Biology subjects

Hernandez, M.

Publications and source records attributed to Hernandez, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Are we serologically prepared against an avian influenza pandemic and could seasonal flu vaccines help us?

The current situation with H5N1 highly pathogenic avian influenza virus (HPAI) is causing a worldwide concern due to multiple outbreaks in wild birds, poultry, and mammals. Moreover, multiple zoonotic infections in humans have been reported. Importantly, HPAI H5N1 viruses with genetic markers of adaptation to mammals have been detected. Together with HPAI H5N1, avian influenza viruses H7N9 (high and low pathogenic) stand out due to their high mortality rates in humans. This raises the question of how prepared we are serologically and whether seasonal vaccines are capable of inducing protective immunity against these influenza subtypes. An observational study was conducted in which sera from people born between years 1925-1967, 1968-1977, and 1978-1997 were collected before or after 28 days or 6 months post-vaccination with an inactivated seasonal influenza vaccine. Then, haemagglutination inhibition, viral neutralization, and immunoassays were performed to assess the basal protective immunity of the population as well as the ability of seasonal influenza vaccines to induce protective responses. Our results indicate that subtype-specific serological protection against H5N1 and H7N9 in the representative Spanish population evaluated was limited or nonexistent. However, seasonal vaccination was able to increase the antibody titers to protective levels in a moderate percentage of people, probably due to cross-reactive responses. These findings demonstrate the importance of vaccination and suggest that seasonal influenza vaccines could be used as a first line of defense against an eventual pandemic caused by avian influenza viruses, to be followed immediately by the use of more specific pandemic vaccines. ImportanceInfluenza A viruses (IAV) can infect and replicate in multiple mammalian and avian species. Avian influenza virus (AIV) is a highly contagious viral disease that occurs primarily in poultry and wild water birds. Due to the lack of population immunity in humans and ongoing evolution of AIV, there is a continuing risk that new IAV could emerge and rapidly spread worldwide causing a pandemic, if the ability to transmit efficiently among humans was gained. The aim of this study is to analyze the basal protection and presence of antibodies against IAV H5N1, and H7N9 subtypes in the population from different ages. Moreover, we have evaluated the h humoral response after immunization with a seasonal influenza vaccine. This study is strategically important to evaluate the level of population immunity that is a major factor when assessing the impact that an emerging IAV strain would have, and the role of seasonal vaccines to mitigate the effects of a pandemic.

immunology↗

bioRxiv2024

Mechanosensing by Piezo1 regulates osteoclast differentiation via PP2A-Akt axis in periodontitis

Mechanical stimulus to the multicellular bone unit (MBU) plays a key role in normal bone remodeling, whereas disuse osteoporosis, for example, represents loss of bone owing to lack of mechanical stresses. The analogy can be applied to a variety of pathogenic bone lytic complications, including periodontitis, in which local mechanical stress appears to be diminished. The activation of mechanosensitive Piezo1 Ca2+ channel expressed by osteoblasts and osteocytes in the MBU elicits the osteogenic signals in those cells. However, since osteoclast (OC)-specific Piezo1-gene knockout mice showed no skeletal phenotype, it has been assumed that Piezo1 might not play any role in OC-mediated bone remodeling. Here, however, we showed that mechanical stimulation of Piezo1 expressed on preosteoclasts (pre-OCs) downmodulates OC formation and, hence, bone resorptive activity in periodontitis, accompanied by significantly reduced expression of NFATc1, a master transcription factor for RANKL-induced OC-genesis. We know that the Ca2+/calcineurin/NFAT axis upregulates NFATc1 activation in pre-OCs. Interestingly, Piezo1-elicited Ca2+ influx did not affect NFATc1 expression. Instead, PP2A-mediated dephosphorylation of Akt downregulated NFATc1 in Piezo1-activated pre-OCs. However, systemic administration with Yoda1, a Piezo1 chemical agonist, or local injection of PP2A agonist, significantly downregulated the bone resorption induced in a mouse model of periodontitis, together with reduced numbers of TRAP+/phospho-Akt+ pre-OCs in local bone. These results suggest that mechanosensing by Piezo1 expressed on pre-OCs can downmodulate the RANKL-induced OC-genesis via the PP2A/Akt-dephosphorylation pathway, but that such Piezo1-mediated downregulation of bone resorption is attenuated in periodontitis. Significance StatementThe mechanosensitive Ca2+ channel Piezo1 plays important regulatory roles in a variety of cellular activities. RANKL-mediated OC-genesis requires permissive co-stimulatory signal from ITAM receptors, such as OSCAR and TREM2, to trigger the calcineurin/calmodulin signaling axis via Ca2+ oscillation, thereby upregulating NFATc1 expression. Activation of Piezo1 remarkably suppressed RANKL-induced NFATc1 activation which, in turn, reduced OC-genesis. Such mechanical activation of Piezo1 expressed on pre-OCs induced intracellular Ca2+ influx. Nonetheless, PP2A-mediated dephosphorylation of Akt, not the calcineurin/calmodulin pathway, suppressed NFATc1 in RANKL-elicited OC-genesis and resultant bone resorption, both in vitro and in vivo. These results indicate that mechanostress applied to pre-OCs can downregulate pathogenic OC-genesis and that Piezo1, as the mediator, is a novel molecular target for the development of anti-osteolytic therapies.

