation showed that on human skin, all viruses were completely inactivated by exposure to 35 w/w % ethanol for 15 s. The high environmental stability of these VOCs could increase transmission risk and contribute to spread. Additionally, the Omicron variant might have been replaced by the Delta variant due to its increased environmental stability and rapid spread. To prevent VOC spread, it is highly recommended that current infection control practices use disinfectants with appropriate ethanol concentrations.


title: High activity of an affinity-matured ACE2 decoy against Omicron SARS-CoV-2 and pre-emergent coronaviruses

abstract:
The viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and particularly its cell-binding spike protein gene, has undergone rapid evolution during the coronavirus disease 2019 (COVID-19) pandemic. Variants including Omicron now seriously threaten the efficacy of therapeutic monoclonal antibodies and vaccines that target the spike protein. Viral evolution over a much longer timescale has generated a wide range of genetically distinct sarbecoviruses in animal populations, including the pandemic viruses SARS-CoV-2 and SARS-CoV-1. The genetic diversity and widespread zoonotic potential of this group complicates current attempts to develop drugs in preparation for the next sarbecovirus pandemic. Receptor-based decoy inhibitors can target a wide range of viral strains with a common receptor and may have intrinsic resistance to escape mutant generation and antigenic drift. We previously generated an affinity-matured decoy inhibitor based on the receptor target of the SARS-CoV-2 spike protein, angiotensin-converting enzyme 2 (ACE2), and deployed it in an adeno-associated viral vector (rAAV) for intranasal delivery and passive prophylaxis against COVID-19. Here, we demonstrate the exceptional binding and neutralizing potency of this ACE2 decoy against SARS-CoV-2 variants including Omicron, as well as binding to diverse ACE2-dependent coronaviruses. We also discuss a strategy of decoy-based treatment and passive protection to mitigate the ongoing COVID-19 pandemic and future airway virus threats.

Author SummaryViral sequences can change dramatically during pandemics lasting multiple years. Likewise, evolution over centuries has generated genetically diverse virus families posing similar threats to humans. This variation presents a challenge to drug development, in both the breadth of achievable protection against related groups of viruses and the durability of therapeutic agents or vaccines during extended outbreaks. This phenomenon has played out dramatically during the coronavirus disease 2019 (COVID-19) pandemic. The highly divergent Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has upended previous gains won by vaccine and monoclonal antibody development. Moreover, ecological surveys have increasingly revealed a broad class of SARS-CoV-2-like viruses in animals, each poised to cause a future human pandemic. Here, we evaluate an alternative to antibody-based protection and prevention--a decoy molecule based on the SARS-CoV-2 receptor. Our engineered decoy has proven resistant to SARS-CoV-2 evolution during the ongoing COVID-19 pandemic and can neutralize all variants of concern, including Omicron. Furthermore, the decoy binds tightly to a broad class of sarbecoviruses related to pandemic SARS-CoV-2 and SARS-CoV-1, indicating that receptor decoys offer advantages over monoclonal antibodies and may be deployed during the COVID-19 pandemic and future coronavirus outbreaks to prevent and treat severe illness.

category: molecular biology


title: Changes in late frontal event-related potentials to self-produced foreign phonemes correlate with improvements in pronunciation

abstract: The pronunciation of foreign phonemes is assumed to involve auditory feedback control processes that compare vocalized phonemes to target sounds. The electrophysiological correlate of this process is known as the speaking-induced suppression (SIS) of early auditory evoked activity. To gain insight into the neural processes that mediate the learning of foreign phoneme pronunciation, we recorded event-related potentials (ERP) when participants (N=19) pronounced either native or foreign phonemes. Analyses of single-trial ERPs revealed no differences in SIS between foreign and native phonemes in early time-windows (approx. 85-290 ms). In contrast, the amplitude of the fronto-centrally distributed late slow wave (LSW, 320-440 ms) was modulated by the pronunciation of foreign phonemes. Whereas the self-produced native phonemes evoked a constant amplitude LSW, the LSW evoked by self-vocalized foreign phonemes shifted towards more positive amplitudes across the experiment. Importantly, the LSW amplitude correlated positively with the improved pronunciation of the foreign phoneme. These results suggest that the LSW may reflect higher-order internal monitoring processes that signal successful pronunciation and enable adjustments to future vocalization.

category: neuroscience


title: High-Throughput Screening for Myelination Promoting Compounds Using Human Stem Cell-derived Oligodendrocyte Progenitor Cells Identifies Novel Targets

abstract: Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for central nervous system demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination promoting compounds, we generated an advanced, genome engineered, human pluripotent stem cell (hPSC) line that consist of three reporters (identification-and-purification tag, GFP, and secreted NanoLuc) driven by the endogenous PDGFR, PLP1 and MBP genes, respectively. Based upon this line, we established a high-throughput drug screening platform and performed a small molecule screen with 2500 bioactive molecules. In addition to a number of previously known pathways, our screening effort identified new pathways whose inhibition enhance oligodendrocyte maturation and myelination. Although further genetic and molecular validation is required, the identified inhibitors could potentially be repurposed to develop remyelination therapy for MS and other demyelinating disorders.


title: Neural hyperactivity is a core pathophysiological change induced by deletion of an autism risk gene Ash1l in the mouse brain

abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disease associated with various gene mutations. Previous genetic and clinical studies report that mutations of the epigenetic gene ASH1L are highly associated with human ASD and intellectual disability (ID). Recent studies demonstrate that loss of Ash1l in the mouse brain is sufficient to induce ASD/ID-like behavioral and cognitive memory deficits, suggesting that disruptive ASH1L mutations are likely to be the causative driver leading to the ASD/ID pathogenesis in human patients. However, the brain pathophysiological changes underlying the Ash1l-deletion-induced ASD/ID-like behavioral and memory deficits remain unknown. Here we show loss of Ash1l in the mouse brain causes locomotor hyperactivity and higher metabolic rates . In addition, the mutant mice display lower thresholds for the convulsant reagent-induced epilepsy and increased neuronal activities in broad brain areas. Thus, our current study reveals that neural hyperactivity is a core pathophysiological change in the Ash1l-deficient mouse brain, which provides a brain-level basis for further studying the cellular and molecular mechanisms underlying the Ash1l-deletion-induced ASD/ID pathogenesis.


title: SARS-CoV-2 Delta variant induces enhanced pathology and inflammatory responses in K18-hACE2 mice

abstract:
The COVID-19 pandemic has been fueled by novel variants of concern (VOC) that have increased transmissibility, receptor binding affinity, and other properties that enhance disease. The goal of this study is to characterize unique pathogenesis of the Delta VOC strain in the K18-hACE2-mouse challenge model. Challenge studies suggested that the lethal dose of Delta was higher than Alpha or Beta strains. To characterize the differences in the Delta strains pathogenesis, a time-course experiment was performed to evaluate the overall host response to Alpha or Delta variant challenge. qRT-PCR analysis of Alpha- or Delta- challenged mice revealed no significant difference between viral RNA burden in the lung, nasal wash or brain. However, histopathological analysis revealed high lung tissue inflammation and cell infiltration following Delta- but not Alpha-challenge at day 6. Additionally, pro-inflammatory cytokines were highest at day 6 in Delta-challenged mice suggesting enhanced pneumonia. Total RNA-sequencing analysis of lungs comparing infected to uninfected mice revealed that Alpha-challenged mice have more total genes differentially activated, conversely, Delta-challenged mice have a higher magnitude of differential gene expression. Delta-challenged mice have increased interferon-dependent gene expression and IFN-{gamma} production compared to Alpha. Analysis of TCR clonotypes suggested that Delta challenged mice have increased T-cell infiltration compared to Alpha challenged. Our data suggest that Delta has evolved to engage interferon responses in a manner that may enhance pathogenesis. The in vivo and in silico observations of this study underscore the need to conduct experiments with VOC strains to best model COVID-19 when evaluating therapeutics and vaccines.

ImportanceThe Delta variant of SARS-CoV-2 is known to be more transmissible and cause severe disease in human hosts due to mutations in its genome that are divergent from previous variants of concern (VOC). Our study evaluates the pathogenesis of Delta in the K18-hACE2 mouse model compared to the Alpha VOC. We observed that relative to Alpha, Delta challenge results in enhanced inflammation and tissue damage with stronger antiviral responses. These observations provide insight into Deltas unique pathogenesis.

category: immunology


title: Hedgehog regulation of epithelial cell state and morphogenesis in the larynx

abstract: The larynx enables speech while regulating swallowing and respiration. Larynx function hinges on the laryngeal epithelium which originates as part of the anterior foregut and undergoes extensive remodeling to separate from the esophagus and form vocal folds that interface with the adjacent trachea. Here we find that Sonic hedgehog (SHH) is essential for epithelial integrity in the larynx as well as the anterior foregut. During larynx-esophageal separation, low Shh expression marks specific domains of actively remodeling epithelium that undergo an epithelial to mesenchymal transition (EMT) characterized by the induction of N-Cadherin and movement of cells out of the epithelial layer. Consistent with a role for SHH signaling in regulating this process, Shh mutants undergo an abnormal EMT throughout the anterior foregut and larynx, marked by a cadherin switch, movement out of the epithelial layer and cell death. Unexpectedly, Shh mutant epithelial cells are replaced by a new population of Pax-1 expressing cells that form a rudimentary epithelium. These findings have important implications for interpreting the etiology of HH- dependent birth defects within the foregut. We propose that SHH signaling has a default role in maintaining epithelial identity throughout the anterior foregut and that regionalized reductions in SHH trigger epithelial remodeling.

category: developmental biology


title: The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

abstract:
Development of the adrenal cortex, a vital endocrine organ, originates in the adrenogonadal primordium, a common progenitor for both the adrenocortical and gonadal lineages in rodents. In contrast, we find that in humans and cynomolgus monkeys, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to- mesenchymal-like transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages provides a molecular framework for understanding human adrenal and gonadal disorders.

