Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 955 records · Page 53Linked to original sources

bioRxiv2019

Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 >2 Mb and genomic completeness [≥] 91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a [~]1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

genomics↗

bioRxiv2019

Base editing in Streptomyces with Cas9-deaminase fusions

Conventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we demonstrate the multiplex genome editing potential of the nicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improved editing specificity. Collectively, this work provides a powerful new tool for genome editing in Streptomyces.

microbiology↗

bioRxiv2019

Sonogenetic modulation of cellular activities using an engineered auditory-sensing protein

Biomolecules that respond to different external stimuli enable the remote control of genetically modified cells. Chemogenetics and optogenetics, two tools that can control cellular activities via synthetic chemicals or photons, respectively, have been widely used to elucidate underlying physiological processes. These methods are, however, very invasive, have poor penetrability, or low spatiotemporal precision, attributes that hinder their use in therapeutic applications. We report herein a sonogenetic approach that can manipulate target cell activities by focused ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an engineered auditory-sensing protein prestin. Heterogeneous expression of mouse prestin containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in prestins from echolocating species endowed transfected mammalian cells with the ability to sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed ultrasound can also non-invasively stimulate target neurons expressing prestin(N7T, N308S) in deep regions of mice brains. Our study delineates how an engineered auditory-sensing protein can cause mammalian cells to sense ultrasound stimulation. Moreover, owing to the great penetration of low-frequency ultrasound ([~]400 mm in depth), our sonogenetic tools will serve as new strategies for non-invasive therapy in deep tissues of large animals like primates.

bioengineering↗

bioRxiv2019

Variation in PU.1 binding and chromatin looping at neutrophil enhancers influences autoimmune disease susceptibility

Neutrophils play fundamental roles in innate inflammatory response, shape adaptive immunity1, and have been identified as a potentially causal cell type underpinning genetic associations with immune system traits and diseases2,3 The majority of these variants are non-coding and the underlying mechanisms are not fully understood. Here, we profiled the binding of one of the principal myeloid transcriptional regulators, PU.1, in primary neutrophils across nearly a hundred volunteers, and elucidate the coordinated genetic effects of PU.1 binding variation, local chromatin state, promoter-enhancer interactions and gene expression. We show that PU.1 binding and the associated chain of molecular changes underlie genetically-driven differences in cell count and autoimmune disease susceptibility. Our results advance interpretation for genetic loci associated with neutrophil biology and immune disease.

genetics↗

bioRxiv2019

Germline-encoded TCR-MHC contacts promote TCR V gene bias in umbilical cord blood T cell repertoire

T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) proteins through T cell receptors (TCRs) on their surface. To recognize a wide range of pathogens, each individual possesses a substantial number of TCRs with an extremely high degree of variability. It remains controversial whether germline-encoded TCR repertoire is shaped by MHC polymorphism and, if so, what is the preference between MHC genetic variants and TCR V gene compatibility. To investigate the \"net\" genetic association between MHC variations and TRBV genes, we applied quantitative trait locus (QTL) mapping to test the associations between MHC polymorphism and TCR {beta} chain V (TRBV) genes usage using umbilical cord blood (UCB) samples of 201 Chinese newborns. We found TRBV gene and MHC loci that are predisposed to interact with one another differ from previous conclusions. The majority of MHC amino acid residues associated with the TRBV gene usage show spatial proximities in known structures of TCR-pMHC complexes. These results show for the first time that MHC variants bias TRBV gene usage in UCB of Chinese ancestry and indicate that germline-encoded contacts influence TCR-MHC interactions in intact T cell repertoires.

immunology↗

bioRxiv2019

Cortical ensemble activity discriminates auditory attentional states

Selective attention modulates sensory cortical activity. It remains unclear how auditory cortical activity represents stimuli that differ behaviorally. We designed a cross-modality task in which mice made decisions to obtain rewards based on attended visual or auditory stimuli. We recorded auditory cortical activity in behaving mice attending to, ignoring, or passively hearing auditory stimuli. Engaging in the task bidirectionally modulates neuronal responses to the auditory stimuli in both the attended and ignored conditions compared to passive hearing. Neuronal ensemble activity in response to stimuli under attended, ignored and passive conditions are readily distinguishable. Furthermore, ensemble activity under attended and ignored conditions are in closer states compared to passive condition, and they share a component of attentional modulation which drives them to the same direction in the population activity space. Our findings suggest that task engagement changes sensory cortical representations across modalities in the same directions, and cross-modality attention may differentially modulates attended and ignored modalities.