cell biology↗

bioRxiv2024

Structural Development of Speech Networks in Young Children at Risk for Speech Disorder

Characterizing the structural development of the neural speech network in early childhood is important to understand speech acquisition. To investigate speech in the developing brain, 94 children aged 4-7-years-old were scanned using diffusion weighted imaging (DWI) magnetic resonance imaging (MRI). In order to increase sample size and performance variability, we included children who were diagnosed with attention-deficit hyperactivity disorder (ADHD) from a larger ongoing study. Additionally, each child completed the Syllable Repetition Task (SRT), a validated measure of phoneme articulation. The DWI data were modeled using restriction spectrum imaging (RSI) to measure restricted and hindered diffusion properties in both grey and white matter. Consequently, we analyzed the diffusion data using both whole brain analysis, and automated fiber quantification (AFQ) analysis to establish tract profiles for each of six fiber pathways thought to be important for supporting speech development. In the whole brain analysis, we found that SRT performance was associated with restricted diffusion in bilateral inferior frontal gyrus, pars opercularis, right pre-supplementary and supplementary motor area, and bilateral cerebellar grey matter (p < .005). Age moderated these associations in left pars opercularis and frontal aslant tract (FAT). However, in both cases only the cerebellar findings survived a cluster correction. We also found associations between SRT performance and restricted diffusion in cortical association fiber pathways, especially left FAT, and in the cerebellar peduncles. Analyses using automated fiber quantification (AFQ) highlighted differences in high and low performing children along specific tract profiles, most notably in left but not right FAT, in bilateral SLFIII, and in the cerebellar peduncles. These findings suggest that individual differences in speech performance are reflected in structural grey and white matter differences as measured by restricted and hindered diffusion metrics, and offer important insights into developing brain networks supporting speech in very young children.

neuroscience↗

bioRxiv2024

Pangenomic analysis of Helcococcus ovis reveals widespread tetracycline resistance and a novel bacterial species, Helcococcus bovis.

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes. Phylogenetic and average nucleotide identity analyses placed four Helcococcus isolates within a cryptic clade-representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. H. ovis was more often isolated from cows with metritis, however, there was no associations between H. ovis gene clusters and uterine infection. The phylogenetic distribution of high-virulence determinants of H. ovis is consistent with convergent gene loss in the species. The majority of H. ovis strains (30/31) contain mobile tetracycline resistance genes, leading to higher minimum inhibitory concentrations of tetracyclines in vitro. In summary, this study showed that the presence of H. ovis is associated with uterine infection in dairy cows, that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov. HighlightsO_LIThe presence of Helcococcus ovis is associated with uterine infection in dairy cows C_LIO_LIMobile genetic element-mediated tetracycline resistance is widespread in H. ovis C_LIO_LICo-occurring virulence factors across clades suggest convergent gene loss in the species C_LIO_LIHelcococcus bovis is a novel species closely related to Helcococcus ovis C_LI

microbiology↗

bioRxiv2024

Minimally invasive serial collection of cerebrospinal fluid reveals sex-dependent differences in neuroinflammation in a rat model of mild traumatic brain injury