One Sentence SummarySpecification of the adrenal cortex occurs in adrenogenic coelomic epithelium independent of gonadogenesis in humans and cynomolgus monkeys


title: Reconstructing the spatiotemporal patterns of admixture during the European Holocene using a novel genomic dating method

abstract:
Recent studies have shown that gene flow or admixture has been pervasive throughout human history. While several methods exist for dating admixture in contemporary populations, they are not suitable for sparse, low coverage data available from ancient specimens. To overcome this limitation, we developed DATES that leverages ancestry covariance patterns across the genome of a single individual to infer the timing of admixture. By performing simulations, we show that DATES provides reliable results under a range of demographic scenarios and outperforms available methods for ancient DNA applications. We apply DATES to ~1,100 ancient genomes to reconstruct gene flow events during the European Holocene. Present-day Europeans derive ancestry from three distinct groups, local Mesolithic hunter-gatherers, Anatolian farmers, and Yamnaya Steppe pastoralists. These ancestral groups were themselves admixed. By studying the formation of Anatolian farmers, we infer that the gene flow related to Iranian Neolithic farmers occurred no later than 9,600 BCE, predating agriculture in Anatolia. We estimate the early Steppe pastoralist groups genetically formed more than a millennium before the start of steppe pastoralism, providing new insights about the history of proto-Yamnaya cultures and the origin of Indo-European languages. Using ancient genomes across sixteen regions in Europe, we provide a detailed chronology of the Neolithization across Europe that occurred from ~6,400-4,300 BCE. This movement was followed by a rapid spread of steppe ancestry from ~3,200-2,500 BCE. Our analyses highlight the power of genomic dating methods to elucidate the legacy of human migrations, providing insights complementary to archaeological and linguistic evidence.

SignificanceThe European continent was subject to two major migrations during the Holocene: the movement of Near Eastern farmers during the Neolithic and the migration of Steppe pastoralists during the Bronze Age. To understand the timing and dynamics of these movements, we developed DATES that leverages ancestry covariance patterns across the genome of a single individual to infer the timing of admixture. Using ~1,100 ancient genomes spanning ~8,000-350 BCE, we reconstruct the chronology of the formation of the ancestral populations and the fine-scale details of the spread of Neolithic farming and Steppe pastoralist-related ancestry to Europe. Our analysis demonstrates the power of genomic dating methods to provide an independent and complementary timeline of population origins and movements using genetic data.

category: evolutionary biology


title: Elucidating gene expression patterns across multiple biological contexts through a large-scale investigation of transcriptomic datasets

abstract: Distinct gene expression patterns within cells are foundational for the diversity of functions and unique characteristics observed in specific contexts, such as human tissues and cell types. Though some biological processes commonly occur across contexts, by harnessing the vast amounts of available gene expression data, we can decipher the processes that are unique to a specific context. Therefore, with the goal of developing a portrait of context-specific patterns to better elucidate how they govern distinct biological processes, this work presents a large-scale exploration of transcriptomic signatures across three different contexts (i.e., tissues, cell types, and cell lines) by leveraging over 600 gene expression datasets categorized into 98 subcontexts. The strongest pairwise correlations between genes from these subcontexts are used for the construction of co-expression networks. Using a network-based approach, we then pinpoint patterns that are unique and common across these subcontexts. First, we focused on patterns at the level of individual nodes and evaluated their functional roles using a human protein-protein interactome as a referential network. Next, within each context, we systematically overlaid the co-expression networks to identify specific and shared correlations as well as relations already described in scientific literature. Additionally, in a pathway-level analysis, we overlaid node and edge sets from co-expression networks against pathway knowledge to identify biological processes that are related to specific subcontexts or groups of them. Finally, we have released our data and scripts at https://zenodo.org/record/5831786 and https://github.com/ContNeXt/, respectively and developed ContNeXt (https://contnext.scai.fraunhofer.de/), a web application to explore the networks generated in this work.

category: bioinformatics


title: Probing the influence of SMA and vmPFC on the motor system with dual-site transcranial magnetic stimulation

abstract: Dual-site transcranial magnetic stimulation (TMS) has been widely exploited to probe the influence of cortical structures on the primary motor cortex (M1). However, several issues remain open, notably regarding the use of this approach on areas of the medial frontal cortex - including the supplementary motor area (SMA) and the ventromedial prefrontal cortex (vmPFC) - known to play a fundamental role in motor behavior. First, the few TMS studies that have targeted SMA have mostly focused on short inter-stimulation intervals (6-8 ms), supposed to recruit cortico-cortical circuits. There is a current lack of data on the nature of the influence (i.e., facilitatory vs. suppressive) of SMA stimulation on M1 when probed with longer intervals (10-15 ms), thought to recruit more indirect cortico-subcortico-cortical circuits. Second, it is unclear whether the facilitatory influence of SMA stimulation previously reported with short intervals reflects the recruitment of cortico-cortical circuits (as commonly assumed) or results from the summation of volleys descending from SMA and M1 at the spinal level. Third, dual-site TMS has never been used to date to probe the influence of the vmPFC on M1, putatively due to the presumed difficulty of reaching the former area with magnetic fields. Here, we show that SMA stimulation facilitates motor activity with a 12 ms interval. Additionally, our data reveal that the facilitatory influence of SMA stimulation observed with short intervals does not result from spinal interactions. Finally, we show that vmPFC stimulation induces a moderate suppressive effect on M1, both with short and with long inter-stimulation intervals.


title: Seeing sounds: Neural mechanisms underlying auditory contributions to visual detection

abstract: Sounds enhance the detection of visual stimuli while concurrently biasing an observers decisions. To investigate the neural mechanisms that underlie such multisensory interactions, we decoded time-resolved signal detection theory (SDT) sensitivity and criterion parameters from neural activity using magnetoencalography, while participants performed a visual detection task. Using temporal generalization analysis, we found that sounds improve visual detection by enhancing the maintenance of the most informative perceptual samples over time. In parallel, criterion decoding analyses revealed that sounds evoke patterns of activity that resembled the patterns evoked by an actual visual stimulus. These two complementary mechanisms of audiovisual interaction differed in terms of their automaticity: Whereas the sound-induced enhancement in visual information maintenance depended on participants being actively engaged in a detection task, sounds evoked visual activity patterns in the visual cortex in a bottom-up fashion, challenging the classical assumption that sound- induced reductions in criterion correspond to decision-level biases.


title: An excitatory-inhibitory fronto-cerebellar loop resolves the Stroop effect

abstract: The Stroop effect is a well-known behavioral phenomenon in humans that refers to robust interference between language and color information. Although this effect has long been studied, it remains unclear when the interference occurs and how it is resolved in the brain. By manipulating the verbality of stimulus perception and response generation, here we show that the Stroop effect occurs during perception of color-word stimuli and is resolved by a cross-hemispheric, excitatory-inhibitory functional loop involving the lateral prefrontal cortex and cerebellum. Humans performed a Stroop task and a control task in which the stimulus did not contain verbal information, and made a response either vocally or manually. The resolution of Stroop interference involved the lateral prefrontal cortex in the left hemisphere and the cerebellum in the right hemisphere, independently of whether the response was made vocally or manually. In contrast, such cross-hemispheric lateralization was absent during the non-verbal control task. Moreover, the prefrontal cortex amplified cerebellar activity, whereas the cerebellum suppressed prefrontal activity, and these effects were enhanced during interference resolution. These results suggest that this fronto-cerebellar loop involving language and cognitive systems regulates goal-relevant information to resolve the interference occurring during simultaneous perception of a word and color.


title: A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants

abstract: The spike glycoprotein of SARS-CoV-2 engages with human angiotensin-converting enzyme 2 (ACE2) to facilitate infection. Here, we describe an alpaca-derived heavy chain antibody fragment (VHH), saRBD-1, that disrupts this interaction by competitively binding to the spike protein receptor-binding domain. We further generated an engineered bivalent nanobody construct engineered by a flexible linker, and a dimeric Fc conjugated nanobody construct. Both multivalent nanobodies blocked infection at picomolar concentrations and demonstrated no loss of potency against emerging variants of concern including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427/429), and Delta (B.1.617.2). saRBD-1 tolerates elevated temperature, freeze-drying, and nebulization, making it an excellent candidate for further development into a therapeutic approach for COVID-19.


