Bipolar Patient-Specific In Vitro Diagnostic Test Reveals Underlying Cardiac Arrhythmia Phenotype Caused by Calcium Channel Genetic Risk Factor.

A common genetic risk factor for bipolar disorder is CACNA1C, a gene that is also critical for cardiac rhythm. The impact of CACNA1C mutations on bipolar patient cardiac rhythm is unknown. Here, we report the cardiac electrophysiological implications of a bipolar disorder-associated genetic risk factor in CACNA1C using patient induced pluripotent stem cell-derived cardiomyocytes. Results indicate that the CACNA1C bipolar disorder-related mutation causes cardiac electrical impulse conduction slowing mediated by impaired intercellular coupling via connexin 43 gap junctions. In vitro gene therapy to restore connexin 43 expression increased cardiac electrical impulse conduction velocity and protected against thioridazine-induced QT prolongation. Patients positive for bipolar disorder CACNA1C genetic risk factors may have elevated proarrhythmic risk for adverse events in response to psychiatric medications that slow conduction or prolong the QT interval. This in vitro diagnostic tool enables cardiac testing specific to patients with psychiatric disorders to determine their sensitivity to off-target effects of psychiatric medications.

Bipolar disorder (BD) is associated with genetic risk factors that present as mutations in specific genes. One gene commonly associated with BD is the calcium channel gene CACNA1C, found in the brain and the heart. The impact of CACNA1C mutation on cardiac function in patients with BD is unclear. Here, we created a BD CACNA1C mutant patient "heart in a dish" using patient-specific stem cells. Gene editing was also used to correct the mutation to create an isogenic control cell line. We found that the BD calcium gene mutation caused slow electrical impulse propagation, reduced the function of the calcium channel, and was associated with low intercellular communication channels called connexin. Using connexin gene therapy in vitro, the the cardiac dysfunction could be corrected and cured. This new approach offers patient-specific hearts-in-a-dish that can be used to ensure that medications will not cause heart racing or arrhythmias.

High-throughput longitudinal electrophysiology screening of mature chamber-specific hiPSC-CMs using optical mapping.

hiPSC-CMs are being considered by the Food and Drug Administration and other regulatory agencies for in vitro cardiotoxicity screening to provide human-relevant safety data. Widespread adoption of hiPSC-CMs in regulatory and academic science is limited by the immature, fetal-like phenotype of the cells. Here, to advance the maturation state of hiPSC-CMs, we developed and validated a human perinatal stem cell-derived extracellular matrix coating applied to high-throughput cell culture plates. We also present and validate a cardiac optical mapping device designed for high-throughput functional assessment of mature hiPSC-CM action potentials using voltage-sensitive dye and calcium transients using calcium-sensitive dyes or genetically encoded calcium indicators (GECI, GCaMP6). We utilize the optical mapping device to provide new biological insight into mature chamber-specific hiPSC-CMs, responsiveness to cardioactive drugs, the effect of GCaMP6 genetic variants on electrophysiological function, and the effect of daily ß-receptor stimulation on hiPSC-CM monolayer function and SERCA2a expression.

Dynamics of oral microbiome acquisition in healthy infants: A pilot study.

Objectives: The human oral microbiota is one of the most complex bacterial communities in the human body. However, how newborns initially acquire these bacteria remains largely unknown. In this study, we examined the dynamics of oral microbial communities in healthy infants and investigated the influence of the maternal oral microbiota on the acquisition of the infant's oral microbiota. We hypothesized that the infant oral microbial diversity increases with age. Methods: One hundred and sixteen whole-salivary samples were collected from 32 healthy infants and their biological mothers during postpartum and 9- and 15-month well-infant visits. Bacterial genomic DNA was extracted and sequenced by Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) methods. The Shannon index was used to measure the microbial diversity of the infant-mother dyads (alpha diversity). The microbial diversity between the mother-infant dyads (beta-diversity) was calculated using the weighted non-phylogenetic Bray-Curtis distance in QIIME 1.9.1. Core microbiome analysis was performed using MicrobiomeAnalyst software. Linear discriminant analysis coupled with effect size analysis was used to identify differentially abundant features between mother and infant dyads. Results: A total of 6,870,571 16S rRNA reads were generated from paired mother-infant saliva samples. Overall, oral microbial profiles significantly differed between the mother and infant groups (p < 0.001). The diversity of the salivary microbiomes in the infants increased in an age-dependent manner, whereas the core microbiome of the mothers remained relatively stable during the study period. Breastfeeding and gender did not affect the microbial diversity in infants. Moreover, infants had a greater relative abundance of Firmicutes and a lower abundance of Actinobacteria, Bacteroidetes, Fusobacteria, and Proteobacteria than their mothers. The SparCC correlation analysis demonstrated constant changes in infants' oral microbial community network (p < 0.05). Conclusions: This study provides new evidence that the oral cavities of infants are colonized by a distinct group of bacterial species at birth. The acquisition and diversity of changes in oral microbial composition are dynamic during the first year of an infant's life. Before reaching the second birthday, the composition of the oral microbial community could be more similar to that of their biological mothers.