Traumatic brain injuries (TBI) are the seventh leading cause of disability globally with 48.99 million prevalent cases and 7.08 million years lived with diability. Approximately 80% of TBI patients are diagnosed with mild TBI (mTBI), or concussion, caused by nonpenetrating mechanical trauma to the head or body along with sudden rotational motion of the head. Studies investigating the temporal dynamics of neuroinflammation after mTBI are greatly needed. Without longitudinal studies, translating preclinical studies to clinical studies remains challenging as the difference in timing remains poorly understood. In this study, we describe a method of minimally invasive serial cerebrospinal fluid (CSF) collection that enables longitudinal investigation of CSF inflammation. The method described in this study can easily be adapted by any laboratory prepared for animal studies. Multiplex immunoassay of serially collected and singly collected CSF samples show collection frequency does not alter protein expression in the CSF. Further, sex-dependent differences in TBI have been reported, but remain poorly understood. This study establishes a framework for assessing sex difference in neuroinflammation after a concussion. We showed that results vary based on the framing of the statistical test. However, it is evident that males experience a more robust inflammatory response to a single concussion than females.

neuroscience↗

bioRxiv2024

Reduced recruitment of inhibitory control regions in very young children with ADHD during a modified Kiddie Continuous Performance Task: a fMRI study.

Attention-Deficit/Hyperactivity Disorder (ADHD) symptom profiles are known to undergo changes throughout development, rendering the neurobiological assessment of ADHD challenging across different developmental stages. Particularly in young children (ages 4 to 7 years), measuring inhibitory control network activity in the brain has been a formidable task due to the lack of child-friendly functional Magnetic Resonance Imaging (fMRI) paradigms. This study aims to address these difficulties by focusing on measuring inhibitory control in very young children within the MRI environment. A total of 56 children diagnosed with ADHD and 78 typically developing (TD) 4-7-year-old children were examined using a modified version of the Kiddie-Continuous Performance Test (K-CPT) during BOLD fMRI to assess inhibitory control. We concurrently evaluated their performance on the established and standardized K-CPT outside the MRI scanner. Our findings suggest that the modified K-CPT effectively elicited robust and expected brain activity related to inhibitory control in both groups. Comparisons between the two groups revealed subtle differences in brain activity, primarily observed in regions associated with inhibitory control, such as the inferior frontal gyrus, anterior insula, dorsal striatum, medial pre-supplementary motor area (pre-SMA), and cingulate cortex. Notably, increased activity in the right anterior insula was associated with improved response time (RT) and reduced RT variability on the K-CPT administered outside the MRI environment, although this did not survive statistical correction for multiple comparisons. In conclusion, our study successfully overcame the challenges of measuring inhibitory control in very young children within the MRI environment by utilizing a modified K-CPT during BOLD fMRI. These findings shed light on the neurobiological correlates of inhibitory control in ADHD and TD children, provide valuable insights for understanding ADHD across development, and potentially inform ADHD diagnosis and intervention strategies. The research also highlights remaining challenges with task fMRI in very young clinical samples.

neuroscience↗

bioRxiv2023

Carboxydovores from the Pseudomonadota colonise volcanic soils during succession

Carbon monoxide (CO) degrading microorganisms are present in volcanic deposits throughout succession, with vegetation and soil influencing the communities present. The carboxydovores are a subset of CO degraders that use CO only as an energy source, raising the question of how the physiological and metabolic features of the carboxydovores can make these bacteria more competitive in harsh volcanic ecosystems. An enrichment strategy was modified, which enabled the isolation of two carboxydovore representatives from genera that were abundant in the native soils, Cupriavidus sp. CV2T (92.3% ANI vs. Cupriavidus basilensis DSM 11853) and a putative strain of Paraburkholderia terrae (Pb. terrae COX) (96.42% ANI vs. Pb. terrae KU-64T). These isolates oxidise CO across a very broad range of concentrations, and genome sequence analysis indicated that they use form-I carbon monoxide dehydrogenase (CODH) to do so. Cupriavidus sp. CV2T and Pb. terrae COX each oxidised CO specifically at stationary phase, but the conditions for induction of CODH expression were distinct. Cupriavidus sp. CV2T expressed CODH only in the presence of CO, while Pb. terrae COX expressed CODH regardless of the presence of CO. Based on metabolic and phylogenetic analyses, Cupriavidus sp. CV2T is recommended as a novel species within the genus Cupriavidus. Therefore, we propose the name Cupriavidus ulmosensis sp. nov. for the type strain CV2T (= NCIMB 15506T, = CECT 30956T). This study provides valuable insights into the physiology and metabolism of carboxydovores, which colonise volcanic ecosystems during succession. ImportanceVolcanic ecosystems harbour many bacteria that contribute to the environmentally important process of carbon monoxide (CO) oxidation. We demonstrate a modified method for isolating bacteria, which consume CO at very low concentrations as a supplementary energy source (carboxydovory), leading to the isolation of two novel strains (Cupriavidus sp. CV2T and Paraburkholderia terrae COX) from volcanic strata that formed in 1917 and 2015, respectively. The conditions under which CO consumption occurs were investigated; each strain consumed CO during stationary phase, but Pb. terrae COX consumed CO regardless of the prior growth conditions while Cupriavidus sp. CV2 was more controlled. Cupriavidus sp. CV2 is a type strain of a new species, Cupriavidus ulmosensis str. CV2, which demonstrates relatively high tolerance for CO. These strains provide the basis for further study of the physiology, metabolism, and genetics of CO oxidation by carboxydovores, and will help us to understand how bacteria colonise harsh volcanic ecosystems.