neuroscience↗

bioRxiv2019

Powerful solution for experimental cerebral malaria treatment: artesunate and tetramethylpyrazine

BackgroundCerebral malaria (CM) is a kind of serious neurological complication caused by the acute Plasmodium falciparum infection. About 300000 patients including children under 5 years old died from this disease every year. Even intravenous artesunate (Art) is employed as the most effictive drug in the treatment of CM, high incidence of death and neurological sequelae are still inevitable. Therefore, we assessed the combination of Art and tetramethylpyrazine (TMP), to treat experimental CM (ECM) in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). A non-biased whole brain quantitative proteomic analysis was also conducted to get some insight of the mechanism of the combinational treatment.\n\nResultsTreatment of (ECM)-C57BL/6 mice with the combination of Art and TMP increased the survival, improved clinical signs and prevented neurological manifestations. These effects were related to reduction of parasitised red blood cells (pRBC) adhesion, sequestration, maintaining brain microvascular integrity, increasing nerve growth factor, neurotrophin levels, and alleviating hippocampal neuronal damage and astrocyte activation. The pharmacological effects of Art-TMP combination therapy were analyzed by ECM mice brain proteomic function enrichment. Based on an isobaric tag for relative and absolute quantitation (iTRAQ) fold-change of 1.2 (P-value < 0.05), 217 down-regulated and 177 up-regulated proteins were identified, presenting a significantly altered proteome profile of the combined Art and TMP group as compared to the group treated with Art or TMP alone. These results suggested that the Art-TMP combination could be used as a powerful solution for CM and its neurologic damage.\n\nConclusionsAn effictive therapy for CM with low mortality rate and protect against ECM-induced neurocognitive impairment has been proposed through the combination of Art and TMP, which can provide an effective adjuvant treatment in the clinic. iTRAQ proteomics provide a resource for further mechanistic studies to examine the synergistic effects of Art and TMP and their potential to serve as an adjunctive treatment method and intervention targets.\n\nAuthor SummaryCerebral malaria (CM) is the most serious neurological complication caused by Plasmodium falciparum infection. Even after antimalarial treatment, severe neurological sequelae still exist. We used tetramethylpyrazine (TMP), the main ingredient of the traditional Chinese medicine Chuanxiong, and artesunate (Art) as a combination of drugs. We found that Art-TMP combination could improve the clinical symptoms of CM and protect the nervous system. At the same time, proteomics was used to analyze the protective mechanism of Art-TMP combination administration on ECM mice. This study suggests that the combination of Art and TMP may be used as an adjuvant therapy for clinical CM and iTRAQ proteomics provides resources for further study of Art-TMP combination and provides potential prognostic biomarkers for this therapeutic intervention.

pathology↗

bioRxiv2019

Interaction between DNA damage response, translation and apoptosome determines cancer susceptibility to TOP2 poisons

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We show that glioblastoma (GBM), the most malignant of all primary brain tumors in adults is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 impaired the induction of pro-apoptotic gene APAF1 following etoposide treatment, and led to resistance to this drug and doxorubicin. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from multiple cancers.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC=\"FIGDIR/small/614024v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (14K):\norg.highwire.dtl.DTLVardef@310f7org.highwire.dtl.DTLVardef@14ed578org.highwire.dtl.DTLVardef@a0d9c0org.highwire.dtl.DTLVardef@37f59a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

Genetic risk loadings influence the susceptibility and severity of systemic lupus erythematosus

