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Abstract Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability. Yet, an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis, due to limited in vitro translational lacrimal gland (LG) models. Consequently, this remains a major roadblock to discover effective therapies for the restoration of tear film secretion. Herein, the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states. Using a proof-of-concept approach, porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform. This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity). DNA damage-induced senescence and cell death was induced with etoposide, and LG organoid hypofunction and senescence-associated pathogenesis were observed. To confer DNA protection against aging, a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group, was applied here to prevent LG cellular senescence for the first time. HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic, metabolomic and proteomic levels. Thus, M3DB platforms could generate functional and DNA damage-induced senescence LG organoids, and this latter damage could be prevented with HMGB1-Box A gene therapy.

Background Antifungal resistance is a major problem, commonly caused by drug-efflux pump overexpression. To evaluate if chitosan could be effective in drug-resistant Candida infections, we investigated the effects of efflux pumps on antifungal activity of chitosan.Materials and Methods The minimal fungicidal concentration (MFC) of oligomer (7–9 kD) and polymer (900–1,000 kD) chitosan against Saccharomyces cerevisiae and Candida albicans were evaluated by broth and agar dilution methods. The MFCs of S. cerevisiae with single deletion of efflux pump genes, with deletion of seven efflux pumps (AD∆), and AD∆ overexpressing C. albicans efflux pump genes (CDR1, CDR2 and MDR1) were determined. C. albicans with homozygous deletions of CDR1 and of CDR2 were generated using CRISPR-Cas9 system and tested for chitosan susceptibility.Results While deleting any individual efflux pump genes had no effect on chitosan susceptibility, simultaneous deletion of multiple pumps (in AD∆) increased sensitivity to both types of chitosan. Interestingly, the overexpression of CDR1, CDR2 or MDR1 in AD∆ barely affected its sensitivity. Moreover, C. albicans with homozygous deletions of CDR1 and/or CDR2 showed similar sensitivity to wildtype.Conclusion Thus, C. albicans susceptibility to chitosan was not affected by drug-efflux pumps. Chitosan may be a promising antifungal agent against pump-overexpressing azole-resistant C. albicans.

Abstract Concerns exist about prolonged breastfeeding increasing dental caries risk, but evidence is mixed. This 2-year cohort study followed 486 toddlers, to examine the association between breastfeeding duration and caries at age 3. Caregivers reported feeding practices and potential confounders every 6 months. “Full breastfeeding” was defined as feeding breastmilk without formula milk regardless of other foods/liquids, whereas “any breastfeeding” was feeding breastmilk with/without formula milk. A calibrated dentist performed dental examinations. We used multivariable log-binomial and negative binomial regressions to estimate relative risks (RRs) and 95% confidence intervals (CIs) for caries prevalence and severity, adjusted for confounders. At 3-year-old, 60.3% of children exhibited caries (mean decayed-and-filled-teeth, dft: 3.3). Notably, full breastfeeding for 6–17 months reduced caries prevalence (RR = 0.84, 95%CI 0.73–0.98 for 6–11 months; RR = 0.78, 95%CI 0.63–0.96 for 12–17 months). Conversely, any breastfeeding ≥ 18 months significantly increased caries risk (RR = 1.45, 95%CI 1.31–1.60). Full breastfeeding ≥ 6 months or any breastfeeding 6–17 months was associated with lower dft scores in children. Our findings suggest a complex relationship between breastfeeding duration and caries. Full breastfeeding for moderate durations (6–17 months) offers protective benefits, while any breastfeeding ≥ 18 months increases risk in this population.

The roles of antibodies secreted by subsets of B cells in dengue virus (DENV) infection have been extensively studied, yet, the contribution of tissue-homing B cells to antiviral immunity remains unclear. In this study, we performed a comprehensive analysis of B cell subpopulations in peripheral blood samples from DENV-infected patients using single-cell RNA-sequencing (scRNA-seq) datasets and flow cytometry. We showed that plasma cells (PCs) and plasmablasts (PBs) were the predominant B cell populations during the acute phase of secondary natural DENV infection, but not in convalescent phase nor in healthy controls. Interestingly, these cells expressed proliferation, adhesion, and tissue-homing genes, including SELPLG, a homing marker of the skin, the initial infected site of DENV. Flow cytometry analysis confirmed a significant upregulation of cell surface expression of a cutaneous lymphocyte-associated antigen (CLA) encoded by SELPLG in PCs and PBs, compared to naive and memory B cells from the same patients. The analysis of an independent single-cell B-cell receptor sequencing (scBCR-seq) dataset of DENV-infected patients revealed that the peripheral blood PCs and PBs exhibited the highest clonal expansion in secondary DENV infection compared to other B cell subsets. These clonally expanded cells also expressed the highest levels of tissue-homing genes, including SELPLG. In addition, by utilizing a public scRNA-seq dataset of SARS-CoV2 infection, we demonstrated the upregulation of several tissue-homing genes in PCs and PBs. Our study provides evidence for the potential roles of tissue-homing B cell subsets in the context of immune responses against viral infections in humans.