microbiology↗

bioRxiv2023

Dictyostelium discoideum-assisted pharmacognosy of plant resources for discovering antivirulence molecules targeting Klebsiella pneumoniae

The rise of antibiotic-resistant bacterial strains poses a significant global health challenge, underscoring the critical need for innovative strategies to address this threat. Natural products and their derivatives have emerged as a promising reservoir for drug discovery. The social amoeba Dictyostelium discoideum is an advantageous model organism in this effort. Using this invertebrate model, we introduce a novel perspective to screen natural plant extracts for molecules with potential antivirulence activity. As a proof of concept, we established a simple high-throughput assay to screen for antivirulence molecules targeting Klebsiella pneumoniae among extracts of Helenium aromaticum. Thus, we aimed to identify compounds attenuating K. pneumoniae virulence without inducing cytotoxic effects on amoeba cells. Notably, the methanolic root extract of H. aromaticum but not other extracts fulfilled these prerequisites. Further analysis via UHPLC-ESI-MS/MS led to the identification of 24 chemical compounds boasting potential antivirulence attributes. This research underscores the potential of employing D. discoideum-assisted pharmacognosy for unearthing novel antivirulence agents against multidrug-resistant pathogens. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/564015v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@17530faorg.highwire.dtl.DTLVardef@e28259org.highwire.dtl.DTLVardef@dbc87eorg.highwire.dtl.DTLVardef@147de2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Antimicrobial resistance in Arctic soils is mediated by competition and facilitation

Antimicrobial resistance (AMR) is widespread in terrestrial ecosystems. However, the natural processes shaping the spatial and temporal dissemination of AMR in soils are not well understood. We aimed to determine whether, how, and why AMR varies in recently deglaciated pioneer and developing Arctic soils. We showed that antibiotic-resistant genes (ARGs), mobile genetic elements (MGEs), and antibiotic-resistant bacteria (ARB) are abundant, exhibit a non-uniform distribution, and generally increase with soil age. Our analyses suggest a strong positive relationship between soil age and ARG and ARB, which we attribute to increased competition between microbes in older soils. We also observed a weak negative relationship between soil age and ARG diversity mediated by soil organic matter - suggesting facilitation due to the alleviation of nutrient limitation. The microbial processes regulating the spread of AMR in Arctic soils may be further susceptible to the effects of future climate change and human activities. TeaserThe spatial and temporal spread of antimicrobial resistance in Arctic soils is dependent on microbial interactions for nutrients

microbiology↗

bioRxiv2023

Effects of Polyphosphate metabolism Mutations on Biofilm, Capsule Formation and Virulence Traits in Hypervirulent ST23 Klebsiella pneumoniae SGH10

The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulent K. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

microbiology↗

bioRxiv2023

The intrinsic clock of the hippocampal subfield CA3 rescues limbic seizures in a biohybrid graft-host interaction in vitro

Hippocampal dysfunction is the hallmark of mesial temporal lobe epilepsy (MTLE), the most common epileptic syndrome in adults and the most often refractory to medical therapy. Deep brain stimulation (DBS) may ameliorate drug-refractory MTLE, but it still cannot guarantee a seizure-free life. One major drawback is that the stimulation policy is informed by trial-and-error rather than by the operating mode of the brain. Thus, optimizing DBS parameters is still an unmet clinical need. Here, we propose the deployment of hippocampal interictal activity in a biohybrid approach to control limbic ictogenesis. Specifically, an electronic bridge establishes a graft-host interaction between the hippocampal subfield CA3 (graft) and the parahippocampal cortex (CTX - host) of distinct rodent brain slices, both treated with 4-aminopyridine; the electronic bridge relays the graft interictal events to the host via electrical pulses. We show that interictal activity generated by the graft CA3 controls limbic ictogenesis in the host CTX even in the absence of feedback from it, thus likely reflecting an intrinsic anti-ictogenic clock of this brain region. This work opens a translational perspective for MTLE treatment via biohybrid neuroprostheses relying on the intrinsic clock of incorporated hippocampal cells.