ObjectiveUsing three European and two Chinese genome-wide association studies (GWAS), we investigated the performance of genetic risk scores (GRS) for predicting the susceptibility and severity of Systemic lupus erythematosus (SLE), using renal disease as a proxy for severity. MethodsWe used four GWASs to test the performance of GRS both cross validating within the European population and between European and Chinese populations. The performance of GRS in SLE risk prediction was evaluated by Receiver Operating Characteristic (ROC) curves. We then analyzed the polygenic nature of SLE statistically. We also partitioned patients according to their age-of-onset and evaluated the predictability of GRS in disease severity in each age group. ResultsWe found consistently that the best GRS in the prediction of SLE used SNPs associated at the level of P<1e-05 in all GWAS datasets and that SNPs with P-values above 0.2 were inflated for SLE true positive signals. The GRS results in an area under the ROC curve ranging between 0.64 and 0.72, within European and between the European and Chinese populations. We further showed a significant positive correlation between a GRS and renal disease in two independent European GWAS (Pcohort1=2.44e-08; Pcohort2=0.00205) and a significant negative correlation with age of SLE onset (Pcohort1=1.76e-12; Pcohort2=0.00384). We found that the GRS performed better in prediction of renal disease in the later onset compared to the earlier onset group. ConclusionThe GRS predicts SLE in both European and Chinese populations and correlates with poorer prognostic factors: young age of onset and lupus nephritis.

genetics↗

bioRxiv2019

HNF4 factors control chromatin accessibility and are redundantly required for maturation of the fetal intestine

As an embryo matures into a fetus, cells undergo remarkable transitions, accompanied by shifts in transcription factor regulatory networks and chromatin landscapes. The mechanisms of these developmental transitions are not completely understood. The embryonic intestine transitions from a rapidly proliferating tube with pseudostratified epithelium prior to embryonic day (E) 14.5, to an exquisitely folded columnar epithelium in the fetus. We sought to define factors that drive fetal maturation of the intestine. ATAC-seq profiling revealed a dramatic restructuring of intestinal chromatin during the embryonic-to-fetal transition, with CDX2 transcription factor motifs abundant at chromatin-accessible regions of the embryo, and hepatocyte nuclear factor 4 (HNF4) transcription factor motifs the most abundant in the fetal stages. Genetic inactivation of Hnf4 and its paralog, Hnf4{gamma}, revealed that HNF4 factors are redundantly and vitally required for fetal maturation. In the embryo, CDX2 binds to and activates Hnf4 gene loci to drive HNF4 expression at fetal stages. HNF4 and CDX2 transcription factors then occupy shared genomic regulatory sites and are required for chromatin accessibility at genes expressed in the maturing fetal intestine. Thus, intestinal transcription factor regulatory networks shift to accompany changing chromatin landscapes and gene expression profiles that occur during the transition of an embryonic tissue to its mature state.

developmental biology↗

bioRxiv2019

Identifying drug combinations of individual cancer patients by personalized drug controller method

Treating cancer in precision medicine, it is important to identify the personalized combinational drugs under consideration of the individual heterogeneity. Many bioinformatics tools for the personalized driver genes identification have presented promising clues in determining candidate personalized drug targets for the personalized drugs discovery. However, it has not been studied how to fill the gap between personalized driver genes identification and personalized combinational drugs discovery. In this work, we developed a novel algorithm of structure network Controllability-based Personalized driver Genes and combinational Drug identification (CPGD), aiming to mine the personalized driver genes and identify the combinational drugs of an individual cancer patient. On two benchmark cancer datasets, the performance of CPGD for predicting the clinical efficacious combinational drugs is superior to that of other state-of-the-art driver gene-focus algorithms in terms of precision accuracy. In particular, by quantifying and referring the relationships between target genes of pairwise combinatorial drugs and disease module genes on breast cancer data set, CPGD can significantly divide patients into the discriminative high-risk and low-risk groups for risk asessment in combination therapy. In addition, CPGD can further enhance cancer subtyping by providing computationally personalized side effect signatures for individual patients. Collectively, CPGD provided a new and effecient bioinformatics tool from structure network controllability perspective for discovering personalized combinational drugs with personalized side effect consideration, so as to effectively support personalized risk assessement and disease subtyping. SignificanceIt is quite challenging to predict personalized combinational drugs rather than patient-cohort s drugs based on cancer omics data. In this work, a novel structure network Controllability-based algorithm (CPGD) from feedback vertex sets control perspective was developed, for discovering efficacious combinational drugs of an individual cancer patient by targeting the personalized driver genes. The CPGD contains three methodological advances by exploring more precise mathematical models on high-throughput personalized multi-omics data. The first is that a proper network structure is constructed to characterize the gene regulatory mechanism of an individual patient. The second is that considering the weight information of network edges/relations improves the performance for predicting clinical efficacious combinational drugs compared with other drivers-focus methods. And the third is that proper evaluation metrics for personalized combinational drugs prioritization, personalized risk assessment and disease subtyping are designed when evaluating the performance of CPGD.