title: SARS-CoV-2 infection results in lasting and systemic perturbations post recovery

abstract: SARS-CoV-2 has been found capable of inducing prolonged pathologies collectively referred to as Long-COVID. To better understand this biology, we compared the short- and long-term systemic responses in the golden hamster following either SARS-CoV-2 or influenza A virus (IAV) infection. While SARS-CoV-2 exceeded IAV in its capacity to cause injury to the lung and kidney, the most significant changes were observed in the olfactory bulb (OB) and olfactory epithelium (OE) where inflammation was visible beyond one month post SARS-CoV-2 infection. Despite a lack of detectable virus, OB/OE demonstrated microglial and T cell activation, proinflammatory cytokine production, and interferon responses that correlated with behavioral changes. These findings could be corroborated through sequencing of individuals who recovered from COVID-19, as sustained inflammation in OB/OE tissue remained evident months beyond disease resolution. These data highlight a molecular mechanism for persistent COVID-19 symptomology and characterize a small animal model to develop future therapeutics.

category: microbiology


title: Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

abstract:
The recognition of microbiome inhabiting the healthy female bladder engendered the need for comprehensive characterization of the female urinary microbiome (FUM) in health and disease. Although previous studies reported FUM composition at different taxonomic levels, progress towards reliable identification at species level is highly required. The aim of this study was to comprehensively characterize bacterial species of FUM of healthy reproductive-age European women by two complementary methodologies i.e., extended culturomics and long-read third generation sequencing of near full-length 16S rRNA gene.

A wide diversity of bacterial species was captured (297 species) with a median of 53 species/sample, including 16 putative uropathogens. Clustering FUM into community structure types revealed high inter-individual differences. Notably, there was not a single species common to all samples, although the Lactobacillus genus was detected in all samples. Lactobacillus crispatus, Lactobacillus iners and Lactobacillus mulieris were observed in high relative abundance in several samples as well as other species (e.g., Streptococcus agalactiae, Atopobium vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while more prevalent species were often low abundant members (e.g., Finegoldia magna). We captured remarkable richness within Corynebacterium spp. (25 species) and Lactobacillaceae (4 genera, 14 species). While amplicon sequencing allowed detection of more anaerobic species (e.g., 11 Peptoniphilus spp.), culturomics enabled the identification of recently recognized Gardnerella species and putative novel Corynebacterium species.

This study provided fine-grained FUM profiling at species level and revealed detailed FUM structure, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders.

IMPORTANCEDespite evidence of the resident microbial community in the female lower urinary tract, bacterial species diversity and abundance in healthy women is still unclear. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported Lactobacillaceae species and putative novel Corynebacterium species. Although some particular species were present in high relative abundance, low-abundant members were more prevalent. FUM classification into community structure types demonstrated high inter-individual differences in urinary microbiome composition among healthy women. We also report moderate correlation between culture-dependent and -independent derived data highlighting drawbacks resulting from each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of healthy FUM required for further exploration of the urinary microbiome role in urinary tract diseases.


title: Doderlin: Isolation and Characterization of a Broad-Spectrum Antimicrobial Peptide from Lactobacillus acidophilus

abstract:
Lactobacillus acidophilus are Gram-positive bacteria distributed in diverse environments, and as being a component of the normal microbiota of gastrointestinal and urogenital tract, being relevant to humans. Classified as lactic acid bacteria, due to the pro-duction of lactic acid, Lactobacillus can also produce antimicrobial peptides (AMPs), which is a compound synthesized by all forms of life aiming for protecting themselves from threats and to increase their competitivity to survive in a specific environment. AMPs are molecules capable of inhibiting the growth of microorganisms and, due to the indiscriminate use of conventional antibiotics and the emergence of multi-resistant bacteria, they have become an alternative, not only for treating multi-resistant infections, but also for probiotic product confection and food conservation. Considering the rampant rise of resistance, the present study aimed to isolate and characterize antimicrobial peptides from Lactobacillus acidophilus extracts. Samples were obtained from Lactobacillus acid extract supernatant which was pre-fractionated on disposable cartridges, followed by a high-performance liquid chromatography (HPLC). The collected fractions were evaluated in a liquid growth inhibition assay where eight fractions antimicrobial activity were obtained. One of them was selected for further characterization by mass spectrometry (MS), due to its antimicrobial activity against Candida albicans and conclusive results in mass spectrometry analysis. This molecule was identified as a peptide having a molecular mass of 1788.01 Da, peptide sequence NEPTHLLKAFSKAGFQ, and named Doderlin. Interestingly, antimicrobial molecules isolated from L. acidophilus have already been described previously, but few reports in the literature describe an AMP effective against C. albicans as reported here. The results obtained suggest that this newly discovered molecule have a biological property with potential to be applied in pharmaceutical and food companies in the fight against contamination and/or for treating infections caused by microorganisms.

IMPORTANCEDoderlin, this newly discovered molecule have a biological property with potential to be applied in pharmaceutical and food companies in the fight against contamination and/or for treating infections caused by microorganisms.


title: Unexpected growth of a classic yeast auxotroph

abstract: In budding yeast, the Met15 enzyme has long been assumed to be the sole homocysteine synthase facilitating de novo synthesis of sulfur-containing organic compounds (organosulfurs) from inorganic precursors. Here we show that an alternative homocysteine synthase encoded by the previously uncharacterized gene YLL058W supports growth of mutants lacking MET15 in the absence of exogenous organosulfurs. This growth is observed specifically when cells are deposited in an automated fashion to seed colonies, but not with traditional cell propagation techniques such as thick patches of cells or liquid cultures. We show that the lack of growth in these contexts, which has historically justified the status of MET15 as a classic auxotrophic marker, is largely due to toxic levels of hydrogen sulfide accumulation rather than an inability to perform de novo homocysteine biosynthesis. These data suggest that cell fate decisions in yeast are influenced by hydrogen sulfide levels in the microenvironment. Our results have broad implications for investigations of sulfur metabolism, including studies of methionine restriction and aging.


title: Decoding the activated stem cell phenotype of the vividly maturing neonatal pituitary

abstract: The pituitary represents the endocrine master regulator. In mouse, the gland undergoes vivid maturation immediately after birth. Here, we in detail portrayed the stem cell compartment of neonatal pituitary. Single-cell RNA-sequencing pictured an active gland, revealing proliferative stem as well as hormonal (progenitor) cell populations. The stem cell pool displayed a hybrid epithelial/mesenchymal phenotype, characteristic of development-involved tissue stem cells. Organoid culturing recapitulated the stem cells phenotype, interestingly also reproducing their paracrine activity. The pituitary stem cell-activating interleukin-6 (IL-6) advanced organoid growth, although the neonatal stem cell compartment was not visibly affected in IL-6-/- mice, likely due to cytokine family redundancy. Further transcriptomic analysis exposed a pronounced WNT pathway in the neonatal gland, shown to be involved in stem cell activation and to overlap with the (fetal) human pituitary transcriptome. Following local damage, the neonatal gland efficiently regenerates, despite absence of additional stem cell proliferation, or upregulated IL-6 or WNT expression, all in line with the already high stem cell activation status, thereby exposing striking differences with adult pituitary. Together, our study decodes the stem cell compartment of neonatal pituitary, exposing an activated state in the vividly maturing gland. Understanding stem cell activation is key to potential pituitary regenerative prospects.


title: Tactical signalling by victims increases bystander consolation in bonobos

abstract: Tactical emotion communication has long been considered uniquely-human. As a species, we readily exaggerate, inhibit and modify emotional expressions according to social context and audience. Notably, emitting emotional displays, such as those pertaining to distress states, can evoke empathic responses in others such as the offering of consolation to victims after a fight. Animal emotion expressions, by contrast, are traditionally viewed as uncontrollable arousal responses. Our study challenges this view by assessing the level of control in the emotional signalling of sanctuary-living bonobo victims following aggressive attacks (N = 27 victims, N = 144 attacks) and its and its corresponding effect on receivers. Results show that the production of paedomorphic signals by adult bonobo victims increased chances of receiving consolation from bystanders and reduced risk of future aggression from former opponents, highlighting a strategic function. Victim signalling also increased with audience size, yet strategies differed by age: immature bonobos were more likely to cease signalling in proximity of close-social partners, whereas adults were more likely to cease signalling after having been consoled. These data suggest that bonobo emotion communication has a developmental trajectory and that tactical emotion signalling is a Pan-human capacity, preceding the split of Homo.