Novel Clustering Methods Identified Three Caries Status-Related Clusters Based on Oral Microbiome in Thai Mother-Child Dyads.

Early childhood caries (ECC) is a disease that globally affects pre-school children. It is important to identify both protective and risk factors associated with this disease. This paper examined a set of saliva samples of Thai mother-child dyads and aimed to analyze how the maternal factors and oral microbiome of the dyads influence the development of ECC. However, heterogeneous latent subpopulations may exist that have different characteristics in terms of caries development. Therefore, we introduce a novel method to cluster the correlated outcomes of dependent observations while selecting influential independent variables to unearth latent groupings within this dataset and reveal their association in each group. This paper describes the discovery of three heterogeneous clusters in the dataset, each with its own unique mother-child outcome trend, as well as identifying several microbial factors that contribute to ECC. Significantly, the three identified clusters represent three typical clinical conditions in which mother-child dyads have typical (cluster 1), high-low (cluster 2), and low-high caries experiences (cluster 3) compared to the overall trend of mother-child caries status. Intriguingly, the variables identified as the driving attributes of each cluster, including specific taxa, have the potential to be used in the future as caries preventive measures.

Mucolytic bacteria license pathobionts to acquire host-derived nutrients during dietary nutrient restriction.

Pathobionts employ unique metabolic adaptation mechanisms to maximize their growth in disease conditions. Adherent-invasive Escherichia coli (AIEC), a pathobiont enriched in the gut mucosa of patients with inflammatory bowel disease (IBD), utilizes diet-derived L-serine to adapt to the inflamed gut. Therefore, the restriction of dietary L-serine starves AIEC and limits its fitness advantage. Here, we find that AIEC can overcome this nutrient limitation by switching the nutrient source from the diet to the host cells in the presence of mucolytic bacteria. During diet-derived L-serine restriction, the mucolytic symbiont Akkermansia muciniphila promotes the encroachment of AIEC to the epithelial niche by degrading the mucus layer. In the epithelial niche, AIEC acquires L-serine from the colonic epithelium and thus proliferates. Our work suggests that the indirect metabolic network between pathobionts and commensal symbionts enables pathobionts to overcome nutritional restriction and thrive in the gut.

Multimodal Data Integration Reveals Mode of Delivery and Snack Consumption Outrank Salivary Microbiome in Association With Caries Outcome in Thai Children.

Early childhood caries (ECC) is not only the most common chronic childhood disease but also disproportionately affects underserved populations. Of those, children living in Thailand have been found to have high rates of ECC and severe ECC. Frequently, the cause of ECC is blamed on a handful of cariogenic organisms, such as Streptococcus mutans and Streptococcus sobrinus. However, ECC is a multifactorial disease that results from an ecological shift in the oral cavity from a neutral pH (~7.5) to an acidic pH (<5.5) environment influenced by the host individual's biological, socio-behavioral, and lifestyle factors. Currently, there is a lack of understanding of how risk factors at various levels influence the oral health of children at risk. We applied a statistical machine learning approach for multimodal data integration (parallel and hierarchical) to identify caries-related multiplatform factors in a large cohort of mother-child dyads living in Chiang Mai, Thailand (N=177). Whole saliva (1 mL) was collected from each individual for DNA extraction and 16S rRNA sequencing. A set of maternal and early childhood factors were included in the data analysis. Significantly, vaginal delivery, preterm birth, and frequent sugary snacking were found to increase the risk for ECC. The salivary microbial diversity was significantly different in children with ECC or without ECC. Results of linear discriminant analysis effect size (LEfSe) analysis of the microbial community demonstrated that S. mutans, Prevotella histicola, and Leptotrichia hongkongensis were significantly enriched in ECC children. Whereas Fusobacterium periodonticum was less abundant among caries-free children, suggesting its potential to be a candidate biomarker for good oral health. Based on the multimodal data integration and statistical machine learning models, the study revealed that the mode of delivery and snack consumption outrank salivary microbiome in predicting ECC in Thai children. The biological and behavioral factors may play significant roles in the microbial pathobiology of ECC and warrant further investigation.

Roles of TroA and TroR in Metalloregulated Growth and Gene Expression in Treponema denticola.