Abstract Background Patients with fixed orthodontic appliances have higher plaque accumulation and gingival inflammation. Our aim was to compare the effectiveness of a light emitting diode (LED) toothbrush with a manual toothbrush in reducing dental plaque and gingival inflammation in orthodontic patients with fixed appliances, and to investigate the effect of the LED toothbrush on Streptococcus mutans (S. mutans) biofilm in vitro. Methods Twenty-four orthodontic patients were recruited and randomly assigned into 2 groups: (1) started with manual and (2) started with LED toothbrushes. After a 28-day usage and 28-day wash-out period, the patients switched to the other intervention. The plaque and gingival indices were determined at baseline and 28 days after each intervention. The patients’ compliance and satisfaction scores were collected using questionnaires. For the in vitro experiments, S. mutans biofilm was divided into 5 groups (n = 6) with 15-, 30-, 60-, or 120-sec LED exposure, and without LED exposure as a control group. Results There was no significant difference in the gingival index between the manual and LED toothbrush groups. The manual toothbrush was significantly more effective in reducing the plaque index in the proximal area on the bracket side (P = 0.031). However, no significant difference was found between the two groups in other areas around the brackets or on the non-bracket side. After LED exposure in vitro, the percentages of bacterial viability after LED exposure for 15–120 s were significantly lower compared with the control (P = 0.006). Conclusion Clinically, the LED toothbrush was not more effective in reducing dental plaque or gingival inflammation than the manual toothbrush in orthodontic patients with fixed appliances. However, the blue light from the LED toothbrush significantly reduced the number of S. mutans in biofilm when it was exposed to the light for at least 15 s in vitro. Clinical Trial Registration Thai Clinical Trials Registry (TCTR20210510004). Registered 10/05/2021.

Abstract Background In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis are not well understood. To address these questions, we performed a comparative analysis of publicly available single-cell transcriptomes of healthy adult human DP to 5 other reference tissues: peripheral blood mononuclear cells, bone marrow, adipose tissue, lung, and skin. Results Our analysis revealed that DP resident cells have a unique gene expression profile when compared to the reference tissues, and that DP fibroblasts are the main cell type contributing to this expression profile. Genes coding for pleiotrophin (PTN) and midkine (MDK), homologous heparin-binding growth-factors, possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP resident cells, including Schwann cells, mesenchymal stem cells and odontoblasts, mediated by PTN and MDK. Conclusions DP fibroblasts emerge as unappreciated players in DP homeostasis, mainly through their crosstalk with glial cells. These findings suggest that fibroblast-derived growth factors possess major regulatory functions and thus have a potential role as dental therapeutic targets.