category: animal behavior and cognition


title: Dopamine ameliorates hyperglycemic memory-induced vascular dysfunction in diabetic retinopathy

abstract: Dopamine is a neurotransmitter that mediates visual function in the retina, and hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic retinopathy (DR); however, the role of dopamine in HGM-induced retinal vascular dysfunction remains unclear. Here, we report a mechanism of HGM-induced retinal vascular dysfunction and the protective effect of dopamine against the HGM-induced DR. HGM induces retinal vascular lesions through persistent oxidative stress, mitochondrial dysfunction, and microvascular abnormalities after blood glucose normalization, and dopamine ameliorates this HGM-induced retinopathy. HGM induced persistent oxidative stress, mitochondrial membrane potential collapse and fission, and adherens junction disassembly and subsequent vascular leakage in the mouse retinas. These persistent hyperglycemic stresses were inhibited by dopamine treatment in human retinal endothelial cells and by intravitreal injection of levodopa in the retinas of HGM mice. Our findings suggest that dopamine alleviates HGM-induced retinal vascular dysfunction by inhibiting persistent mitochondrial dysfunction and microvascular abnormalities.

category: biochemistry


title: Broad-host-range mutagenesis with CRISPR-associated transposase

abstract:
Transposons have been instrumental tools in microbiology enabling random mutagenesis, with transposons like Tn5 and Mariner, and site-specific DNA integrations with Tn7. However, programmable targeting of transposons was impossible until CRISPR-associated transposase (CasTn) systems were described. Like other CRISPR-derived systems, CasTn can be programmed with a short DNA encoded sequence that is transcribed into a guide-RNA. Here we describe a broad-host-range CasTn system and demonstrate its function in bacteria from three classes of the Proteobacteria. The CasTn genes are expressed from a broad-host-range replicative plasmid, while the guide-RNA and transposon are provided on a high-copy pUC plasmid that is suicidal in most bacteria outside of E. coli. Using our CasTn system, single-gene disruptions were performed with on-target efficiencies approaching 100% in the Beta- and Gammaproteobacteria, Burkholderia thailandensis, and Pseudomonas putida, respectively. The results were more modest in the Alphaproteobacterium Agrobacterium fabrum, with a peak efficiency of 45%, though for routine single-gene disruptions, this efficiency is adequate. In B. thailandensis, the system allowed simultaneous co-integration of transposons at two different target sites. The CasTn system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in P. putida. Given the iterative capabilities and large payload size, this system will be helpful for genome engineering experiments across several fields of research.

SignificanceThe genetic modification of bacteria to disrupt native genes and integrate recombinant genes is necessary for basic and applied research. Traditional methods for targeted disruptions and insertions are often cumbersome and inefficient, limiting experiments' scale and throughput. This work developed a system for targeted transposon mutagenesis that is easy to use, iterative, and efficient. We demonstrate that the system functions across three different classes of the Proteobacteria in species widely used in research and biotechnology. Moreover, the framework of the system and accompanying plasmids that we developed will facilitate porting the system to other bacteria. Our system provides a fast and efficient protocol to genetically modify these bacteria by inserting desired genetic cargo into specific genomic targets.

category: synthetic biology


title: NRP1 and furin as putative mediators of SARS-CoV-2 entry into human brain cells

abstract:
COVID-19 has prominent neurological manifestations including psychiatric symptoms, indicating significant synaptic pathology. Surprisingly, existing evidence suggests negligible expression of the key SARS-CoV-2 host cell entry mediators ACE2 and TMPRSS2 in human brain, which complicates understanding of the pathomechanisms of the neuropsychiatric manifestations in COVID-19. Recent studies suggested that an alternative host-cell entry receptor, NRP1, can mediate entry of furin cleaved SARS-CoV-2 spike proteins into the host cells. However, the role of NRP1 and furin in mediating SARS-CoV-2 entry in human brain cells has been least explored and remains a lacuna in the literature. We performed an in silico analysis of the transcriptomic and proteomic expressions of SARS-CoV-2 host-cell entry receptors and associated tissue proteases in human brain tissue, using the publically available databases. Based on the expression analysis, SARS-CoV-2 entry in human brain cells is likely to be mediated through NRP1 and furin.

Graphical Abstract

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title: Molecular basis of broad neutralization against SARS-CoV-2 variants including Omicron by a human antibody

abstract:
Omicron, a newly emerging SARS-CoV-2 variant, carried a large number of mutations in the spike protein leading to an unprecedented evasion from many neutralizing antibodies (nAbs). Here, we performed a head-to-head comparison of Omicron with other existing highly evasive variants in terms of their reduced sensitivities to antibodies, and found that Omicron variant is significantly more evasive than Beta and Mu variants. Of note, some key mutations occur in the conserved epitopes identified previously, especially in the binding sites of Class 4 nAbs, contributing to the increased Ab evasion. We also reported a broadly nAb (bnAb), VacW-209, which effectively neutralized all tested SARS-CoV-2 variants and even SARS-CoV. Finally, we determined six cryo-electron microscopy structures of VacW-209 complexed with the spike ectodomains of wild-type, Delta, Mu, C.1.2, Omicron, and SARS-CoV, and revealed the molecular basis of the broadly neutralizing activities of VacW-209 against SARS-CoV-2 variants. Overall, Omicron has once again raised the alarm over virus variation with significantly compromised neutralization. BnAbs targeting more conserved epitopes among variants will continue to play a key role in pandemic control and prevention.

One sentence summaryStructural and functional analyses reveal that a human antibody named VacW-209 confers broad neutralization against SARS-CoV-2 variants including Omicron by recognizing a highly conserved epitope.


title: Enterococcus faecalis antagonizes Pseudomonas aeruginosa growth in polymicrobial biofilms

abstract:
Enterococcus faecalis is often co-isolated with Pseudomonas aeruginosa in polymicrobial biofilm-associated infections of wounds and the urinary tract. As a defense strategy, the host innately restricts iron availability at infection sites. Despite their co-prevalence, the polymicrobial interactions of these two pathogens in iron- restricted conditions, such as those found in the host, remains unexplored. Here we show that E. faecalis inhibits P. aeruginosa growth within biofilms when iron is restricted. E. faecalis lactate dehydrogenase (ldh1) gives rise to L-lactate production during fermentative growth. We find that E. faecalis ldh1 mutant fails to inhibit P. aeruginosa growth. Additionally, we demonstrate that ldh1 expression is induced in iron-restricted conditions, resulting in increased lactic acid exported and consequently, a reduction in pH. Together, our results suggest that E. faecalis synergistically inhibit P. aeruginosa growth by decreasing environmental pH and L-lactate-mediated iron chelation. Overall, this study highlights that the microenvironment in which the infection occurs is important for understanding its pathophysiology.

IMPORTANCEMany infections are polymicrobial and biofilm-associated in nature. Iron is essential for many metabolic processes and plays an important role in controlling infections, where the host restricts iron as a defense mechanism against invading pathogens. However, polymicrobial interactions between pathogens are underexplored in iron- restricted conditions. Here, we explore the polymicrobial interactions between commonly co-isolated E. faecalis and P. aeruginosa within biofilms. We find that E. faecalis modulates the microenvironment by exporting lactic acid which further chelates already limited iron, and also lowers the environmental pH to antagonize P. aeruginosa growth in iron-restricted conditions. Our findings provide insights into polymicrobial interactions between pathogens in an infection-relevant condition and how manipulating the microenvironment can be taken advantage of to better control infections.


title: Novel Internalin P homologs in Listeria

abstract:
Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis in immunocompromised individuals, particularly pregnant women. Several virulence factors support the intracellular lifecycle of Lm and facilitate cell-to-cell spread, allowing it to occupy multiple niches within the host and cross protective barriers, including the placenta. One family of virulence factors, internalins, contributes to Lm pathogenicity by inducing specific uptake and conferring tissue tropism. Over 25 internalins have been identified thus far, but only a few have been extensively studied. Internalins contain leucine-rich repeat (LRR) domains which enable protein-protein interactions, allowing Lm to bind host proteins. Notably, other Listeria species express internalins but cannot colonize human hosts, prompting questions regarding the evolution of internalins within the genus Listeria. Internalin P (InlP) promotes placental colonization through interaction with the host protein afadin. Though prior studies of InlP have begun to elucidate its role in Lm pathogenesis, there remains a lack of information regarding homologs in other Listeria species. Here, we have used a computational evolutionary approach to identify InlP homologs in additional Listeria species. We found that L. ivanovii londoniensis (Liv) and L. seeligeri (Ls) encode InlP homologs. We also found InlP-like homologs in L. innocua and the recently identified species L. costaricensis. All newly identified homologs lack the full-length LRR6 and LRR7 domains found in Lms InlP. These findings inform on the evolution of one key Lm virulence factor, InlP, and serve as a springboard for future evolutionary studies of Lm pathogenesis as well as mechanistic studies of Listeria internalins.