The availability of divalent metal cations required as cofactors for microbial metabolism is severely limited in the host environment. Bacteria have evolved highly regulated uptake systems to maintain essential metal homeostasis to meet cellular demands while preventing toxicity. The Tro operon (troABCDR), present in all sequenced Treponema spp., is a member of a highly conserved family of ATP-binding cassette transporters involved in metal cation uptake whose expression is controlled by TroR, a DtxR-like cation-responsive regulatory protein. Transcription of troA responds to divalent manganese and iron (T. denticola) or manganese and zinc (T. pallidum), and metal-dependent TroR binding to the troA promoter represses troA transcription. We report here the construction and complementation of defined T. denticola ΔtroR and ΔtroA strains to characterize (i) the role of TroA in metal-dependent T. denticola growth and (ii) the role of TroR in T. denticola gene expression. We show that TroA expression is required for T. denticola growth under iron- and manganese-limited conditions. Furthermore, TroR is required for the transcriptional regulation of troA in response to iron or manganese, and deletion of troR results in significant differential expression of more than 800 T. denticola genes in addition to troA These results suggest that (i) TroA-mediated cation uptake is important in metal homeostasis in vitro and may be important for Treponema survival in the host environment and (ii) the absence of TroR results in significant dysregulation of nearly one-third of the T. denticola genome. These effects may be direct (as with troA) or indirect due to dysregulation of metal homeostasis.IMPORTANCETreponema denticola is one of numerous host-associated spirochetes, a group including commensals, pathobionts, and at least one frank pathogen. While most T. denticola research concerns its role in periodontitis, its relative tractability for growth and genetic manipulation make it a useful model for studying Treponema physiology, metabolism, and host-microbe interactions. Metal micronutrient acquisition and homeostasis are highly regulated both in microbial cells and by host innate defense mechanisms that severely limit metal cation bioavailability. Here, we characterized the T. denticolatroABCDR operon, the role of TroA-mediated iron and manganese uptake in growth, and the effects of TroR on global gene expression. This study contributes to our understanding of the mechanisms involved in cellular metal homeostasis required for survival in the host environment.

Reciprocal interactions between cancer and Schwann cells contribute to oral cancer progression and pain.

Pain associated with oral squamous cell carcinoma (oral SCC) decreases quality of life and survival. The interaction between cancer and the peripheral nerves is known to initiate and amplify pain and contribute to carcinogenesis. Schwann cells envelop peripheral nerves and are activated in response to neuronal damage. The contributions of Schwann cells to oral SCC progression and pain are unknown. Using a non-contact co-culture model, we demonstrate that Schwann cells (RSC-96) and oral SCC cells (HSC-3) reciprocally interact to promote proliferation, migration, and invasion. Schwann cell-oral SCC interaction leads to increased production of adenosine, which stimulates cell proliferation and migration of both cell types. The adenosine receptor A2B (ADORA2B) is expressed on RSC-96 cells. We show that supernatant from the RSC-96 cells co-cultured with HSC-3 cells induces increased mechanical hypersensitivity in mice compared to supernatant from control RSC-96 cells. Treatment with the ADORA2B antagonist PSB603 significantly inhibits co-culture interactions - proliferation and migration, and co-culture supernatant induced mechanical hypersensitivity. RSC-96 cells co-cultured with HSC-3 cells secrete increased amounts of the pronociceptive mediator, interleukin-6 (IL-6), which can be reduced by adding PSB603 into the co-culture. Our data support a reciprocal interaction between oral SCC and Schwann cells mediated by adenosine with potential to promote oral SCC progression and pain via increased secretion of IL-6.

Comparison of Real-Time Quantitative PCR with a Chairside Test for Streptococcus Mutans Assessment.

OBJECTIVE: To compare two methods for Streptococcus mutans detection and quantification in the human oral cavity: a chairside commercial test and a molecular-based real-time quantitative polymerase chain reaction (qPCR) method. METHODS: A total of 688 whole saliva samples were collected from 344 children aged 3 and 5 and their biological mothers. Caries status was examined using a World Health Organisation survey method. S. mutans levels were measured using the Dentocult SM Strip mutans test and scored as colony forming units per millilitre of saliva. Meanwhile, bacterial genomic DNA was extracted from the saliva, qPCR was performed with S. mutans species-specific primers, and absolute S. mutans DNA concentrations were obtained and scored as micrograms of DNA per millilitre of saliva. The two methods were compared for sensitivity, specificity, agreement and correlation with caries status. RESULTS: Significantly more participants tested positive for S. mutans by qPCR than in the chairside SM Strip test (82.4% vs 71.4%). When only the highest and lowest test scores were considered, the agreement between the two methods assessing S. mutans colonisation was 0.956. Children with high levels of S. mutans in their saliva were six to eight times more likely to develop dental caries at 5 years old. CONCLUSION: The study provides new evidence supporting the use of the chairside SM Strip test or the qPCR assay for the detection and quantification of S. mutans colonisation in saliva as the analytical approach of choice for caries risk assessment in clinical and epidemiological studies.

