Exploration of Extracellular Vesicle Long Non-Coding RNAs in Serum of Patients with Cervical Cancer.

INTRODUCTION: Cervical cancer (CC) is the fourth most common cancer type and a leading cause of cancer-related deaths in women worldwide. Its underlying molecular mechanisms are unclear. Cancer cell-derived extracellular vesicles (EVs) are involved in cancer development and progression by delivering regulatory factors, including microRNAs and long non-coding RNAs (lncRNAs). METHODS: Here, we identified the EV lncRNA expression profiles associated with different developmental stages of CC using next-generation sequencing. EVs from the serum of patients with stages I-III CC and healthy donors were characterized using EV marker immunoblotting and transmission electron microscopy. RESULTS: The EV concentration increases with progression of the disease. Most particles had a 100-250-nm diameter, and their sizes were similar in all groups. We identified many lncRNAs that were uniquely and differentially expressed (DE) in patients with different stages of CC. The pathway analysis results indicated that the upregulated DE EV lncRNAs abundant in stages I and II were associated with cell proliferation and inflammation and cancer progression pathways, respectively. LINC00941, LINC01910, LINC02454, and DSG2-AS1 were highly expressed, suggesting poor overall survival of CC patients. Interestingly, DSG2-AS1 was associated with the human papillomavirus infection pathway through AKT3, DLG1, and COL6A2 genes. CONCLUSION: This is the first study that reports the levels of EVs and their lncRNA contents change during cancer development, demonstrating the existence of a unique vesicle-mediated cell-to-cell communication network underlying cancer progression.

A novel, 4-h DNA extraction method for STR typing of casework bone samples.

Bones are often found in mass grave crime scene. To increase DNA identification success rates, a highly efficient DNA extraction method should be selected. Several DNA extraction methods for human bones have been published yet never been systematically compared, and some are time-consuming or complex. As such, a quick and highly efficient DNA extraction method was developed and compared with three published methods (Hi-Flow silica-based, total demineralization (TD) and PrepFiler BTA) using 70 fresh and 22 casework bones from different body parts. The highest median DNA concentrations were obtained from developed method (135.85 ng/µL and 0.224 ng/µL for fresh and casework bones, respectively). For residual PCR inhibitors, the threshold cycle (Ct) of the internal positive control (IPC) showed that developed method and PrepFiler BTA removed most PCR inhibitors. Similarly, 95.45% of casework STR profiles obtained using the developed protocol meet the standard requirements for Australian National Criminal Investigative DNA Database (NCIDD) entry, followed by 86.35% using TD, 81.82% using PrepFiler BTA, and 45.45% using Hi-Flow. Additionally, DNA extracts from seven different bones revealed that the 1st distal phalange of the hand contained the highest DNA concentration of 338.43 ng/µL, which was three times higher than the tibia and femur. Our findings suggest that developed method was highly efficient for casework bone analysis. It significantly reduced the extraction processing time down to 4 h and is two to four times cheaper compared with other methods. In practice, both the extraction method and the bone sampling must be considered by a forensic DNA analyst to increase the chances of successful identification.

Lovastatin Production by Aspergillus sclerotiorum Using Agricultural Waste.

RESEARCH BACKGROUND: Lovastatin is a well-known drug used to reduce hypercholesterolaemia. However, the cost of lovastatin production is still high. Therefore, alternative low-cost carbon sources for the production of lovastatin are desirable. EXPERIMENTAL APPROACH: Four different agricultural wastes, namely corn trunks, rice husks, wild sugarcane, and soya bean sludge, were tested separately as substrates to produce lovastatin using a new fungal strain, Aspergillus sclerotiorum PSU-RSPG 178, under both submerged and solid-state fermentation (SSF). RESULTS AND CONCLUSIONS: Of these substrates and cultivation systems, soya bean sludge gave the highest lovastatin yield on dry mass basis of 0.04 mg/g after 14 days of SSF at 25 °C. Therefore, the soya bean sludge was separately supplemented with glucose, wheat flour, trace elements, palm oil, urea and molasses. The addition of the palm oil enhanced the lovastatin yield to 0.99 mg/g. In addition, the optimum conditions, which gave a lovastatin yield of (20±2) mg/g after 18 days of SSF, were soya bean sludge containing 80% moisture (dry basis) at a ratio of soya bean sludge (g) to mycelial agar plugs of 1:4, and a ratio of soya bean sludge (g) to palm oil (mL) of 1:2. Besides, the lovastatin yields obtained from SSF using fresh or dry soya bean sludge were not significantly different. NOVELTY AND SCIENTIFIC CONTRIBUTION: We conclude that A. sclerotiorum PSU-RSPG 178 has a good potential as an alternative strain for producing lovastatin using soya bean sludge supplemented with palm oil as a carbon source.