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Summary: Here, we present a computational approach for investigating highly variable genes (HVGs) associated with biological pathways of interest, across multiple time points and cell types in single-cell RNA-sequencing (scRNA-seq) data. Using public dengue virus and COVID-19 datasets, we describe steps for using the framework to characterize the dynamic expression levels of HVGs related to common and cell-type-specific biological pathways over multiple immune cell types.For complete details on the use and execution of this protocol, please refer to Arora et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Enterococcus faecalis and Candida albicans are frequently found in persistent endodontic infection and could remain in dentinal tubules despite intracanal medication with calcium hydroxide (Ca(OH)2), a commonly used medication. Thus, an effective and safe antimicrobial medication against such refractory infection is necessary in endodontic retreatment, so we aimed to test the efficacy of chitosan paste against these microorganisms compared with Ca(OH)2 in root canals of extracted human teeth. Methods Thirty-six sterilized human root samples prepared from extracted premolars and upper maxillary incisors were infected with E. faecalis for 14 days, while 32 were infected with C. albicans for 48 h, for mature biofilm formation. The samples were assigned to 6 groups of intracanal medications: Group 1: no medication (negative control); Group 2: 20% Polyethylene glycol (PEG); Group 3: 20% Propylene glycol (PG); Group 4: Ca(OH)2; Group 5: Chitosan + PEG; and Group 6: Chitosan + PG. After 7 days, intracanal surface dentin was harvested using Protaper next, resuspended, serially diluted and spread on Brain–Heart-Infusion agar (for E. faecalis) and Yeast Extract-Peptone-Dextrose agar (for C. albicans) for colony count. Antimicrobial efficacy was determined as percentage of remaining colony forming unit (CFUs) relative to negative control and analyzed using One-way ANOVA and post-hoc Games-Howell test. The significance level was set at 0.05. Results For E. faecalis, chitosan + PG had significantly higher antibacterial activity than Ca(OH)2 (P = 0.039). Chitosan + PEG and chitosan + PG medication significantly reduced viable bacteria compared with negative control, PEG and PG (P = 0.001, 0.003, 0.024, respectively for chitosan + PEG; P = 0.002, 0.003, 0.014, respectively for chitosan + PG). For C.albicans, chitosan + PEG and chitosan + PG were not significantly different from Ca(OH)2. However, Chitosan + PEG and chitosan + PG, but not Ca(OH)2, showed a significantly lower level of remaining CFUs compared with negative control (P = 0.013 and 0.005, respectively). Conclusion Chitosan paste showed better efficacy in reducing viable E. faecalis biofilm when compared to Ca(OH)2 after 7-day intracanal medication in this in vitro root canal model. It could also significantly reduce viable C. albicans, but was not significantly different from Ca(OH)2.

Abstract Background Although topical steroids are an effective treatment for oral lichen planus, they can have suppressive effects on oral immunity and predispose the patients to Candida overgrowth. Lactoferrin is a crucial local immunity protein in the oral cavity with important antimicrobial activity. The aim of this study was to prospectively investigate salivary lactoferrin secretion levels and Candida colonization in oral lichen planus patients treated with fluocinolone acetonide 0.1% in orabase. Methods Saliva samples were collected from 15 oral lichen planus subjects who had never received topical steroid treatment prior to this study and 15 healthy volunteers to determine their salivary lactoferrin levels using an enzyme-linked immunosorbent assay and to investigate the presence of oral Candida species at baseline and 3 months after treatment with fluocinolone acetonide 0.1% in orabase. Statistical analysis was performed to compare lactoferrin secretion and Candida colonization levels between the groups using the Mann–Whitney U test for independent data or the Wilcoxon Signed-Rank test for paired data. Results The salivary lactoferrin secretion level was not significantly different between the control group and oral lichen planus patients or between before and after treatment with fluocinolone acetonide 0.1% in orabase (P > 0.05). Candida was detected in 11 (73.33%) healthy volunteers, 8 (53.33%) oral lichen planus patients before treatment, and 9 (60%) oral lichen planus patients after treatment with fluocinolone acetonide 0.1% in orabase. There was no significant difference in Candida counts between the groups (P > 0.05). Conclusion Our study indicates that using fluocinolone acetonide 0.1% in orabase to treat oral lichen planus for 3 months did not affect salivary lactoferrin protein secretion or Candida carriage. Trial registration The trial was registered at the Thai Clinical Trials Registry (TCTR identifier: TCTR20200723002).

Objectives: Virgin coconut oil (VCO) pulling has antimicrobial activity and has been promoted as beneficial to oral health; however, limited information exists on its clinical effectiveness. This study aimed to compare the microbiological effects of VCO with palm oil (PO) pulling when used as an adjunctive oral hygiene care. Materials and Methods: Thirty-six volunteers with gingival inflammation were randomly assigned to start with (1) VCO (test) and (2) PO (control) interventions in a crossover design. Oil pulling was performed for 28 days adjunctively to oral hygiene routine. After a 21-day wash-out period, the participants switched the oil type and restarted the protocol. Plaque samples were collected for microbial culture at baseline, after the first oil pulling period, after washout, and after the second oil pulling period. The total, aerobic, and anaerobic bacteria and Mutans streptococci (MS) counts were recorded. The mean differences between VCO and PO were compared by paired t-test. Results: The number of total, aerobic, or anaerobic bacteria after 28 days of oil pulling was not significantly different from baseline in both PO and VCO groups. However, PO pulling demonstrated a significant reduction from baseline of MS count (P = 0.010), while VCO pulling showed no significant reduction. There was no statistically significant difference in the mean changes of any microbiological parameters between the two treatments. Conclusions: VCO pulling did not show statistically significant superior benefit against plaque bacteria over PO pulling. Using PO pulling as an adjunctive oral hygiene care may reduce the number of MS, but this requires further investigations.

