Beneficial Effects of Indigenous Probiotics in High-Cholesterol Diet-Induced Hypercholesterolemic Rats.

Hypercholesterolemia is a significant risk factor for cardiovascular disease and metabolic disorders. Probiotics are the essential constituents of the gastrointestinal microbiota that provide health-promoting effects. Cholesterol-lowering activity is a specific property of probiotics, improving the cholesterol metabolism without adverse effects. Thus, the purpose of this study was to investigate the hypocholesterolemic effect of single and mixed cholesterol-lowering probiotic strains (including Limosilactobacillus reuteri TF-7, Enterococcus faecium TF-18, and Bifidobacterium animalis TA-1) in high-cholesterol diet (HCD)-induced hypercholesterolemic rats. The results showed that the administration of single probiotics contributed to a reduction in the body weight gain, visceral organ indexes, hyperlipidemia, and hepatic steatosis and also an improvement in the gastrointestinal microbiota. Besides the effect of single cholesterol-lowering probiotics, three probiotics strains could also synergize their hypocholesterolemic effect when administered simultaneously. These findings indicate that three cholesterol-lowering probiotic strains are suitable for development as probiotic supplements to reduce the risk of diseases caused by cholesterol and exert health benefits with synergistic effect when administered simultaneously.

Identification of Pathogenic and Opportunistic Yeasts in Pigeon Excreta by MALDI-TOF Mass Spectrometry and Their Prevalence in Chon Buri Province, Thailand.

Pigeon excreta can cause environmental and public health issues, particularly in urban and public areas. They are reservoirs of several human pathogens including fungi, bacteria, and viruses. Epidemiological data of pathogenic and opportunistic yeasts in pigeon droppings in Chon Buri, one of the most reputable tourist cities of Thailand, are scarce. The present study aimed to identify yeasts in pigeon droppings by MALDI-TOF mass spectrometry, and to study their prevalence in Chon Buri, Thailand. A total of 200 pigeon fecal samples were collected randomly from all 11 districts of Chon Buri. A sum of 393 yeast-like colonies were isolated on Sabourand's dextrose agar and CHROMagar media. These isolates were further confirmed for their species by MALDI-TOF MS. Twenty-four yeast species belonging to 11 different genera were identified in pigeon fecal samples. Candida spp., predominantly C. krusei (14.32%), were the most prevalent yeast species. Other yeast species, including C. glabrata (12.73%), C. metapsilosis (11.93%), Lodderomyces elongisporus (10.87%), C. tropicalis (7.16%), C. albicans (5.83%), and Cryptococcus neoformans (4.77%) were identified. This study provides valuable epidemiological data and diversity of yeasts in pigeon droppings in Chon Buri, Thailand, and also supports the use of MALDI-TOF MS for yeast identification and epidemiological surveillance.

Novel Application of 3D Scaffolds of Poly(E-Caprolactone)/Graphene as Osteoinductive Properties in Bone Defect.

OBJECTIVE: Scaffolds provided a surface on which cells could attach, proliferate, and differentiate. Nowadays, bone tissue engineering offers hope for treating bone cancer. Poly(e-caprolactone) (PCL)/graphene have capability as an osteogenic and regenerative therapy. It could be used to produce bone tissue engineering scaffolds. The purpose of this study was to investigate the ability of PCL/graphene to enhance the osteoinductive mechanism. MATERIALS AND METHODS: The PCL/graphene scaffold was developed utilizing a particulate-leaching process and cultured with osteoblast-like cells MG63 at 0.5, 1.5, and 2.5 wt% of graphene. We evaluated the porosity, pore size, migratory cells, and cell attachment of the scaffold. STATISTICAL ANALYSIS: Data was expressed as the mean ± standard error of the mean and statistical analyses were performed using one-way analysis of variance and Tukey's post hoc at a level of p-value < 0.05. RESULTS: Porosity of scaffold with various percentage of graphene was nonsignificant (p > 0.05). There were differences in the acceleration of cell migration following wound closure between groups at 24 hours (p < 0.01) and 48 hours (p < 0.00). Adding the graphene on the scaffolds enhanced migration of osteoblast cells culture and possibility to attach. Graphene on 2.5 wt% exhibited good characteristics over other concentrations. CONCLUSION: This finding suggests that PCL/graphene composites may have potential applications in bone tissue engineering.

Optimizing Concentration of Polyethelene Glycol for Exosome Isolation from Plasma for Downstream Application.