Induction of vitellogenesis by glass bottom boat in the female banana shrimp, Fenneropenaeus merguiensis de Man.

In shrimp aquaculture, eyestalk ablation is the only technique that is widely used to accelerate ovarian development. Alternative methods for producing improved ovarian development in broodstock are currently being investigated. Several factors involved in the regulation of ovarian development in shrimp have been investigated. Among these factors, growth factors in the transforming growth factor beta (TGF-ß) superfamily have been implicated as playing potential roles in the regulation of gonad development. In this work, a member of the TGF-ß superfamily known as glass bottom boat (GBB), an ortholog of bone morphogenetic protein (BMP), was investigated to uncover its role in ovarian development in the banana shrimp Fenneropenaeus merguiensis. Full-length cDNA of FmGBB was obtained from transcriptome data. Phylogenetic analysis indicated that the sequence of FmGBB from banana shrimp was similar to those of other arthropods and vertebrate BMP 5/6/7, but was different from those of decapentaplegic proteins and vertebrate BMP 2/4. The FmGBB transcript was found to be widely expressed in shrimp tissues, and its expression in the ovary was dramatically increased in early and late vitellogenic stages during ovarian development and decreased in the mature stage, suggesting its role in vitellogenesis. To study the effects of FmGBB, a soluble recombinant mature FmGBB peptide (His-TF-rgbb) containing both monomers and homodimers was successfully expressed in Escherichia coli. The His-TF-rgbb peptide triggered oocyte proliferation in both cultured ovarian explants and in previtellogenic shrimp upon injection. Interestingly, the injection of His-TF-rgbb into previtellogenic female shrimp stimulated an increase in Vg expression in their ovaries while suppressing production of 20-hydroxyecdysone. Our results suggest the potential role of FmGBB in oocyte proliferation and vitellogenesis; this novel finding can be utilized to stimulate ovarian development in cultured shrimp.

Semiquantitative dot-blot immunogold assay for specific detection of white spot syndrome virus.

A dot-blot immunogold assay (DBIA) was developed to detect white spot syndrome virus (WSSV) using the polyclonal antibody VP26 (anti-VP26). The anti-VP26 was immobilized on gold nanoparticles (Ab-AuNPs), and a nitrocellulose membrane was used as a detection pad. When the target WSSV bound to the Ab-AuNPs a reddish dot appeared on the surface of the membrane used within 2-5 Min, which could be seen with the naked eye. The test was able to detect WSSV at concentrations as low as 105 copies µL-1 of WSSV. The DBIA developed had good specificity, and the colloidal gold probe can be applied within 2-3 days when stored at 4 °C. For real sample analysis, the DBIA was applied to samples of seawater used for shrimp cultivation without sample preparation. The results indicate that sample 1 showed a positive result, whereas samples 2 and 3 produced negative results. Then, samples 2 and 3 were spiked with WSSV for method validation. To confirm the performance of the DBIA developed, polymerase chain reaction (PCR) was conducted and the PCR results were the same as those found by the DBIA. Therefore, the DBIA developed could be applied for WSSV detection in real water samples.

Characterization of a Chikungunya virus strain isolated from banked patients' sera.

BACKGROUND: Chikungunya virus (CHIKV) is a prevalent mosquito-borne pathogen that is emerging in many parts of the globe causing significant human morbidity. Here, we report the isolation and characterization of an infectious CHIKV from banked serum specimens of suspected patients from the 2009 epidemic in Thailand. METHODS: Standard plaque assay was used for CHIKV isolation from the banked serum specimens. Isolated CHIKV was identified base on E1 structural gene sequence. Growth kinetic, infectivity, cell viability and cytokine gene expression throughout CHIKV infection in a permissive cell line, 293T cells, was performed using several approaches, including standard plaque assay, immunofluorescence assay, classical MTT assay, and quantitative real-time PCR. Two tailed Student's t test was used for evaluation statistically significance between the mean values of the groups. RESULTS: Based on the E1 structural gene sequence and phylogenetic analysis, we identified the virus as the CHIK/SBY8/10 isolate from Indonesia. Assessment of the growth kinetics, cytopathic effects as well as its ability to induce cellular immune responses suggested that the currently isolated CHIK/SBY8/10 virus is relatively more virulent than a known CHIKV vaccine strain, which also induces more dramatic proinflammatory responses. CONCLUSIONS: Our studies further add to the infectivity of a less-studied yet infectious CHIKV isolate as well as underscored the importance and utility of 293T cells as an excellent cell culture model for studying viral growth, CHIKV-induced inflammatory cellular responses and cell death. Together, these studies provide novel information on the CHIKV biology, infectivity and virus-cell interaction, which would help develop novel interventions against the infection.