Summary: Effective clinical management of acute dengue virus (DENV) infection relies on the timing of suitable treatments during the disease progression. We analyzed single-cell transcriptomic profiles of the peripheral blood mononuclear cell samples from two DENV patients, collected daily during acute phase and also at convalescence. Key immune cell types demonstrated different dynamic responses over the course of the infection. On the day before defervescence (Day −1), we observed the peak expression of several prominent genes in the adaptive immunological pathways. We also characterized unique effector T cell clusters that expressed skin-homing signature genes at Day −1, whereas upregulation of skin and gut homing genes was also observed in plasma cells and plasmablasts during the febrile period. This work provides an overview of unique molecular dynamics that signify the entry of the critical phase, and the findings could improve the patient management of DENV infection.

A comprehensive understanding of dental pulp cellular compositions and their molecular responses to infection are crucial for the advancement of regenerative dentistry. Here, we presented a pilot study of single-cell transcriptomic profiles of 6,810 pulpal cells isolated from a sound human maxillary third molar and three carious teeth with enamel and deep dental caries. We observed altered immune cell compositions of the dental pulp in deep, but not enamel ones. Differential expression analysis revealed up-regulation of several pro-inflammatory, anti-inflammatory, and mineralization-related genes in the immune and stromal cells of the deep dental caries. Making use of an algorithm for predicting cell-to-cell interactions from single-cell transcriptomic profiles, we showed an increase in cell-cell interactions between B cells, plasma cells and macrophages, and other cell types in deep dental caries, including those between TIMP1 (odontoblasts)—CD63 (myeloid cells), and CCL2 (macrophages)—ACKR1 (endothelial cells). Collectively, our work highlighted the single-cell level gene regulations and intercellular interactions in the dental pulps in health and disease.

Background: Dengue infection is a global health threat. While symptomatic cases contribute to morbidity and mortality, the majority of infected people are asymptomatic but serve as an important reservoir. However, the kinetics of viremia in asymptomatic infections remains unknown. Methods: We enrolled 279 hospital-based symptomatic index cases and quantified dengue virus (DENV) RNA at enrollment and at the day of defervescence. To identify asymptomatic cases, 175 household members of index cases were monitored for clinical symptoms during follow-up, and blood was taken twice weekly to test for and quantify DENV RNA until cleared. Results: We detected DENV in thirteen asymptomatic household members (7.43%). Their DENV serotypes were primarily the same as those of their family index cases. The median peak DENV viremia in asymptomatic subjects was lower than that of symptomatic individuals during the febrile phase, and the viral decay rate was slower in asymptomatic infections. Conclusions: DENV level and kinetics in asymptomatic individuals differed significantly from those of symptomatic cases. Despite the lower viremia, the slower decay rate in asymptomatic infections could lead to their prolonging the infectious reservoir. The improvement of transmission control to prevent such long-lived asymptomatic infections from transmitting the DENV is needed.

BackgroundDengue virus (DENV) infection has a global impact on public health. The clinical outcomes (of DENV) can vary from a flu-like illness called dengue fever (DF), to a more severe form, known as dengue hemorrhagic fever (DHF). The underlying innate immune mechanisms leading to protective or detrimental outcomes have not been fully elucidated. Helper innate lymphoid cells (hILCs), an innate lymphocyte recently discovered, functionally resemble T-helper cells and are important in inflammation and homeostasis. However, the role of hILCs in DENV infection had been unexplored.MethodsWe performed flow cytometry to investigate the frequency and phenotype of hILCs in peripheral blood mononuclear cells from DENV-infected patients of different disease severities (DF and DHF), and at different phases (febrile and convalescence) of infection. Intracellular cytokine staining of hILCs from DF and DHF were also evaluated by flow cytometry after ex vivo stimulation. Further, the hILCs were sorted and subjected to transcriptome analysis using RNA sequencing. Differential gene expression analysis was performed to compare the febrile and convalescent phase samples in DF and DHF. Selected differentially expressed genes were then validated by quantitative PCR.ResultsPhenotypic analysis showed marked activation of all three hILC subsets during the febrile phase as shown by higher CD69 expression when compared to paired convalescent samples, although the frequency of hILCs remained unchanged. Upon ex vivo stimulation, hILCs from febrile phase DHF produced significantly higher IFN-γ and IL-4 when compared to those of DF. Transcriptomic analysis showed unique hILCs gene expression in DF and DHF, suggesting that divergent functions of hILCs may be associated with different disease severities. Differential gene expression analysis indicated that hILCs function both in cytokine secretion and cytotoxicity during the febrile phase of DENV infection.ConclusionsHelper ILCs are activated in the febrile phase of DENV infection and display unique transcriptomic changes as well as cytokine production that correlate with severity. Targeting hILCs during early innate response to DENV might help shape subsequent immune responses and potentially lessen the disease severity in the future.