systems biology↗

bioRxiv2019

EPIC: MHC-I epitope prediction integrating mass spectrometry derived motifs and tissue-specific expression profiles

BackgroundAccurate prediction of epitopes presented by human leukocyte antigen (HLA) is crucial for personalized cancer immunotherapies targeting T cell epitopes. Mass spectrometry (MS) profiling of eluted HLA ligands, which provides high-throughput measurements of HLA associated peptides in vivo, can be used to faithfully model the presentation of epitopes on the cell surface. In addition, gene expression profiles measured by RNA-seq data in a specific cell/tissue type can significantly improve the performance of epitope presentation prediction. However, although large amount of high-quality MS data of HLA-bound peptides is being generated in recent years, few provide matching RNA-seq data, which makes incorporating gene expression into epitope prediction difficult. MethodsWe collected publicly available HLA peptidome and matching RNA-seq data of 34 cell lines derived from various sources. We built position score specific matrixes (PSSMs) for 21 HLA-I alleles based on these MS data, then used logistic regression (LR) to model the relationship among PSSM score, gene expression and peptide length to predict whether a peptide could be presented in each of the cell line. We further built a universal LR model, termed Epitope Presentation Integrated Prediction (EPIP), based on more than 180,000 unique HLA ligands collected from public sources and ~3,000 HLA ligands generated by ourselves, to predict epitope presentation for 66 common HLA-I alleles. ResultsWhen evaluating EPIP on large, independent HLA eluted ligand datasets, it performed substantially better than other popular methods, including MixMHCpred (v2.0), NetMHCpan (v4.0), and MHCflurry (v1.2.2), with an average 0.1% positive predictive value (PPV) of 52.01%, compared to 37.24%, 36.96%, 24.90% and 23.76% achieved by MixMHCpred, NetMHCpan-4.0 (EL), NetMHCpan-4.0 (BA) and MHCflurry, respectively. It is also comparable to EDGE, a recent deep learning-based model that is not publicly available, on predicting epitope presentation and selecting immunogenic cancer neoantigens. However, the simplicity and flexibility of EPIP makes it easier to be applied in diverse situations, and we demonstrated this by generating MS data for the HCC4006 cell line and adding the support of HLA-A*33:03 to EPIP. EPIP is publicly available as a web tool < http://epip.genomics.cn/>. Conclusionswe have developed an easy to use, publicly available epitope prediction tool, EPIP, that incorporates information from both MS and RNA-seq data, and demonstrated its superior performance over existing public methods.

bioinformatics↗

bioRxiv2019

Neural circuits mediating visual stabilization during active motion in zebrafish

Visual stabilization is an inevitable requirement for animals during active motion interaction with the environment. Visual motion cues of the surroundings or induced by self-generated behaviors are perceived then trigger proper motor responses mediated by neural representations conceptualized as the internal model: one part of it predicts the consequences of sensory dynamics as a forward model, another part generates proper motor control as a reverse model. However, the neural circuits between the two models remain mostly unknown. Here, we demonstrate that an internal component, the efference copy, coordinated the two models in a push-pull manner by generating extra reset saccades during active motion processing in larval zebrafish. Calcium imaging indicated that the saccade preparation circuit is enhanced while the velocity integration circuit is inhibited during the interaction, balancing the internal representations from both directions. This is the first model of efference copy on visual stabilization beyond the sensorimotor stage.

neuroscience↗

bioRxiv2019

Molecular Phenotyping of Oxidative Stress in Diabetes Mellitus with Point-of-care NMR system