Emilia sonchifolia extract activity against white spot syndrome virus and yellow head virus in shrimp cell cultures.

Emilia sonchifolia (L.) DC is a plant used in traditional medicine to treat several viral and bacterial diseases. The antiviral activities of selected Sephadex LH-20 column fractions and HPLC subfractions of an acetone extract of E. sonchifolia leaves were determined in shrimp Penaeus merguiensis primary lymphoid cells infected with either white spot syndrome virus (WSSV) or yellow head virus (YHV). WSSV and YHV replication was quantified using quantitative real-time PCR tests targeted to the VP19 and ORF1b gene transcripts, respectively. In lymphoid organ cells exposed to 100 µg ml⁻¹ of either the Sephadex fraction F14 or the HPLC F14 subfraction SF4, both fractions caused reduced replication, but YHV replication was reduced only by SF4. In the asthiazolyl blue mitochondrial enzyme activity assays to assess extract cytotoxicity, >60% of primary lymphoid organ cells remained viable following exposure to 100 µg ml⁻¹ of either F14 or SF4. GC-MS analysis of the HPLC F14 subfraction SF4 showed that it contained 2,4-di-tert-butylphenol. This study is the first to show that E. sonchifolia leaf extracts might be useful as bioactive agents to protect shrimp against viruses such as WSSV and YHV.

Molecular evolution of the odorant and gustatory receptor genes in lepidopteran insects: implications for their adaptation and speciation.

Lepidoptera (comprised of butterflies and moths) is one of the largest groups of insects, including more than 160,000 described species. Chemoreception plays important roles in the adaptation of these species to a wide range of niches, e.g., plant hosts, egg-laying sites, and mates. This study investigated the molecular evolution of the lepidopteran odorant (Or) and gustatory receptor (Gr) genes using recently identified genes from Bombyx mori, Danaus plexippus, Heliconius melpomene, Plutella xylostella, Heliothis virescens, Manduca sexta, Cydia pomonella, and Spodoptera littoralis. A limited number of cases of large lineage-specific gene expansion are observed (except in the P. xylostella lineage), possibly due to selection against tandem gene duplication. There has been strong purifying selection during the evolution of both lepidopteran odorant and gustatory genes, as shown by the low ω values estimated through CodeML analysis, ranging from 0.0093 to 0.3926. However, purifying selection has been relaxed on some amino acid sites in these receptors, leading to sequence divergence, which is a precursor of positive selection on these sequences. Signatures of positive selection were detected only in a few loci from the lineage-specific analysis. Estimation of gene gains and losses suggests that the common ancestor of the Lepidoptera had fewer Or genes compared to extant species and an even more reduced number of Gr genes, particularly within the bitter receptor clade. Multiple gene gains and a few gene losses occurred during the evolution of Lepidoptera. Gene family expansion may be associated with the adaptation of lepidopteran species to plant hosts, especially after angiosperm radiation. Phylogenetic analysis of the moth sex pheromone receptor genes suggested that chromosomal translocations have occurred several times. New sex pheromone receptors have arisen through tandem gene duplication. Positive selection was detected at some amino acid sites predicted to be in the extracellular and transmembrane regions of the newly duplicated genes, which might be associated with the evolution of the new pheromone receptors.

Development of an immuno-based colorimetric assay for white spot syndrome virus.

White spot syndrome virus (WSSV) is a major cause of infectious disease in cultured shrimp. A fast and reliable method for detecting and monitoring the amount of WSSV during farming would be extremely useful. This work describes a sandwich immunoassay that uses anti-GST-VP26, a WSSV-binding protein (WBP), and modified streptavidin magnesphere paramagnetic particles (SMPPs) to develop the technique. The WBP was immobilized on SMPPs and later bound to different copies of WSSV. The binding was detected using anti-GST-VP26 conjugated to alkaline phosphatase. This enzymatic reaction successfully changed the test solution to a concentration-dependent yellow color that was measured at 405 nm. The sensitivity of this method was between 1.6 × 10(4) and 1.6 × 10(7) copies µL(-1) of WSSV. In this study, the color for detection and semiquantitative analysis is easily observed and measured and can lead to the development of a test kit for screening WSSV during shrimp farming.

Effect of novel chitosan-fluoroaluminosilicate resin modified glass ionomer cement supplemented with translationally controlled tumor protein on pulp cells.