Background: Mosquito allergy is common in tropical countries but remains under-diagnosed. This may be due to the lack of knowledge and diagnostic tools for tropical mosquito allergens. Objective: We aimed to characterize allergens from tropical mosquito species and investigate IgE reactivity in mosquito-allergic patients to the salivary gland proteins from these mosquitoes. Methods: Salivary gland extract (SGE) from 4 mosquito species, highly distributed in the tropics, including Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles dirus b, were studied. SGE-specific IgE and IgG ELISA were developed, and serum from 64 mosquito-allergic and 22 non-allergic healthy control subjects was assayed. Further investigations using IgE-immunoblots followed by mass spectrometry analysis were performed to identify and characterize allergens from each species. Results: Mosquito-allergic subjects have detectable serum IgE to SGE derived from local mosquito species, while the IgE levels to Aedes communis using commercially available ELISA were mostly minimal. IgE-immunoblot analysis and mass spectrometry identified 5 novel mosquito allergens from A. albopictus (Aed al 2, Aed al 3), C. quinquefasciatus (Cul q 2.01, Cul q 3), and A. dirus b (Ano d 2). Interestingly, 4 of the 5 new allergens belong to the D7 protein family. Conclusions & clinical relevance: Five novel allergens from 3 tropical mosquito species were characterized. The majority of mosquito-allergic subjects who live in the tropics have IgE reactivity to these allergens. Our study paves the way for the development of diagnostic tests, component-resolved diagnostics, and future immunotherapy for mosquito allergy in tropical countries. Keywords: Allergens, Insect allergy, IgE, Mosquito, Tropical species

The aim of this study was to describe the changes in oral health policies and guidelines in response to the Coronavirus disease 2019 (COVID-19) pandemic in different countries and regions around the world. Information on oral health policies and guidelines from 9 countries (Canada, China including Hong Kong, Egypt, India, Japan, New Zealand, Nigeria, Switzerland, and Thailand) were summarized, and sources of the information were mostly the national or regional health authorities and/or dental council/associations. The changes made to the oral health guidelines depended on the severity of the COVID-19 pandemic. This included suspension of non-emergency dental care services at the peak of the COVID-19 outbreak, and easing the restrictions on non-essential and elective dental care when the pandemic became under control. The COVID-19 risk mitigation strategies include strict adherence to infection control practices (use of hand sanitizers, facemask and maintaining social distancing), reducing the amount of aerosol production in the dental setting, and managing the quality of air in the dental treatment rooms by reducing the use of air conditioners and improving air exchange. The COVID-19 pandemic has shown a major impact on dental practice. Dental professionals are trying to adapt to the new norms, while the medium to long-term impact of COVID-19 on dentistry needs further investigation.

When the dose of conventional disease-modifying anti-rheumatic drugs (cDMARDs) is tapered in rheumatoid arthritis (RA) patients who achieve sustained remission, biomarkers for predicting disease relapse may be needed. A prospective, unblinded cohort study was conducted in nine RA patients with remission. Peripheral blood samples were collected at baseline and at 6, 12, and 24 weeks after cDMARD dose reduction (dose of combination regimens reduced to 50%) to determine the number of regulatory Foxp3+T cells (Tregs) and other T cell subpopulations as well as Treg suppressive activity. Additionally, plasma levels of 14 cytokines at each time-point were measured via flow cytometry. Univariate and multivariate analyses were performed to identify the factor(s) associated with RA relapse during the observational period. In univariate analysis, Treg suppression and DAS28 and VAS scores were associated with RA relapse after cDMARD dose tapering. However, in multivariate analysis, only Treg suppressive activity (