Diabetes mellitus is one of the fastest growing health burdens globally. Oxidative stress which has been implicated to the pathogenesis of diabetes complication (e.g., cardiovascular event) were, however, poorly understood. We report a novel approach to rapidly manipulate the redox chemistry (in a single drop) of blood using point-of-care NMR system. We exploit the fact that oxidative stress changes the subtle molecular motion of water-proton in the blood, and thus inducing a measurable shift in magnetic resonance relaxation properties. This technique is label-free and the whole assays finish in a few minutes. Various redox states of the hemoglobin were mapped out using our newly proposed two-dimensional map, known as T1-T2 magnetic state diagram. We demonstrated the clinical utilities of this technique to rapidly sub-stratify diabetes subjects based on their oxidative status (in conjunction to the traditional glycemic level), to improve the patient risk stratification and thus the overall outcome of clinical diabetes care and management. (155 words) Key Points for SummariesO_LIA novel approach to rapidly manipulate the redox chemistry (in a single drop) of blood using point-of-care NMR system. C_LIO_LIAssessment of the oxidative status, in conjunction to their glycemic level allows sub-stratification of diabetes subjects which was demonstrated clinically. C_LI Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/565325v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@123d111org.highwire.dtl.DTLVardef@72e4e9org.highwire.dtl.DTLVardef@112b9bforg.highwire.dtl.DTLVardef@60e498_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Clinical Significance of C4d Deposition in Renal Tissues from Patients with Primary Sj&oumlgren's Syndrome--A Preliminary Study

ObjectivesTo evaluate renal expression of C4d, a complement component in the classical/mannose binding lectin (MBL) pathway, in patients with primary Sjogrens syndrome (pSS)-associated renal impairments. MethodsWe retrospectively reviewed the clinical and pathological data from 39 patients with pSS presenting with renal impairments. C4d was examined in paraffin-embedded biopsy tissues using immunohistochemistry. Glomerular C4d positive was defined when >75% glomeruli were globally stained. Tubulointerstitial C4d (TI-C4d) were scored semi-quantitatively as 0 (absent), 1 (spotty or weak), 2 (patchy) and 3 (diffuse). A TI-C4d score [≥]2 was considered TI-C4d positive and included in the TI-C4d+ group and vice versa. Peritubular capillary (PTC) C4d was scored as 0 (absent), 1 (0[~]10%, minimal), 2 (10%[~]50%, focal), and 3 (>50%, diffuse). ResultsGlomerular C4d deposition was observed in all 8 patients with pSS-related membranous nephropathy (MN) without obvious C1q deposition. Two of 5 patients with mesangial proliferative glomerulonephritis and 1 of 2 patients with IgA nephropathy had mild mesangial C4d deposition. Sixteen patients (6 glomerular dominant and 10 tubulointerstitial dominant) presented TI-C4d score [≥]2. Patients in the TI-C4d+ group exhibited a higher serum creatinine level at the time of renal biopsy (TI-C4d+ 132.5 [89.7, 165.5] vs. TI-C4d- 83.0 [70.7, 102.0] mol/L, P=0.008). PTC C4d was observed in 12 patients, with each of minimal, focal and diffuse staining being noted in 4 patients. ConclusionsThe MBL pathway of complement activation was potentially involved in pSS-related MN. Tubulointerstitial C4d might be a pathological marker of severe renal injury in patients with pSS-related renal impairments.

pathology↗

bioRxiv2019

Prototheca zopfii Genotype II induces mitochondrial apoptosis in models of bovine mastitis

Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production by unknown mechanisms. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance ({Delta}{Psi}m) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-, IL-1{beta} and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis. Author summaryBovine mastitis (inflammation of the udder) reduces milk production and quality, causing huge economic losses in the dairy industry worldwide. Although the alga Prototheca zopfii is a major cause of mastitis in dairy cows, mechanisms by which it damages mammary tissues are not well known. Here, we used cell cultures and a mouse model of mastitis to determine how Prototheca caused inflammation and cell death in mammary tissues. Prototheca invaded mammary gland cells, from cattle and mice, as well as macrophages (white cells that take up and kill pathogens) and caused cell death by interfering with mitochondria. Furthermore, Prototheca causes severe inflammation and tissue damage when injected into the mammary glands of mice. Although there are two genotypes of P. zopfii, only genotype II causes tissue damage, whereas gentotype I, common in farm environments, does not damage mammary tissues. Since P. zopfii is an alga and not a bacterium, antibiotic treatments, frequently used to treat mastitis in cattle, are not effective against this organism. Understanding how P. zopfii damages mammary tissue and causes mastitis is important new knowledge to promote future development of evidence-based approaches to prevent and treat mammary gland infections with this organism.