Midgut bacterial communities in the giant Asian honeybee (Apis dorsata) across 4 developmental stages: A comparative study.

Bacterial communities are known to play important roles during the developmental stages of insects, but current knowledge of bacteria associated with the midgut of Apis dorsata, the giant Asian honeybee, is limited. Using polymerase chain reaction-denaturing gradient gel electrophoresis analysis (PCR-DGGE) and 16S rRNA sequencing, the aim of this study was to determine the dynamics of bacterial community structure across four A. dorsata life stages in different geographical locations. The results reveal that bacterial diversity increased as the bee progressed through larval stage to newly emerged worker and old worker. However, in the pupal stage, no bands identified as bacteria could be observed. Overall, 2 bacterial phyla (Proteobacteria and Firmicutes) and 4 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Bacilli) were identified, but the frequency varied among the different stages and locations. The classes of Gammaproteobacteria and Bacilli dominated among larval, newly emerged worker and old worker developmental stages.

Characterizing Diversity of Lactobacilli Associated with Severe Early Childhood Caries: A Study Protocol.

Lactobacilli have been consistently associated with dental caries for decades; however, knowledge of this group of bacteria in the etiology of the disease is limited to quantitative elucidation. Nowadays, explicit identification of oral Lactobacillus species is possible, despite their taxonomic complexity. Here we describe a combined approach involving both cultivation and genetic methods to ascertain and characterize the diversity and abundance of the Lactobacillus population in the oral cavities of children with severe early childhood caries (S-ECC). Eighty 3- to 6-year-old children (40 S-ECC and 40 caries free) who were seeking dental care at the Pediatric Dental Clinic of Bellevue Hospital in New York City were invited to participate in this study. Clinical data on socio-demographic information and oral health behavior were obtained from the primary caregiver. The data included a detailed dental examination, children's medical history, and a questionnaire survey. Combined non-stimulated saliva and supra-gingival plaque samples were collected from each child and cultivated on selective media for quantitative measures of lactobacilli levels. The procedure for Lactobacillus species screening will include the random selection of 50 colonies per plate, extraction of DNA from each colony, and genotyping by arbitrarily primed polymerase chain reaction (AP-PCR). Each unique Lactobacillus AP-PCR genotype will be selected for taxonomic assessment by 16S rRNA gene sequencing analysis. Lactobacillus species will be identified by comparing the 16S rRNA sequences with the Ribosomal Database and the Human Oral Microbiome Database. Meanwhile, the same set of clinical samples will be independently subjected to genomic DNA isolation, 16S rRNA amplification with Lactobacillus genus-specific primers, sequencing, and taxonomic identification, both at genus and species levels with a customized pipeline. The distribution and phylogenetic differences of these Lactobacillus species will be compared between children with or without S-ECC. One of the main objectives of this study is to establish a study protocol for the identification and characterization of lactobacilli in the oral cavity. Future caries risk assessments can include lactobacilli counts (quantitative) and the presence/absence of specific cariogenic genetic signatures of a Lactobacillus species (qualitative) associated with S-ECC.

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol-chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae.

Aggregatibacter actinomycetemcomitans serotypes, the JP2 clone and cytolethal distending toxin genes in a Thai population.

AIM: To examine the genetic diversity of Aggregatibacter actinomycetemcomitans in Thai adults. MATERIALS AND METHODS: Subgingival plaque samples from 453 subjects were analysed for A. actinomycetemcomitans serotypes, the presence of the high leukotoxin-producing JP2 clone and cytolethal distending toxin genes (cdtABC) using the polymerase chain reaction technique. In subjects who were positive for cdtABC, restriction fragment length polymorphism analysis was used to identify a single nucleotide polymorphism (SNP) in the cdtB gene at amino acid position 281. The extent and severity of periodontal disease were compared between subjects harbouring different A. actinomycetemcomitans genotypes. RESULTS: Eighty six subjects (19%) were positive for A. actinomycetemcomitans. The JP2 clone was not detected. Serotype c was the most prevalent (57%), followed by serotypes a (33%) and b (7%). Among A. actinomycetemcomitans-positive subjects, 27% were positive for cdtABC. All cdtABC-positive subjects possessed the SNP in the cdtB, which is involved with increased toxin activity. The presence of A. actinomycetemcomitans, but not a specific genotype, was significantly related to increased probing depth and periodontal attachment loss. CONCLUSIONS: Our results confirm the previous findings that genotype distribution of A. actinomycetemcomitans varies between ethnic groups. However, no clear relationship between a specific genotype and periodontal conditions was observed.

Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction.

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.