Hyposalivation is an important problem in elders and could interfere with several oral functions and microbial ecology. While the number of independent elders who retain more natural teeth increases worldwide, few studies examined hyposalivation in this population. Thus, this study aims to examine relationships between hyposalivation, oral health conditions and oral Candida colonization in independent dentate elders and evaluate factors associated with salivary flow and Candida carriage. We conducted a cross-sectional study in fifty-three dentate elders (≥65 years old with at least 4 pairs of posterior occlusal contacts) with no, or well-controlled, systemic conditions. Participants were interviewed for medical history, subjective dry mouth symptoms, oral hygiene practices and denture information. Unstimulated and stimulated salivary flow rates, objective dry mouth signs, gingival, tongue-coating, and root-caries indices were recorded. Stimulated saliva was cultured on Sabouraud-dextrose agar for Candida counts. Candida species were identified using chromogenic Candida agar and polymerase chain reaction. Statistical significance level was set at p

BackgroundAggregatibacter actinomycetemcomitans, a periodontal pathogen, secretes a cytolethal distending toxin (AaCDT) that causes host cell cycle arrest and cell death. Although CDT could be an important virulence factor, it is unclear how it enters the nucleus to exert its cytotoxicity.ObjectiveTo investigate the mechanisms of AaCDT by genome-wide screening for host mutations that confer resistance to the catalytic subunit, AaCdtB, in a Saccharomyces cerevisiae model.MethodsWe transformed the yeast haploid deletion library, a collection of yeast mutants with single gene deletions of virtually all non-essential ORFs in the genome, with plasmids carrying galactose-inducible AaCdtB. Yeast mutants that showed resistance to AaCdtB were selected and rescreened by a spotting assay. AaCdtB expression was confirmed by western blot analysis; any mutants that showed no or weak expression of AaCdtB were omitted from the analysis. The lists of genes whose mutations confer resistance to AaCdtB were analyzed for Gene Ontology (GO) term enrichments. Localization of AaCdtB-EGFP was examined using fluorescent microscopy. Nuclear localization relative to EGFP control was calculated and compared to wild-type.ResultsOut of approximately 5,000 deletion mutants, we isolated 243 mutants that are resistant to AaCdtB. GO analyses indicated that genes associated with organic anion transport are significantly enriched (16 genes). Furthermore, several genes associated with the nucleus and endoplasmic reticulum (ER) were identified. Localization studies of AaCdtB, in mutants with the deletion of genes associated with the GO term organic anion transport, showed lower nuclear localization than wild-type. The results suggest that these genes may be required for AaCdtB translocation into the nucleus and its cytotoxicity.ConclusionThe genome-wide screen in the yeast deletion library allowed us to identify a large number of host genes required for AaCdtB cytotoxicity. Further investigation could lead to more insights into the mechanisms of CdtB intoxication.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form—dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.

We prepared a monoclonal antibody (mAb 1C1) showing specificity for artemisinin (AM) and artesunate (AS), and we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) using this novel mAb. Moreover, we prepared a recombinant antibody derived from mAb 1C1 in order to overcome insufficient mAb production by hybridoma culture. A recombinant antigen-binding fragment (Fab) was easily constructed using antibody manipulation technologies and was produced by microorganisms in high yield. We herein review immunochemical approaches for analysis of the antimalarial drugs AM and AS that were able to yield analysis results for multiple samples in a short period of time using simple and reliable protocols.

Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

BACKGROUND:Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection. METHODS:Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured. RESULTS:iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated. CONCLUSION:iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future.

Immune cells called mast cells can hinder rather than help the body's response to dengue virus, which suggests that mast cell products could be used as biomarkers to identify severe forms of the disease.

Without exposure to any DNA-damaging agents, non-dividing eukaryotic cells carry endogenous DNA double-strand breaks (EDSBs), or Replication-Independent (RIND)-EDSBs. In human cells, RIND-EDSBs are enriched in the methylated heterochromatic areas of the genome and are repaired by an ATM-dependent non-homologous end-joining pathway (NHEJ). Here, we showed that Saccharomyces cerevisiae similarly possess RIND-EDSBs. Various levels of EDSBs were detected during different phases of the cell cycle, including G0. Using a collection of mutant yeast strains, we investigated various DNA metabolic and DNA repair pathways that might be involved in the maintenance of RIND-EDSB levels. We found that the RIND-EDSB levels increased significantly in yeast strains lacking proteins involved in NHEJ DNA repair and in suppression of heterochromatin formation. RIND-EDSB levels were also upregulated when genes encoding histone deacetylase, endonucleases, topoisomerase, and DNA repair regulators were deleted. In contrast, RIND-EDSB levels were downregulated in the mutants that lack chromatin-condensing proteins, such as the high-mobility group box proteins, and Sir2. Likewise, RIND-EDSB levels were also decreased in human cells lacking HMGB1. Therefore, we conclude that the genomic levels of RIND-EDSBs are evolutionally conserved, dynamically regulated, and may be influenced by genome topology, chromatin structure, and the efficiency of DNA repair systems.