BACKGROUND: Exosomes are ubiquitous extracellular nanovesicles secreted from almost all living cells that are thought to be involved in several important cellular processes, including cell-cell communication and signaling. Exosomes serve as a liquid biopsy tool for clinical and translational research. Although many techniques have been used to isolate exosomes, including ultracentrigation, size-exclusion chromatography, and immunocapturing-based techniques, these techniques are not convenient, they require expensive instrumentation, and they are unhandy for clinical samples. Precipitation techniques from available commercial kits that contain polyethelene glycol (PEG) are now widely used, but these kits are expensive, especially if a large number of biological samples are to be processed. OBJECTIVE: the purpose of this study is to compare and optimize the efficacy of different concentrations of PEG with two commercial kits ExoQuick (SBI) and Total Exosome Isolation (TEI) from Invitrogen in human plasma. METHODS AND MATERIALS: we determined exosome quantity, size distribution, marker expression, and downstream application. RESULTS: among the precipitation methods, we found the size of particles and concentrations with 10-20% PEG are similar to ExoQuick and better than TEI. Interestingly, we detected cfDNA with ExoQuick and 10-20% PEG but not TEI and 5% PEG. Moreover, 10% PEG detection of miR-122 and miR-16 expression was superior to ExoQuick and TEI. Furthermore, in proteomics results it also found the identified proteins better than commercial kits but there was a high level of contamination of other proteins in serum. CONCLUSIONS: together, these findings show that an optimal concentration of 10% PEG serves as a guide for use with clinical samples in exosome isolation for downstream applications.

Genome Sequence of Streptomyces cavourensis BUU135, Isolated from Soil from a Tropical Fruit Farm in Thailand.

Streptomyces cavourensis BUU135 is a bacterial species isolated from the soil of a tropical fruit farm. The genome of S. cavourensis BUU135 comprises a gene encoding nebramycin 5' synthase, which produces nebramycin 5' by catalyzing the O-carbamoylation reaction of tobramycin. The newly sequenced 7.66-Mb draft genome of S. cavourensis BUU135 may contribute to the discovery of novel natural products derived from this organism.

Isolation of fungal communities and identification of Scedosporium species complex with pathogenic potentials from a pigsty in Phra Nakhon Si Ayutthaya, Thailand.

Soil fungal communities play an important role in regulating biogeochemical transformations, yet soil-related fungal pathogens are emerging threats to humans. Our previous studies have revealed the pathogenic Scedosporium species in soils samples from public parks with high human activities in Thailand. However, measurement and survey of soil fungal communities in other areas with high human/animal activities, such as the pigsty, are poorly determined. In this study, soil fungal pathogens from a pigsty were isolated and identified. Soil samples were collected from the surrounding drainage areas. Fungal species were identified using morphological and molecular analyses. Isolation of soil samples from the pigsty revealed at least 11 species that have been identified. The most abundant fungal species belonged to genera Aspergillus and Penicillium. Moreover, Scedo-Select III culturing and phylogenetic analysis with ß-tubulin gene sequencing revealed the three environmental isolates of Scedosporium species, which were consistent with the S.apiospermum. These three Scedosporium isolates were susceptible to voriconazole and caused pathological characteristics of scedosporiosis similar to S. apiospermum in vivo. In conclusion, our findings contribute towards a better understanding of soil-borne pathogenic fungi in the pigsty. The isolation of Scedosporium species with pathogenic potentials in the present study can be beneficial for the management of public health surveillance, epidemiologists, as well as physicians to reduce the risk of soil fungal contamination among pigsty workers.

Simple Colorimetric Assay for Vibrio parahaemolyticus Detection Using Aptamer-Functionalized Nanoparticles.

Simple, rapid, and sensitive screening methods are the key to prevent and control the spread of foodborne diseases. In this study, a simple visual colorimetric assay using magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) was developed for the detection of Vibrio parahaemolyticus. First, the aptamer responding to V. parahaemolyticus was conjugated onto the surface of MNPs and used as a specific magnetic separator. In addition, the aptamer was also immobilized on the surface of AuNPs and used as a colorimetric detector. In the presence of V. parahaemolyticus, a sandwich structure of MNP-aptamer-bacteria-aptamer-AuNPs is formed through specific recognition of the aptamer and V. parahaemolyticus. The magnetic separation technique was then applied to generate a detection signal. Owing to the optical properties of AuNPs, a visual signal could be observed, resulting in an instrument-free colorimetric detection. Under optimal conditions, this assay shows a linear response toward V. parahaemolyticus concentration through the range of 10-106 cfu/mL, with a limit of detection of 2.4 cfu/mL. This method was also successfully applied for V. parahaemolyticus detection in spiked raw shrimp samples.