neuroscience↗

bioRxiv2023

Identification of diverse antibiotic resistant bacteria in agricultural soil with H218O stable isotope probing and metagenomics

BackgroundIn this study, we aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil, by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled "heavy" and the unlabelled "light" SIP fractions were sequenced. ResultsAfter incubations, an increase of the 16S rRNA copy numbers in the "heavy" fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) are highly abundant after two days of incubation with antibiotics. Several Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. On the other hand, only two ARGs from the unbinned-assembled light fractions were identified. ConclusionsThe results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil, and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

microbiology↗

bioRxiv2022

An Experimental Analysis of the Fine-Scale Effects of Nest Ectoparasites on Incubation Behavior

Avian incubation is a highly complex, adjustable behavior essential to embryo development and survival. When incubating, parents face a tradeoff between investing in incubation to maintain optimal temperatures for egg development or in self-maintenance behaviors to ensure their own survival and future reproduction. Because nest ectoparasites are costly and can reduce nestling quality and survival, infections could shift parental investment in current vs. future offspring. However, it is not well understood whether birds change investment in incubation in response to nest ectoparasitism, particularly in the context of other factors that are known to influence incubation behavior, such as ambient temperature, clutch size, and embryo development. We hypothesized that parents could respond to nest parasites by 1) investing more in incubation to promote the development of high-quality offspring to help offset the cost of parasites, 2) investing less in incubation or even abandoning their nest during incubation to save resources for future reproduction, or 3) being unresponsive to parasite infections, as incubation is more constrained by other factors. We tested these hypotheses by experimentally removing and adding mites in barn swallow nests at the start of incubation and deploying thermocouple eggs to measure egg temperatures at one-minute intervals until hatching. We found that while ambient temperature, clutch initiation date, embryo age, and clutch size were the main factors driving variation in egg conditions and parental incubation behavior, parasitized nests had higher mean egg temperatures, which could aid in nestling immune development. However, despite more optimal developmental temperatures, eggs in parasitized nests also had lower hatching success. Our results indicate that incubation is a dynamic behavior that is largely driven by the constraints of maintaining appropriate egg temperatures for development. Although quite costly upon hatching, ectoparasites appear to play a relatively minor role in driving variation in parental incubation investment. LAY SUMMARYO_LIIncubation is a complex behavior shaped by both internal and external factors. C_LIO_LIEctoparasites often decrease quality and survival of nestlings and could influence investment in incubation because of tradeoffs in how parents spend energy. C_LIO_LIWe manipulated parasites in barn swallow nests and used fake eggs with temperature sensors to collect data on egg temperatures and parental behavior throughout incubation. C_LIO_LIWe found that other factors were the main drivers of variation in incubation behavior, but that eggs in parasite nests were warmer on average, which could help nestlings better cope with parasites upon hatching. C_LIO_LIDespite more optimal developmental temperatures, eggs in parasitized nests had lower hatching success. C_LIO_LIAlthough quite costly to nestlings, ectoparasites played a minor role in shaping parental incubation behavior. C_LI

animal behavior and cognition↗

bioRxiv2022

Wnt/PCP signaling mediates breast cancer metastasis by promoting pro-invasive protrusion formation in collectively motile leader cells

As evidence supporting essential roles for collective cell migration in carcinoma metastasis continues to accumulate, a better understanding of the underlying cellular and molecular mechanisms will be critical to translating these findings to the treatment of advanced cancers. Here we report that Wnt/PCP, a non-canonical Wnt signaling pathway, mediates breast cancer collective migration and metastasis. We observe that mammary gland-specific knockout of Vangl2, a tetraspanin-like scaffolding protein required for Wnt5a-induced signaling and motility in cultured breast cancer cell lines, results in a striking decrease in metastatic efficiency but not primary tumor growth in the MMTV-NDL transgenic mouse model of HER2-positive breast cancer. We also observe that expression levels of core Wnt/PCP components Wnt5a, Vangl1 and Vangl2 are selectively elevated in K14-positive leader cells relative to follower cells within a collectively migrating cohort, and that Vangl2 expression selectively promotes RhoA activation in leading edge cells. Moreover, Vangl expression drives collective migration in three-dimensional ex vivo tumor organoids, and Vangl protein specifically accumulates within pro-migratory filamentous actin-rich protrusions of leader cells. Together, our observations point to a model whereby Wnt/PCP upregulation facilitates breast tumor collective cell motility by selectively augmenting the formation pro-migratory protrusions within leader cells.