To investigate applications of chitosan as antibacterial agent for endodontic treatments, we tested its activity against Enterococcus faecalis standard strain (ATCC29212) and clinical isolates. We determined the minimum bactericidal concentration (MBC) of 6 types of chitosan against ATCC29212; the most effective types were selected for further tests. Four clinical isolates were cultured from endodontically treated-teeth and identified by biochemical assays and polymerase chain reactions. Bacterial cultures were exposed to 1,700 and 2,100 kDa chitosan at MBC for 1, 3, 5, 10, and 60 min in time-kill assays and plated on brain-heart-infusion (BHI) agar for colony counts. Both types of chitosan showed significantly lower numbers of remaining bacteria (log colony forming units per millimeter, logCFUs/mL) than negative controls (0.1% acetic acid and BHI) at 10 min, and completely eliminated the bacteria at 60 min for all strains. Thus, chitosan could be developed as alternative biocompatible antimicrobial irrigant/medication for endodontic treatments. [ABSTRACT FROM AUTHOR]

Objective: To study the kinetics of endocrine changes in adult patients with dengue hemorrhagic fever (DHF) and dengue fever (DF) and association to disease severity. Materials and Methods: This hospital-based observational clinical study involved 48 adult patients, 32 with DHF and 16 with DF, who presented with clinical features and positive serological testing for dengue infection on the day of diagnosis (D1). Serial circulating ACTH and cortisol concentrations were determined on D1, day of defervescence (Ddef), day 1 of convalescence (DC1 or 24 h after Ddef), day 2 of convalescence (DC2 or 48 h after Ddef), at 2-week follow-up (F1), and 2-month follow-up (F2). Results: The median cortisol concentration in the DHF group was higher on D1 than that at F2. This was not found in patients with DF who had no difference of median cortisol concentration at D1 and F2. The median ACTH concentrations in both DHF and DF groups were low on D1 and trended toward recovery at F2. There was association between circulating ACTH and cortisol concentrations at D1 in patients with the severity of DHF (r = 0.309, p = 0.042) but not of DF. Conclusion: The ACTH and cortisol responses were associated with severity of dengue infection and recovery after 2 months. [ABSTRACT FROM AUTHOR]

Background: Spontaneous abortion, or miscarriage, is a complication of pregnancy which can severely affect women both physically and psychologically. We investigated the associations of periodontitis and periodontopathic bacteria with spontaneous abortion.Methods: We conducted a matched case-control study in two tertiary hospitals in Khon Kaen, Thailand. Cases were 85 women with spontaneous abortion at <20 weeks of gestation matched to 85 controls on age, gestational age, and hospital. Full-mouth periodontal examinations were performed. Periodontitis was defined as at least one site with probing depth ≥5 mm and clinical attachment level ≥2 mm at the same site. Subgingival plaque samples were collected to determine the levels of Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum using real time polymerase chain reaction.Results: The cases were significantly more likely to have periodontitis (50.6%) than the controls (21.2%; P = 0.007). Conditional logistic regression revealed a crude odds ratio (OR) of 4.1 for the association between periodontitis and spontaneous abortion (95% confidence interval [CI] = 1.9-8.9, P = 0.001). The OR decreased, but was still significant, after controlling for previous miscarriage (OR = 3.3, 95% CI = 1.4-7.8, P = 0.006). There was no significant association between the levels of periodontopathic bacteria and spontaneous abortion. Increased levels of P. gingivalis and F. nucleatum were associated with periodontitis in both case and control groups. Association between increased T. forsythia levels and periodontitis was observed only in the case women.Conclusions: Periodontitis was more common in women with spontaneous abortions as compared with matched controls. Levels of periodontopathic bacteria was not associated with spontaneous abortion in this population. [ABSTRACT FROM AUTHOR]