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

Prevalence of azole-resistant Aspergillus fumigatus in the environment of Thailand.

Occurrence of azole-resistant Aspergillus fumigatus (ARAF) in the environment is an emerging problem worldwide, likely impacting on patient treatment. Several resistance mutations are thought to have initially arisen through triazole-based fungicide use in agriculture and subsequently being propagated in a similar manner. Here we investigated the prevalence of ARAF in the environment of Thailand and characterized their susceptibility profiles toward clinically used azole compounds along with underlying resistance mutations. Three hundred and eight soil samples were collected and analyzed, out of which 3.25% (n = 10) were positive for ARAF. All isolates obtained were resistant to itraconazole (MIC ≥ 8 µg/ml), two showed additional increased MIC values toward posaconazole (MIC = 0.5 µg/ml), and one other toward voriconazole (MIC = 2 µg/ml). Sequencing of the respective cyp51A genes revealed that eight of the isolates carried the TR34/L98H allele and those two with elevated MIC values to posaconazole the G54R substitution. Although a clear correlation between the use of triazole-based fungicides and isolation of ARAF strains from agricultural lands could not be established for Thailand, but this study clearly demonstrates the spread of globally observed ARAF strains to the environment of South East Asia.

Diversity and Antifungal Drug Susceptibility of Cryptococcus Isolates in Thailand.

Yeasts of the Cryptococcus species complex are the causative agent of cryptococcosis, especially in human immunodeficiency virus (HIV) positive individuals. Cerebral or disseminated cryptococcosis has a very high mortality rate worldwide, including in Thailand. Additionally, an increasing rate of antifungal drug resistant cryptococcal isolates has been reported in several neighboring countries, complicating therapeutic approaches. To understand the situation of this infection in Thailand, we retrospectively investigated the molecular epidemiology and antifungal drug resistance in a collection of 74 clinical, 52 environmental and two veterinary isolates using the URA5-RFLP for typing and the EUCAST guideline for susceptibility testing. Where no EUCAST breakpoints (AMB and 5FC) were available, CLSI epidemiologic cutoff values were used for interpretation. Cryptococcal molecular type diversity showed most isolates were C. grubii, molecular type VNI. One clinical isolate was C. deuterogattii (mol. type VGII) and another C. grubii (mol. type VNII). One strain from environment was classified as C. grubii (mol. type VNII). No resistant strains were detected in this retrospective study for either of the antimycotics tested; however, monitoring of the epidemiology of Cryptococcus species in infected patients in Thailand needs to be continued to detect emergence of resistance.

Environmental isolates of azole-resistant Aspergillus fumigatus in Germany.

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

Prevalence and antifungal susceptibility of Cryptococcus neoformans isolated from pigeon excreta in Chon Buri Province, Eastern Thailand.

 The prevalence of cerebral meningitis caused by Cryptococcus neoformans in HIV-infected patients in Eastern Thailand is high. However, little is known about the occurrence of this pathogenic yeast in the environment of this region.  The aim of our study was to characterize the prevalence of C. neoformans, its serotypes and antifungal drug susceptibilities in environmental isolates from Chon Buri, Eastern Thailand.  C. neoformans was isolated from 10% of fifty pigeon excreta examined from this province. All C. neoformans isolates were of serotype A and although the isolates displayed slightly decreased susceptibility towards fluconazole, all tested sensitive to amphotericin B, fluconazole and itraconazole. This study is the first report of the occurrence of C. neoformans in pigeon excreta in eastern Thailand.

Gross karyotypic and phenotypic alterations among different progenies of the Candida glabrata CBS138/ATCC2001 reference strain.