Impact StatementThe intracellular bacterial pathogen Listeria monocytogenes can breach protective barriers in the pregnant host, allowing for the colonization of the placenta in pregnant women and resulting in numerous adverse pregnancy outcomes. Previous studies aimed at delineating the mechanisms behind placental colonization of L. monocytogenes identified a key virulence factor, internalin P (InlP). The internalin family of proteins has been studied extensively due to their conservation in the Listeria genus and their contribution to virulence and pathogenicity in L. monocytogenes. Still, many questions remain regarding the evolution of internalins and their potential roles in non-pathogenic Listeria. Our work addresses this gap in knowledge by 1) identifying additional InlP homologs in Listeria, including L. ivanovii, L. seeligeri, L. innocua, and L. costaricensis, and 2) characterizing these homologs using computational evolutionary methods to compare their primary sequences, domain architectures, and structural models. Together, our findings contribute to the field by providing insights into the evolution of one key member of the internalin family as well as serving as a catalyst for future studies of InlP and its role in Listeria pathogenesis.


title: Ex Vivo Validation of Six FDA-Approved Non-Receptor Tyrosine Kinase Inhibitors (NRTKIs) as Antivirals to Pandemic and Seasonal Influenza A Viruses

abstract: Influenza viruses are important respiratory pathogens that cause substantial morbidity and mortality annually. In addition to seasonal influenza outbreaks, new emerging influenza A viruses (IAV) can cause pandemic influenza outbreaks. Apart from effective vaccines, there is a need for better treatment options to combat infections with these viruses when vaccines are not available or show reduced efficacy (e.g., in immmunocompromised patients). The limited range of licensed antiviral drugs and emergence of drug-resistance mutations highlight the need for novel intervention strategies like host-targeted antivirals. Repurposing FDA-approved kinase inhibitors may offer a fast-track for a new generation of host-targeted antivirals. Small molecule kinase inhibitors (SMKIs) can inhibit replication of viruses and improve survival in vivo; however, no SMKI has been approved for clinical use against IAV infections. In the present study, we tested eight non-receptor tyrosine kinase-inhibitors (NRTKIs) used to treat cancer and autoimmune diseases for their antiviral potential. Six of those potently inhibited virus replication ([≥]1,000-fold) in A549 cells infected with either A(H1N1)pdm09 or seasonal A(H3N2) strains. These compounds were validated in a biologically relevant ex vivo model of human precision-cut lung slices (hPCLS) to provide proof of principle and show efficacy against contemporary seasonal and pandemic IAVs. We identified the steps of the virus infection cycle affected by these inhibitors and assessed the effect of these NRTKIs on the host response. Considering their established safety profiles, our studies show that the use of these NRTKI shows promise and warrants further development as an alternative strategy to treat influenza virus infections.


title: Inflammation in the COVID-19 airway is due to inhibition of CFTR signaling by the SARS-CoV-2 Spike protein

abstract:
BackgroundSARS-CoV-2-contributes to sickness and death in COVID-19 patients partly by inducing a hyper-proinflammatory immune response in the host airway. This hyper-proinflammatory state involves activation of signaling by NF{kappa}B and ENaC, and expression of high levels of cytokines and chemokines. Post-infection inflammation may contribute to "Long COVID", and there are also other long term consequences of acute severe COVID-19, which double or triple the chances of dying from any cause within a year. Enhanced signaling by NF{kappa}B and ENaC also marks the airway of patients suffering from cystic fibrosis, a lethal proinflammatory genetic disease due to inactivating mutations in the CFTR gene. We therefore hypothesized that inflammation in the COVID-19 airway might similarly be due to inhibition of CFTR signaling by SARS-CoV-2 Spike protein.

MethodsThis hypothesis was tested using the hTERT-transformed BCi-NS1.1 basal stem cell, previously derived from small airway epithelia, which were differentiated into a model of small airway epithelia on an air-liquid-interface (ALI). Cyclic AMP-activated CFTR chloride channel activity was measured using an Ussing Chamber. Cell surface-CFTR was labeled with the impermeant biotin method.

ResultsExposure of differentiated airway epithelia to SARS-CoV-2 Spike protein resulted in loss of CFTR protein expression. As hypothesized, TNF/NF{kappa}B signaling was activated, based on increased protein expression of TRADD, the first intracellular adaptor for the TNF/TNFR1 complex, TNFR1, the TNF receptor, phosphorylated I{kappa}B, and the chemokine IL8. ENaC activity was also activated, based on specific changes in molecular weights for  and {gamma} ENaC. Exposure of the epithelia to viral Spike protein suppressed cAMP-activated CFTR chloride channel activity. However, addition of 30 nM concentrations of cardiac glycoside drugs ouabain, digitoxin and digoxin, prevented loss of channel activity. ACE2 and CFTR were found to co-immunoprecipitate (co-IP) in both basal cells and epithelia, suggesting that the mechanism for Spike-dependent CFTR loss might involve ACE2 as a bridge between Spike and CFTR. In addition, Spike exposure to the epithelia resulted in failure of endosomal recycling to return CFTR to the plasma membrane, suggesting that failure of CFTR recovery from endosomal recycling might be a mechanism for Spike-dependent loss of CFTR.

ConclusionBased on experiments with this model of small airway epithelia, we predict that inflammation in the COVID-19 airway may be mediated by inhibition of CFTR signaling by SARS-CoV-2 Spike protein, thus inducing a CFTR-null, cystic fibrosis-like clinical phenotype. Descriptions of COVID-19 in CF carriers with only one copy of wildtype CFTR suggest that this model-based conclusion might be consistent with patient-based experience.


title: Environmental and taxonomic drivers of bacterial extracellular vesicle production in marine ecosystems

abstract:
Extracellular vesicles are small ([~]50-250 nm diameter) membrane-bound structures released by cells into their surrounding environment. Vesicles are abundant in the global oceans and likely play a number of ecological roles in these microbially dominated ecosystems, yet we know nothing about what influences their production and distributions. Here we examine how vesicle production varies among different strains of cultivated marine microbes and explore the degree to which this is influenced by some key environmental variables. We show that vesicle production rates - the number of vesicles produced per cell per generation - vary across an order of magnitude in cultures of marine Proteobacteria, Cyanobacteria, and Bacteroidetes. Vesicle production rates further differ among strains of the cyanobacterium Prochlorococcus, and vary across temperature and light gradients. These data suggest that both community composition and local environmental conditions modulate the production and standing stock of vesicles in the oceans. Examining samples from the oligotrophic North Pacific Gyre, we show depth-dependent changes in the abundance of vesicle-like particles in the upper water column in a manner broadly consistent with culture observations: highest vesicle abundances are found near the surface, where light irradiances and temperatures are greatest, and then decrease with depth. This work represents the beginnings of a quantitative framework for describing extracellular vesicle dynamics in the oceans - essential as we begin to incorporate vesicles into our ecological and biogeochemical understanding of marine ecosystems.

ImportanceBacteria secrete extracellular vesicles containing a wide variety of cellular compounds, including lipids, proteins, nucleic acids, and small molecules, into their surrounding environment. These structures are found in diverse microbial habitats, including the oceans, where their distributions vary throughout the water column. Differences in vesicle abundances likely affect their functional impacts within microbial ecosystems, but the factors influencing vesicle distributions in the environment remain poorly understood. Using quantitative analysis of marine microbial cultures, we show that bacterial vesicle production in the oceans is shaped by a combination of biotic and abiotic factors. Our data indicate that different marine taxa release vesicles at rates varying across an order of magnitude, and that vesicle production can change dynamically as a function of environmental conditions. Taken together with direct measurements of vesicle concentrations in the oceans, these culture-based measurements further provide a window into estimating vesicle loss rates. These findings represent a step forward in our understanding of marine vesicle distributions and provide a basis for quantitatively exploring vesicle dynamics in natural ecosystems.


title: Metagenomics revealing molecular profiling of microbial community structure and metabolic capacity in Bamucuo, Tibet

abstract:
We performed a survey of the microorganisms in Bamucuo, Tibet, resulting in 160,212 (soil) and 135,994 (water) contigs by shotgun metagenomic methods. We discovered 74 new bacterial species and reconstructed their draft genomes, which were obtained from the 75 reconstructed almost complete metagenomic assembly genomes (MAG) in the soil and water samples. Proteobacteria and Actinobacteria were found to be the most dominant bacterial phyla, while Euryarchaeota was the most dominant archaeal phylum. To our surprise, Pandoravirus salinus was found in the soil microbial community. We concluded that the microorganisms in Bamucuo fix carbon mainly through the 3-hydroxypropionic bi-cycle pathway.

IMPORTANCEThe Qinghai-Tibet Plateau (QTP) is the highest plateau in the world, and the microorganisms there play vital ecological roles in the global biogeochemical cycle; however, detailed information on the microbial communities in QTP is still lacking, especially in high altitude areas above 4500 meters. This study, for the first time, characterized the microbial community composition and metabolic capacity in QTP high-altitude areas (with an altitude of 4,555 meters), confirmed that QTP is a huge and valuable resource bank in which more new non-resistant antibiotics and many other bioactive substances could be developed. In addition, the discovery of Pandoravirus salinus in the soil provides important information for further exploring this unique microorganism, and many draft genomes and the genome annotation information obtained in this study have laid the foundation for further in-depth study of the microbial ecology in Qinghai-Tibet Plateau.


title: SARS-CoV-2 Omicron efficiently infects human airway, but not alveolar epithelium

abstract: In late 2021, the highly mutated SARS-CoV-2 Omicron variant emerged, raising concerns about its potential extensive immune evasion, increased transmissibility and pathogenicity. Here, we used organoids of the human airways and alveoli to investigate Omicrons fitness and replicative potential in comparison with earlier SARS-CoV-2 variants. We report that Omicron replicates more rapidly in the airways and has an increased fitness compared to the early 614G variant and Delta. In contrast, Omicron did not replicate productively in human alveolar type 2 cells. Mechanistically, we show that Omicron does not efficiently use TMPRSS2 for entry or spread through cell-cell fusion. Altogether, our data show that Omicron has an altered tropism and protease usage, potentially explaining its higher transmissibility and decreased pathogenicity.


title: Genomic diversity of Helicobacter pylori populations from different regions of the human stomach.

abstract:
Individuals infected with Helicobacter pylori harbour unique and diverse populations of quasispecies, but diversity between and within different regions of the human stomach and the process of bacterial adaptation to each location are not yet well understood.