Mannan (mannosylated glycoproteins) in the outermost layer of the Candida cell wall may be the first molecules that interact with host dendritic cells (DCs) and activate immune responses that determine disease outcomes. However, little is known about how different mannan structures of common oral Candida species affect DC activation. The effects of heat-inactivated (HI) yeast cells and soluble mannan of Candida albicans, Candida parapsilosis, and Candida dubliniensis on bone marrow-derived DC (BMDC) responses were compared. HI Candida and the mannan exhibited different effects on BMDC activation and functions, which could be due to other carbohydrate compositions in the yeast cell wall. Among Candida mannan, the C. albicans mannan was the weakest stimulus and induced only interferon (IFN)-γ production. This suggests the possibility that C. albicans mannan may skew T helper (Th) responses from protective Th17 toward Th1. In contrast, C. parapsilosis mannan caused strong BMDC activation and high production of several proinflammatory cytokines which possibly promote hyperinflammation. Meanwhile, C. dubliniensis mannan induced moderate BMDC responses, which may correlate with its lower pathogenicity. Therefore, mannan of each Candida species play distinct roles in DC responses and may be involved in the immunopathogenesis and disease severity of oral candidiasis as well as other Candida infection. [ABSTRACT FROM AUTHOR]

Objective: To compare the effect of simulated bleaching with a 10% carbamide peroxide (CP) or a 40% hydrogen peroxide (HP) system on surface roughness of resin composite and resin-modified glass ionomer cement (RMGI) and streptococcal biofilm formation on these surfaces. Methods and Materials: Specimens of nanofilled resin composite and RMGI (n=108 each) were randomly divided into three groups (n=36 each): no treatment control, 10% CP, and 40% HP. The surface roughness values (Ra) were measured before and after treatments. The specimens in each group were randomly divided into three subgroups (n=12) and incubated with Streptococcus mutans, Streptococcus sanguinis, and trypticase soy broth control for 24 hours. Biofilm formation was quantified by crystal violet staining, and the structure was visualized by scanning electron microscopy. The differences between the mean changes in Ra between the 10% CP and 40% HP groups of each material were evaluated with an independent t-test. The quantity of biofilm formation on each material was analyzed with one-way analysis of variance with the post hoc Tukey test (α=0.05). Results: Surface roughness significantly increased after bleaching in all groups. There was no significant difference between the 10% CP and 40% HP groups of each material. For S. mutans biofilm formation, bleaching with 10% CP and 40% HP increased biofilm on both materials compared to controls. However, S. sanguinis biofilm formation was significantly higher on bleached resin composite but not on RMGI specimens. Conclusions: Simulated bleaching with 10% CP or 40% HP increased both surface roughness and biofilm formation on resin composite and RMGI, except for S. sanguinis biofilm on RMGI. [ABSTRACT FROM AUTHOR]

Objective: To compare the effectiveness of a motionless ultrasonic toothbrush to a manual toothbrush in reducing dental plaque, gingival inflammation, and mutans streptococci in patients with fixed orthodontic appliances. Materials and Methods: Twenty-five orthodontic patients were recruited to this crossover study. The patients were randomized into two groups starting with manual or motionless ultrasonic toothbrushes for 30 days. After a 30-day washout period, the patients switched to the other toothbrush type for 30 days. Plaque and gingival indices were evaluated by two calibrated-blinded examiners before and after each 30-day period of brushing. Salivary samples were also collected for quantification of mutans streptococci. Results: On the bracket side, the motionless ultrasonic toothbrush showed a significantly higher mean plaque index bracket score after 30-day usage than baseline ( P = .049), while the manual toothbrush group showed no difference between the before and after brushing periods ( P = .10). The changes in plaque index bracket score were significantly more favorable in the manual toothbrush group than in the ultrasonic toothbrush group ( P = .04). In contrast, no difference was observed on the nonbracket side. There was no significant difference in the changes of gingival index or the numbers of mutans streptococci between the two groups. Conclusion: Manual toothbrushing performed better than brushing with the motionless ultrasonic toothbrush in plaque removal on the bracket side in orthodontic patients. However, no difference was observed in terms of gingival status and the numbers of mutans streptococci. [ABSTRACT FROM AUTHOR]

The macrophage migration inhibition techique was emplyed to study the debvelopment of human maternal cell-met\dilated immune reactions to plecental antigens during the firs pregnancy. Cell-mediated immune reaction to pooled antigens from five placentas could not be demonstrated during the first trimester, In the th month. peripheral blood leucocyte's from seven out of eight primigravidous women tested were ravtive t placental antigens, The one nonreactor became reactive by the th month. All of the primigravidous women tested during subsequent months were immune reactivity in the course of the first pregancy. [ABSTRACT FROM AUTHOR]