microbiology↗

bioRxiv2019

Tbx1 interacts genetically with Vegfr3 to regulate cardiac lymphangiogenesis in mice

The transcription factor TBX1 is the major gene implicated in 22q11.2 deletion syndrome. The complex clinical phenotype includes vascular anomalies and a recent report presented new cases of primary lymphedema in 22q11.2DS patients. We have previously shown that Tbx1 activates Vegfr3 gene expression in lymphatic endothelial cells and that this activation is critical for lymphatic vessel development in prenatal mice and for their survival post-natally. Using loss-of-function genetics and transgenesis, we show a strong genetic interaction between Tbx1 and Vegfr3 in cardiac lymphangiogenesis that causes cardiac lymphatic vessel anomalies in compound heterozygotes. Intriguingly, different aspects of the cardiac lymphatic phenotype were regulated independently by the two genes. Tbx1Cre-activated Vegfr3 transgene expression was able to rescue the morphological abnormalities in the cardiac lymphatic vessels of compound heterozygotes, but it did not rescue the severe cardiac lymphatic vessel hypoplasia observed in Tbx1 homozygotes. Moreover, our study revealed a differential sensitivity between the ventral and dorsal cardiac lymphatic networks to the effects of altered Tbx1 and Vegfr3 gene dosage. Overall, our study demonstrates that a fine dosage balance between Tbx1 and Vegfr3 is required to regulate the number and morphology of cardiac lymphatic vessels.

developmental biology↗

bioRxiv2019

Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci and Streptococcus mutans

N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) enhance the competitiveness of the laboratory strain DL1 of Streptococcus gordonii against the caries pathogen Streptococcus mutans. Here we examine how amino sugars affect the interaction of five low-passage clinical isolates of abundant commensal streptococci with S. mutans utilizing a dual-species biofilm model. Compared to glucose, growth on GlcN or GlcNAc significantly reduced the viability of S. mutans in co-cultures with most commensals, shifting the proportions of species. Consistent with these results, production of H2O2 was increased in most commensals when growing on amino sugars, and inhibition of S. mutans by Streptococcus cristatus, Streptococcus oralis, or S. gordonii was enhanced by amino sugars on agar plates. All commensals except S. oralis had higher arginine deiminase activities when grown on GlcN, and in some cases GlcNAc. In ex vivo biofilms formed using pooled cell-containing saliva (CCS), the proportions of S. mutans were drastically diminished when GlcNAc was the primary carbohydrate. Increased production of H2O2 could account in large part for the inhibitory effects of CCS biofilms. Surprisingly, amino sugars appeared to improve mutacin production by S. mutans on agar plates, suggesting that the commensals have mechanisms to actively subvert antagonism by S. mutans in co-cultures. Collectively, these findings demonstrate that amino sugars can enhance the beneficial properties of low-passage commensal oral streptococci and highlight their potential for moderating the cariogenicity of oral biofilms. SIGNIFICANCEDental caries is driven by dysbiosis of oral biofilms in which dominance by acid-producing and acid-tolerant bacteria results in loss of tooth mineral. Our previous work demonstrated the beneficial effects of amino sugars, GlcNAc and GlcN, in promoting the antagonistic properties of a health-associated oral bacterium, Streptococcus gordonii, in competition with the major caries pathogen Streptococcus mutans. Here we investigated 5 low-passage clinical isolates of the most common streptococcal species to establish how amino sugars may influence the ecology and virulence of oral biofilms. Using multiple in vitro models, including a human saliva-derived microcosm biofilm, experiments showed significant enhancement by at least one amino sugar in the ability of most of these bacteria to suppress the caries pathogen. Therefore, our findings demonstrated the mechanism of action by which amino sugars may affect human oral biofilms to promote health.

microbiology↗