We applied whole-genome deep sequencing to characterise the within- and between- stomach region genetic diversity of H. pylori populations from paired antrum and corpus biopsies of 15 patients, along with single biopsies from one region of 3 patients, by scanning allelic diversity. We combined population deep sequencing with more conventional sequencing of multiple H. pylori single colony isolates from individual biopsies to generate a unique dataset. Single colony isolates were used to validate the scanning allelic diversity pipelines.

We detected extensive population allelic diversity within the different regions of each patients stomach. Diversity was most commonly found within non-coding, hypothetical, outer membrane, restriction modification system, virulence, lipopolysaccharide biosynthesis, efflux systems and chemotaxis-associated genes. Antrum and corpus populations from the same patient grouped together phylogenetically, indicating that most patients were initially infected with a single strain, which then diversified. Single colonies from the antrum and corpus of the same patients grouped into distinct clades, suggesting mechanisms for within-location adaptation across multiple H. pylori isolates from different patients. Recombination was observed both within and between different regions of the same stomach.

The comparisons made available by combined sequencing and analysis of isolates and populations enabled comprehensive analysis of the genetic changes associated with H. pylori diversification and stomach region adaptation.


title: Identification of a biosynthetic gene cluster encoding a novel lanthanide chelator in Methylorubrum extorquens AM1

abstract: While lanthanide-dependent metabolism is widespread in nature and has been proven to drive one-carbon metabolism in bacteria, details about the machinery necessary to sense, sequester, and traffic lanthanides (Ln) remain unknown. This gap in knowledge is in part because nearly all bacterial growth studies with Ln to date have used soluble chloride salts, compounds that do not reflect the insoluble Ln sources common in the natural environment. Here, we describe the changes in the metabolic machinery of Methylorubrum extorquens AM1 in response to poorly soluble Nd2O3, including 4-fold increases in orphan pqqA genes and the Ln-dependent ADHs xoxF1 and exaF compared to growth with soluble NdCl3. We report the first description of a Ln-chelator biosynthetic gene cluster, encoded by META1p4129 through META1p4138 that we named the Lanthanide Chelation Cluster (LCC). The LCC encodes a TonB dependent receptor and NRPS biosynthetic enzymes and is predicted to produce a metal-chelating molecule. As some LCC protein sequences share similarity to biosynthetic enzymes producing the Fe-chelating siderophore aerobactin, the capacity of aerobactin for binding Ln was tested. It was found that while aerobactin can bind lanthanum (La), neodymium (Nd) and lutetium (Lu) at physiological pH, providing only exogenous aerobactin did not affect growth rate or yield. The LCC was highly upregulated when M. extorquens AM1 was grown using Nd2O3 and expression in trans enabled an increase of Nd bioaccumulation by over 50%. Expression of the LCC in trans did not affect iron bioaccumulation, providing further evidence that its product is a novel Ln-chelator. Finally, expression of the LCC in trans increased Nd, dysprosium (Dy), and praseodymium (Pr) bioaccumulation from the complex Ln source NdFeB magnet swarf by over 60%, opening new strategies for sustainable recovery of these critical Rare Earth Elements.


title: Low diversity and instability of the sinus microbiota over time in adults with cystic fibrosis

abstract:
BackgroundChronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). There are currently no standard of care guidelines for the management of CF CRS, but treatment of upper airway disease may ameliorate lower airway disease. We sought to inform future treatment guidelines by determining whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation.

MethodsWe performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS at the University of Pittsburghs CF Sinus Clinic. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation.

FindingsSinus community diversity was low and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and Staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased IL-1{beta}). Increased IL-1{beta} was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics.

InterpretationTo our knowledge, this is the largest longitudinal study of microbial communities and cytokine secretion in CF CRS. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease.

FundingCystic Fibrosis Foundation and US National Institutes of Health.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSA search of the PubMed database on October 11, 2021 with the terms [cystic fibrosis sinus microbiome] yielded 16 results, and we have identified seven primary research articles on the CF CRS microbiome (including re-analyses of existing datasets). Most are cross-sectional cohort analyses, along with one prior longitudinal study of four adults at the University of Auckland, New Zealand. Together, these prior studies reveal similarities between CF CRS and CF sputum microbiomes, including low community diversity associated with sinus disease, the presence of common CF-associated microbes in the sinuses, and prevalence of sinus communities dominated by P. aeruginosa or Staphylococcus aureus. High levels of IL-1{beta} are linked to the presence of nasal polyps in CF CRS, and polymorphisms in the IL-1 receptor antagonist gene are associated with risk of CRS outside of the context of CF. Two prior studies of this cohort have been performed by our laboratory. One describes clinical indicators of CF sinus disease and the other links sinus infection biogeography to P. aeruginosa evolutionary genomics.

Added value of this studyOur study is the first to examine longitudinal relationships between the host immune response (through cytokine profiling) and microbiota dynamics in CF CRS, including linking elevated IL-1{beta} to worse sinus disease through reduced sinus microbial community diversity. The longitudinal nature of our study also allowed us to uncover striking temporal instability of microbial communities in approximately half of our cohorts sinus microbial communities over two years, including switching between communities dominated by P. aeruginosa and Staphylococcus spp. This instability could hinder attempts to link the relative abundance of taxa to clinical outcomes of interest in cross-sectional studies (e.g., markers of disease progression). We also identified patterns of synergy and antagonism between specific taxa, and impacts of the host immune response in the sinuses on community composition.

Implications of all the available evidenceTogether with prior CF CRS microbiome studies, our study underscores similarities between sinus and lower respiratory tract microbial community structure in CF, and we show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1{beta} in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1{beta}. Finally, the widespread use of highly effective CFTR modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.


title: Proton-pumping rhodopsins in marine diatoms

abstract: Diatoms are a major phytoplankton group responsible for about 20% of Earths primary production. They carry out photosynthesis inside the plastid, an organelle obtained through eukaryote-eukaryote endosymbiosis. Recently, microbial rhodopsin, a photoreceptor distinct from chlorophyll-based photosystems, has been identified in certain diatoms. However, the physiological function of diatom rhodopsin is not well understood. Here we show that the diatom rhodopsin acts as a light-driven proton pump and localizes to the outermost membrane of the four membrane-bound complex plastids. Heterologous expression techniques were used to investigate the protein function and subcellular localization of diatom rhodopsin. Using model simulations, we further evaluated the physiological role of the acidic pool in the plastid produced by proton-transporting rhodopsin. Our results propose that the rhodopsin-derived acidic pool may be involved in a photosynthetic CO2-concentrating mechanism and assist CO2 fixation in diatom cells.


title: RNF213 reshapes vascular transcriptome and epigenome; A potential role of Alternative splicing in Moyamoya pathology

abstract:
RationaleMoyamoya disease (MMD) is a rare cerebrovascular occlusive disease that affects Asian population more often. The pathogenesis of MMD is related to mutation in RNF213 gene. However, why, and how RNF213 mutation leads to MMD is still not fully understood.

ObjectiveAnalyze the impact of RNF213 loss of function on vascular cells and the observed changes correlate with MMD.

Methods and resultsRNF213 KD was conducted in HUVEC and vascular smooth muscle cells (vSMCs). First, HUVEC cells showed alteration of angiogenesis, migration under LPS stimulation, Leukocyte endothelial transmigration, and endothelial-to-vSMCs communication.

Transcriptome analysis revealed downregulation of genes regulating cell division and mitosis. Interestingly, Alternative splicing (AS) analysis revealed hundreds of AS events to occur after RNF213 KD in various types of AS. The alternatively spliced genes showed minimal overlap with transcriptome profiling. Pathway analysis revealed many processes and pathways to be regulated by AS events observed. Transcriptome profiling was also performed after LPS treatment and revealed the basis for increased sensitivity to LPS observed in our analysis. AS changes were also observed after LPS treatment in RNF213 KD HUVEC. Different types of AS showed different patterns of convergence or divergence in terms of the regulated pathways after LPS treatment. Finally, transcriptome and AS analysis was performed in vSMCs and showed various processes that impact vSMCs function and phenotype in RNF213 loss of function.