cancer biology↗

bioRxiv2021

Generation of functional ciliates cholangiocytes from human pluripotent stem cells

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) would provide a model for studying the pathogenesis of cholangiopathies and for developing novel therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we describe a new strategy to generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of CFTR and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of new cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies showed that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice providing the first indication that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

cell biology↗

bioRxiv2020

Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection

BackgroundSARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential. MethodsWe developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD). ResultsTo diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values -0.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months. ConclusionThis SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness. One Sentence SummaryIn contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.

immunology↗

bioRxiv2020

Disproportionate CH4 sink strength from an endemic, sub-alpine Australian soil microbial community

Soil-to-atmosphere methane (CH4) fluxes are dependent on opposing microbial processes of production and consumption. Here we use a soil-vegetation gradient in an Australian sub-alpine ecosystem to examine links between composition of soil microbial communities, and the fluxes of greenhouse gases they regulate. For each soil-vegetation type (forest, grassland, and bog), we measured carbon dioxide (CO2) and CH4 fluxes and their production/consumption at 5-cm intervals to a depth of 30 cm. All soils were sources of CO2, ranging from 49-93 mg CO2 m-2 h-1. Forest soils were strong net sinks for CH4 at rates up to -413 {micro}g CH4 m-2 h-1. Grassland soils varied with some soils acting as sources and some as sinks, but overall averaged -97 {micro}g CH4 m-2 h-1. Bog soils were net sources of CH4 (+340 {micro}g CH4 m-2 h-1). Methanotrophs were dominated by USC in forest and grassland soils, and Candidatus Methylomirabilis sp. in the bog soils. Methylocystis were also detected at relatively low abundance. The potential disproportionately large contribution of these ecosystems to global CH4 oxidation, and poorly understood microbial community regulating it, highlight our dependence on soil ecosystem services in remote locations can be driven by a unique population of soil microbes. Originality-Significance Statement(Identify the key aspects of originality and significance that place the work within the top 10% of current research in environmental microbiology) Novel methanotrophic bacteria have been discovered in recent years, but few studies have examined the total known diversity of methanotrophs together with the net flux of CH4 from soils. We used an ecosystem with a vegetation-soil gradient in the sub-alpine regions of Australia (with extremely strong consumption of atmospheric CH4) to examine microbial and abiotic drivers of CH4 fluxes across this gradient. Recently characterized methanotrophs, either USC in forest and grassland soils, or oxygenic Candidatus Methylomirabilis sp. in the bog soil were dominant. Methanotrophs belonging to the families Methylococcaceae and Methylocystaceae represented only a small minority of the methanotrophs in this ecosystem.

microbiology↗

bioRxiv2020

Resuscitation of soil microbiota after > 70-years of desiccation

The abundance and diversity of bacteria in 24 historical soil samples under air-dried storage conditions for more than 70 years were assessed by quantification and high-throughput sequencing analysis of 16S rRNA genes. All soils contained a measurable abundance of bacteria varying from 103 to 108 per gram of soil and contrasting community compositions were observed in different background soils, suggesting that the bacteria detected were indigenous to the soil. Following a 4-week soil rewetting event, the bacterial abundance significantly increased in soils, indicating strong adaptation of soil bacteria to extreme osmotic change and high resuscitation potential of some bacteria over long periods of desiccation. Paenibacillus, Cohnella and two unclassified Bacillales genera within the phylum Firmicutes represented the most ubiquitously active taxa, which showed growth in the highest number of soils ([≥]12 soils), while genera Tumebacillus, Alicyclobacillus and Brevibacillus in the phylum Firmicutes displayed the highest growth rates in soils (with >1000-fold average increase) following rewetting. Additionally, some Actinobacteria and Proteobacteria genera showed relatively high activity following rewetting, suggesting that the resilience to long-term desiccation and rewetting is a common trait across phylogenetically divergent microbes. The present study thus demonstrated that diversified groups of microbes are present and potentially active in historically desiccated soils, which might be of importance in the context of microbial ecology.

microbiology↗