Genomic plasticity is a mechanism for adaptation to environmental cues such as host responses and antifungal drug pressure in many fungi including the human pathogenic yeast Candida glabrata. In this study we evaluated the phenotypic and genotypic stability of the world-wide used C. glabrata reference strain CBS138/ATCC2001 under laboratory conditions. A set of ten lineages of this wild type strain and genetically modified progenies were obtained from different scientific laboratories, and analyzed for genotypic and phenotypic alterations. Even though the derivates were indistinguishable by multi locus sequence typing, different phenotypic groups that correlated with specific karyotypic changes were observed. In addition, modifications in the adherence capacity to plastic surface emerged that were shown to correlate with quantitative changes in adhesin gene expression rather than subtelomeric gene loss or differences in the number of macrosatellite repeats within adhesin genes. These results confirm the genomic plasticity of C. glabrata and show that chromosomal aberrations and functional adaptations may occur not only during infection and under antimicrobial therapy, but also under laboratory conditions without extreme selective pressures. These alterations can significantly affect phenotypic properties such as cell surface attributes including adhesion and the cell wall carbohydrate composition and therefore, if unnoticed, may adulterate the outcome of genetic studies.

Association of IL-18 gene polymorphism (-137C) with arthritis manifestations in SLE: combined effect with IFN gamma gene polymorphism (+874A).

We analyzed the association between single nucleotide polymorphisms in IL-12 and IL-18 genes in disease susceptibility and severity of SLE in Thais. A weak association was observed between A allele of the IL-12 gene at the 3' untranslated region in SLE patients with proteinuria (OR = 1.89, 95% CI = 1.05-3.40, P = 0.02, Pc = 0.06). In addition, we found a significant association between C allele of IL-18 (-137) with arthritis (OR = 6.88, 95% CI = 1.54-42.93, P = 0.003, Pc = 0.009). The presence of one C allele (C/C+C/G) was associated with significant OR of 8.72 (95% CI = 1.83-56.71, P = 0.001, Pc = 0.003). Interestingly, we found the combined effect between the G/C genotype of IL-18 (-137) and the A/A genotype of IFNG (+874) gene causing susceptibility of arthritis in SLE patients (OR = 13.22, 95% CI = 1.56-291.66, P = 0.004).

Association of interferon-gamma gene polymorphism (+874A) with arthritis manifestation in SLE.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease in which genetic factors strongly influence susceptibility. Cytokines such as the interferon-gamma (IFNG) gene play a key role in controlling the immunity and inflammation, and therefore their polymorphisms may affect these genes' expression levels among individuals. We investigated the frequency of IFNG gene intron (+874) polymorphism, previously reported to be associated with IFNG production, in SLE patients compared to a control group. This population-based case-control study includes 154 SLE patients and 154 healthy control subjects with similar ethnic backgrounds. The genotyping was determined by polymerase chain reaction sequence-specific primer method and using the Chi-squared test for analyzing the association between this single-nucleotide polymorphism and SLE. The allele frequencies of the IFNG (+874) gene polymorphism were not significantly different between SLE patients and control subjects (72.7 vs 77%). However, there was a significant association between A dominance model of inheritance with arthritis (odds ratio = 7.64, 95% confidence interval = 1.56-41.64, P = 0.006, P(c) = 0.03). The result suggested that the +874 intron polymorphism of IFNG can be used as the marker for SLE susceptibility with arthritis in the Thai population.

The distribution of IL-10 promoter polymorphism in Thais.

IL-10 is a regulatory cytokine, which plays important roles in the pathogenesis of many diseases polymorphism of IL-10 promoter influences the phenotypic expressions such as the variation of IL-10 production among individuals and is subjected to the genetic susceptibility study of many diseases. However, there is no information about the frequencies of IL-10 promoter polymorphism in a Thai population. To determine the distribution of IL-10 promoter polymorphism in unrelated healthy Thais, genomic-DNA from 160 unrelated healthy volunteers were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The functional single-nucleotide polymorphisms (SNPs) in IL-10 promoter (positions -1082 (G/A), -819 (C/T), -592 (C/A) were included. The allele frequencies of -1082*A (93.4%), -819*T (71.9%), and -592*A (71.9%) were significantly higher than the allele frequencies of -1082*G (6.6%), -819*C (28.1%), and -592*C (28.1%) respectively in a Thai population similar to other Asian populations (Korean, Japanese, Chinese). As for the haplotype analysis, the ATA haplotype (72%) was significantly higher in Thais and other Asian populations compared to non-Asian populations; whereas, GCC haplotype (6.6%) was significantly lower in Thais. Additionally, two rare haplotypes of IL-10 promoter (ATC and ACA) which were previously reported only in the Chinese Han people, were found with similar frequencies (0.6%) in the present study. In conclusion, the distribution of IL-10 promoter polymorphisms in Thais was comparable to other Asian populations but distinct from Non-Asian populations. At least five haplotypes existed in an unrelated healthy Thai population as ACC, GCC, ATA, ATC, and ACA haplotypes.