ConclusionOur data provide a wealth of information on RNF213 gene function in vascular tissues and shed important light on different processes that contribute to MMD pathogenesis. Our results signify an immune-driven and hemodynamically linked process of MMD initiation and progression.


title: RNF213 loss of function reshapes vascular transcriptome and spliceosome leading to disrupted angiogenesis and aggravated vascular inflammatory responses.


title: Linking Dynamic DNA Secondary Structures to Genome Instability

abstract: Genomic double-stranded DNA (dsDNA) becomes single-stranded (ssDNA) during replication, transcription, and DNA repair. ssDNA is therefore believed to be transient, occurring in only a fraction of the genome at a given time, and variable amongst a population of cells. These transiently formed ssDNA segments can also adopt alternative, dynamic DNA conformations, such as cruciform DNA, triplexes, quadruplexes and others. To determine whether there are stable and conserved regions of ssDNA, we utilized our previously developed method S1-END-seq 1 to convert ssDNA to DNA double strand breaks (DSBs), which are then processed for high-throughput sequencing. This approach revealed two predominant dynamic DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulated uniquely in microsatellite unstable human cancer cell lines, and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine (hPu/hPy) mirror repeats common across a variety of human cell lines. Triplex-forming repeats accumulated during replication, blocked DNA synthesis and were hotspots of somatic mutation. In contrast, pathologically expanded (hPu/hPy) repeats in Friedreichs ataxia patient cells formed a replication-independent and transcription-inducible DNA secondary structure. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.


title: Structural insights into G protein activation by D1 dopamine receptor

abstract: G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors and are the most important drug targets. An agonist-bound GPCR engages heterotrimeric G proteins and triggers the exchange of GDP with GTP to promote G proteins activation. A complete understanding of the molecular mechanisms of G proteins activation has been hindered by a lack of structural information of GPCR-G protein complex in nucleotide-bound states. Here, we present the cryoelectron microscopy (cryo-EM) structures of D1 dopamine receptor (D1R)-Gs in the nucleotide-free state, the GDP-bound state and the GTP-bound state with endogenous ligand dopamine. These structures reveal important conformational changes accounting for the release of GDP and the GTP-dependent dissociation of G from G{beta}{gamma} subunits. Combining mutagenesis functional studies, we also identified an important sequence motif in D1R that determines its G protein selectivity. Taken together, these results shed light into the molecular basis of G protein selectivity and the entire molecular signaling events of GPCR-mediated G protein activation.


title: CDC50 orthologues in Plasmodium falciparum have distinct roles in merozoite egress and trophozoite maturation

abstract: In model organisms P4-ATPases require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite, P. falciparum, guanylyl cyclase alpha (GC) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GC is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (denoted A, B and C) encoded by P. falciparum are required for GC function. Using gene tagging and conditional gene disruption, we demonstrate that both CDC50B and CDC50C are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GC whereas CDC50C is the binding partner of another putative P4-ATPase, ATP2. Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GC for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell haemoglobin and consequently hemozoin formation.


title: Merkel cell polyomavirus regulates miR183 cluster and piR62011 in Merkel cell carcinoma

abstract: Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer, a major subset of which is caused by the clonal integration of Merkel Cell Polyomavirus (MCV). Recent studies by Cheng et al. (2017) reported that virus-derived small T antigen protein-bound EP400 complex drives expression of genes essential for cellular transformation. On close analysis of their ChIP-Seq data, we uncovered that the complex binds to the promoter region of the microRNA-183 cluster. The miRNA183 cluster is a cluster of 3 miRNAs (miR183, 182 & 96) expressed and regulated together. These miRNAs are conserved across species, highly expressed in human embryonic stem cells and necessary for sensory/ mechanosensory organ development. We hypothesized that the MCV oncoproteins regulate host miRNA expression directly; an interaction novel in polyomaviruses. We tested miRNA expression via qPCR in both virus positive and negative MCC cell lines and found the former showed a much higher level. Further, fibroblasts expressing T antigens displayed an increase in miR182 expression in comparison to control. Knock-down of T antigens in MCC cells correspondingly decreased miR182 levels. To investigate its regulation we performed luciferase assays for the miRNA predicted promoter that showed increased activity in the presence of T antigens. Intriguingly, the seed sequence of miR182 completely matches to a piRNA called piR62011. Upon reanalysis of a MCC small RNA library, piR62011 emerged as the highest expressed. We found it expressed in MKL-1, a MCV positive cell line as well. Finally, to translate our findings into therapy for MCC, we screened small molecule (CMBL) library by performing surface plasmon resonance (SPR) assay and identified small molecules that binds to pre-miRNA182 and are testing them for their activity to kill MCC.


title: Lamin A/C phosphorylation at serine 22 is a conserved heat shock response to regulate nuclear adaptation during stress

abstract: The heat shock (HS) response is crucial for cell survival in harmful environments. Nuclear lamin A/C, encoded by LMNA gene, has been shown to contribute towards altered gene expression during heat shock, but the underlying mechanisms are poorly understood. Here we show that reversible lamin A/C phosphorylation at Ser22 upon HS is an evolutionary conserved stress response that is triggered in concert with HSF1 activation in human and mouse cells and can also be observed in D. melanogaster in vivo. Consequently, the phosphorylation increase facilitated nucleoplasmic localization of lamin A/C and nuclear rounding in response to HS. The importance of lamin phosphorylation equilibria in HS was confirmed by lamin A/C knock-out (KO) cells that showed deformed nuclei after HS and were rescued by ectopic expression of wild-type, but not by a phosphomimetic (S22D) lamin A mutant. Furthermore, HS triggered release of lamina-associated protein 2 (Lap2) from its association with lamin A/C and concurrently its downregulation, a response that was perturbed in lamin A/C KO cells and in LMNA mutant patient fibroblasts. The abrogated Lap2 response resulted in impaired cell cycle arrest under HS and compromised survival at the recovery. Taken together, our results suggest that the altered phosphorylation stoichiometry of lamin A/C provides an evolutionary conserved mechanism to regulate lamin structure and serve nuclear adaptation and cell survival during HS.


title: HuR-dependent expression of Wisp1 is necessary for TGF????-induced cardiac myofibroblast activity

abstract:
Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGF{beta}-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGF{beta}-induced myofibroblast activation upon HuR inhibition. To delineate HuR-dependent mechanisms, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify eleven mRNA transcripts that showed enriched HuR binding following TGF{beta} treatment as well as significant co-expression correlation with HuR, SMA, and periostin using single-cell RNA-sequencing from ischemic-zone isolated fibroblasts. Of these, Wnt1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 is able to partially rescue myofibroblast contractile function following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGF{beta}. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGF{beta}, in part through post-transcriptional regulation of Wisp1.

HIGHLIGHTSO_LIThe RNA binding protein HuR is highly expressed in cardiac fibroblasts and its expression strongly correlates with markers of active myofibroblasts
C_LIO_LIHuR inhibition reduces migration, contraction, and ECM production activity of cardiac fibroblasts
C_LIO_LIExpression of the secreted matricellular protein, Wisp1, is increased in a HuR-dependent manner following TGF{beta} treatment
C_LIO_LIRecombinant Wisp1 rescues myofibroblast contractile function following HuR inhibition
C_LIO_LIHuR-dependent expression of Wisp1 is necessary for myofibroblast activation
C_LI


title: Increased early sodium current provokes familial atrial fibrillation and reduces effectiveness of sodium channel block

abstract:
Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the -subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF. Designing a new murine model carrying the Scn5a-M1875T mutation enabled us to study the effects of the Nav1.5 mutation in detail.

Atrial cardiomyocytes from newly generated Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, measured by patch clamp. Atrial microelectrode recordings revealed larger action potential amplitudes and a faster peak upstroke velocity. Conduction was studied using optical mapping. Conduction slowing by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. In vivo analysis using electrocardiograms and echocardiography, as well as cardiac histology, excluded overt hypertrophy or heart failure in young adult Scn5a-M1875T+/- mice.

The Scn5a-M1875T point mutation causes a cardiac sodium channel gain-of-function. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability and atrial fibrillation.

Statement of SignificanceThe observed changes induced by this point mutation suggest a selective increase in peak sodium current as a cause of familial AF. Our findings provide a possible explanation for the variable effectiveness of sodium channel blockers in patients with AF. Carriers of such sodium channel gain-of-function mutations may benefit more from tailored treatments.


title: Increased early sodium current causes familial atrial fibrillation and dampens effect of flecainide


title: Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites

abstract: We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

category: cell biology


title: Genetic control of KRAB-ZFP genes explains distal CpG-site methylation which associates with human disease phenotypes.

abstract: Kruppel-associated box zinc finger proteins (KRAB-ZFPs) are the largest gene family of transcriptional regulators in higher vertebrates. We have developed a method for inferring these factors DNA binding-sites in vivo. We achieved this by combining the genome-wide association of methylation at 573,027 CpG-sites (meQTL) in blood and KRAB-ZFP RNA expression changes (eQTL). This methods efficacy is clearly demonstrated by showing that the CpG-sites whose methylation is affected by KRAB-ZFP expression changes occur preferentially near KRAB-ZFP binding-sites in the HEK293T cell line. We found such an enrichment to be significant for 31% of factors tested. In addition, we found that binding-sites of many KRAB-ZFPs are significantly enriched (or sometimes depleted) in disease-associated CpG-sites. In total, 9% (11 of 125) of the traits tested, and 14% (32 of 222) of the factors (KRAB-ZFPs and TIF1-beta) tested, were enriched or depleted in one or more trait-factor pairing. Rheumatoid arthritis and human immunodeficiency virus (HIV) infection were associated with the largest number of KRAB-ZFP enrichments. There have been variable reports on the effects on HIV infection dynamics of transcription intermediary factor 1-beta (TIF1-beta, also known as KAP1 or TRIM28), a nuclear corepressor for KRAB-ZFPs. We provide evidence that KRAB-ZFP-independent effects of TIF1-beta are responsible for the decreased methylation of CpG-sites within its binding-sites that is observed in HIV infection. In conclusion, KRAB-ZFPs often affect CpG-site methylation within the proximity of their binding-sites, and CpG-sites within the binding-sites of KRAB-ZFPs are enriched for association with many traits, including rheumatoid arthritis and HIV infection.

category: genomics


title: Probing the proton release by Photosystem II in the S1 to S2 high-spin transition

abstract: The stoichiometry and kinetics of the proton release were investigated during each transition of the S-state cycle in Photosystem II (PSII) from Thermosynechococcus elongatus containing either a Mn4CaO5 (PSII/Ca) or a Mn4SrO5 (PSII/Sr) cluster. The measurements were done at pH 6.0 and pH 7.0 knowing that, in PSII/Ca at pH 6.0 and pH 7.0 and in PSII/Sr at pH 6.0, the flash-induced S2-state is in a low-spin configuration (S2LS) whereas in PSII/Sr at pH 7.0, the S2-state is in a high-spin configuration (S2HS) in half of the centers. Two measurements were done; the time-resolved flash dependent i) absorption of either bromocresol purple at pH 6.0 or neutral red at pH 7.0 and ii) electrochromism in the Soret band of PD1 at 440 nm. The fittings of the oscillations with a period of four indicate that one proton is released in the S1 to S2HS transition in PSII/Sr at pH 7.0. It has previously been suggested that the proton released in the S2LS to S3 transition would be released in a S2LSTyrZ[bullet] [->] S2HSTyrZ[bullet] transition before the electron transfer from the cluster to TyrZ[bullet] occurs. The release of a proton in the S1TyrZ[bullet] [->]S2HSTyrZ transition would logically imply that this proton release is missing in the S2HSTyrZ[bullet] to S3TyrZ transition. Instead, the proton release in the S1 to S2HS transition in PSII/Sr at pH 7.0 was mainly done at the expense of the proton release in the S3 to S0 and S0 to S1 transitions. However, at pH 7.0, the electrochromism of PD1 seems larger in PSII/Sr when compared to PSII/Ca in the S3 state. This points to the complex link between proton movements in and immediately around the Mn4 cluster and the mechanism leading to the release of protons into the bulk.


title: Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinantleucine-rich repeat proteins and protein complexes

abstract:
The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins.

HighlightsO_LIImproved yields and purity of LRR proteins and multiprotein complexes through chaperone co-expression, DNA construct design, and use of polycistronic baculovirus.
C_LIO_LIOver 300-fold yield increase of SMP protein complex from 0.1 mg/L to 32.6 mg/L.
C_LIO_LISUGT1 co-expression is a highly effective technique for recombinant LRR protein expression and purification.
C_LIO_LIPolycistronic baculovirus infection is ideal for production of multiprotein complexes
C_LI


title: Structural analysis of Red1 as a conserved scaffold of the RNA-targeting MTREC/PAXT complex

abstract: To eliminate specific or aberrant transcripts, eukaryotic cells use nuclear RNA-targeting complexes that deliver them to the exosome for degradation. S. pombe MTREC complex, and its human counterpart PAXT, are key players in this mechanism. Red1 and hZFC3H1 function as scaffolds of these respective complexes. Here, we present an NMR structure of a helix-turn-helix domain of Red1 in complex with the N-terminus of Iss10 and show this interaction is required for proper cellular growth and meiotic mRNA degradation. We also report a crystal structure of a Red1-Ars2 complex that explains the mutually exclusive interactions of hARS2 with various "ED/EGEI/L" motif-possessing RNA regulators such as hZFC3H1, hFLASH or hNCBP3. Finally, we show that both Red1 and hZFC3H1 homo-dimerize via their coiled-coil regions indicating that MTREC and PAXT likely function as dimers. Our results, combining structures of three Red1 binding interfaces with in vivo studies, provide mechanistic insights into conserved features of MTREC/PAXT architecture.


title: Structural features of Dnase1L3 responsible for serum antigen clearance

abstract: Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 does not rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are several key differences between the two enzymes. Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. The CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA to prevent autoimmune pathology. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.


title: Ubiquitinome analysis of articular cartilage post mechanical injury reveals a differential ubiquitination pattern of a subset of DUBs and proteins linked to the ERAD cellular response

abstract: Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to osteoarthritis. Here we identified a ubiquitin signature that is unique to injured articular cartilage tissue (the "injury ubiquitinome"). A total of 408 ubiquitinated peptides mapped to 114 proteins were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum(ER), also known as the ER-associated degradation(ERAD) response, including YOD1, BRCC3, ATXN3 and USP5 as well as the ER stress regulators, RAD23B, VCP/p97 and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB specific activity probes. Inhibition of DUBs using a broad-spectrum inhibitor caused a reduction in the injury-induced inflammatory response in a zebrafish tail fin injury model. These results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and osteoarthritis. This link through the ERAD pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.


title: The accuracy of protein structures in solution determined by AlphaFold and NMR

abstract: In the recent CASP (Critical Assessment of Structure Prediction) competition, AlphaFold2 performed outstandingly. Its worst predictions were for NMR structures, which has two alternative explanations: either the NMR structures were poor, implying that AlphaFold may be more accurate than NMR; or there is a genuine difference between crystal and solution structures. Here, we use the program ANSURR, which measures the accuracy of solution structures, and show that one of the NMR structures was indeed poor. We then compare AlphaFold predictions to NMR structures, and show that AlphaFold tends to be more accurate than NMR ensembles, in particular correctly more rigid in loops. There are however some cases where the NMR ensembles are more accurate. These tend to be dynamic structures where AlphaFold had low confidence. We suggest that AlphaFold could be used as the model for NMR structure refinements, and that AlphaFold structures validated by ANSURR require no further refinement.

category: biophysics


title: Macroscopic chemical to electrical signal converter in microbial communities

abstract: Excitable cells can convert electrical signals into chemical outputs to facilitate the active transport of information across larger distances. This electrical-to-chemical conversion requires a tightly regulated expression of ion channels. Alterations of ion channel expression provide landmarks of numerous pathological diseases, such as cardiac arrhythmia, epilepsy, or cancer. Although the activity of ion channels can be locally regulated by external light or chemical stimulus, it remains challenging to coordinate the expression of ion channels on extended spatial-temporal scales in a non-invasive manner. Here, we have engineered yeast S. cerevisiae to read and convert local chemical concentrations into a dynamic electrical field distributed across cell populations. The core mechanism encodes a chemically-excitable dual-feedback gene circuit that precisely tunes the expression domain of potassium channels, globally coordinating cyclic firing of the plasma membrane potential (PMP). We demonstrate that this mechanism leverages an engineered constitutively open bacterial potassium channel KcsA to directly couple chemical stimuli with ion flux through gene expression and it can interface with the host ion channels through the pulsatile production of toxins. Our study provides a robust synthetic transcriptional toolbox underlying the conversion of local chemical environments into spatiotemporally organized electrical impulses for various cellular engineering, synthetic biology, and potential therapeutic applications.


title: Macroscopic control of synchronous electrical signaling with chemically-excited gene expression


title: The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus

abstract:
Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. The ChvG/ChvI (ChvGI) TCS conserved in -proteobacteria is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to regulate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and found that ChvI regulates cell envelope architecture by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. In addition, we found that ChvI phosphorylation is also activated upon antibiotic treatment with vancomycin. We also challenged chv mutants with other cell envelope related stress and found that treatment with antibiotics targeting transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon. Finally, we observed that the sensor kinase ChvG relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer to maintain cell envelope homeostasis.

ImportanceThe cell envelope is the first barrier protecting cells from harsh environmental conditions, such as temperature, pH, oxidative and osmotic imbalances. It is also an obstacle for the intake of antibiotics targeting essential cellular processes. Therefore, molecular components and systems responding to cell envelope stress and maintaining cell envelope homeostasis are important targets for drug therapy. Here we show that the two-component system ChvGI, highly conserved in free-living and pathogenic -proteobacteria, is activated upon osmotic upshift and treatment with antibiotics targeting peptidoglycan synthesis to activate the transcription of multiple genes involved in cell envelope homeostasis in Caulobacter crescentus. We also show that the kinase sensor ChvG displays a dynamic localisation pattern that changes depending on osmotic imbalance. To our knowledge this is the first two-component system reported to change his cellular localisation upon environmental stress.