Dental materials that can promote cell proliferation and function is required for regenerative pulp therapy. Resin modified glass ionomer cement (RMGIC), a broadly used liner or restorative material, can cause apoptosis to pulp cells mainly due to HEMA (2-hydroxyethyl methacrylate), the released residual monomer. Recent studies found that chitosan and albumin could promote release of protein in GIC while translationally controlled tumor protein (TCTP) has an anti-apoptotic activity against HEMA. The aim of this study was to examine the effect of chitosan and albumin modified RMGIC (Exp-RMGIC) supplemented with TCTP on pulp cell viability and mineralization. Exp-RMGIC+TCTP was composed of RMGIC powder incorporated with 15 % of chitosan, 5 % albumin and supplemented with TCTP mixed with the same liquid components of RMGIC. The effect of each specimen on pulp cells was examined using the Transwell plate. From the MTT assay, Exp-RMGIC+TCTP had the highest percentages of viable cells (P < 0.05) at both 24 and 74 h. Flow cytometry revealed that, after 24 h, Exp-RMGIC+TCTP gave the lowest percentages of apoptotic cells compared to other groups. There was no difference in alkaline phosphatase (ALP) activity among different formula of the specimens, while cells cultured in media with TCTP had higher ALP activity. Von Kossa staining revealed that RMGIC+TCTP, and Exp-RMGIC+TCTP had higher percentages of calcium deposit area compared to those without TCTP. It was concluded that Exp-RMGIC supplemented with TCTP had less cytotoxicity than RMGIC and can protect cells from apoptosis better than RMGIC supplemented with TCTP.

Expression profile of ribosomal protein L10a throughout gonadal development in rainbow trout (Oncorhynchus mykiss).

Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24 h. Among them, 11-ketotestosterone at 100 ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17ß-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.

Forensic animal DNA analysis using economical two-step direct PCR.

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 ± 180.84 ng/µL. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire(®) Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community.

Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA.

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 µg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 µg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.

Differentially expressed genes in the testes from early to mature development of banana shrimp (Fenneropenaeus merguiensis).

Banana shrimp (Fenneropenaeus merguiensis) is an economically important species in Thailand owing to the high value of globally exported frozen brine shrimps. However, the regulatory mechanisms governing spermatogenesis and testicular development in this species are poorly understood. High-throughput RNA sequencing was used to investigate the mechanisms and regulated genes involved in testis development using transcriptome profiling of juvenile and adult banana shrimp testes. Differentially expressed genes (DEGs) in these two libraries were identified and quantified to confirm gene expression. DEGs were found in 7,347 genes, with 4,465 upregulated and 2,882 downregulated. Some of these genes were designated as candidate genes, and six specific DEGs, including PRM1, SPATA20, Sry, SSRF, Sxl, and Tra-2c, were selected to confirm the reliability of the RNA-seq data using qPCR. Moreover, six non-DEGs were chosen based on testis-specific and regulatory genes that support a specific function in spermatogenesis and testis development in this species, including Dsx, Gfra2, IAG, Sox9, Sox13, and Sox14A. Furthermore, Sry, Sox14A, Sox14B and SPATA20 were identified in early stages (nauplius-postlarvae) of shrimp development to provide more information involving testes formation and development. The transcript data from this study could differentiate a group of genes required at the early and late stages of testis development and both sets of testis development. Therefore, this information would help in manipulating each stage of testicular development.

Transcriptome profiling of banana shrimp (Fenneropenaeus merguiensis) ovaries and testes: Insights into FoxL2.

The banana shrimp is found in the Pacific and Indian Oceans. Female shrimp are preferred for consumption because they are larger than males. Understanding the mechanism of sex differentiation is important for developing techniques to increase the number of female shrimp for economic benefits. This study investigates the reproductive development of F. merguiensis using transcriptome analysis. Sxl2, dsx, AGH, FEM-1, and Nrg-X2 were classified as essential genes for testes development during the juvenile stage. Several genes were required for both juvenile and adult male development. Additionally, the expression of several genes was shown to be required for juvenile and adult ovarian development, including SOP1, SOP2, Ptgr1, EST, Vgr, Vmol1, and TR-beta A. Interestingly, high levels of FoxL2 expression were observed in the testes, in contrast to previous studies in humans and other mammals. The binding of FoxL2 to the Vtg promoter was demonstrated in silico with the highest relative binding score (RS = 0.89) using the JASPAR program. Knock-down of the FoxL2 gene with dsRNA significantly suppressed FoxL2 at 2, 4, and 6 d. As a result, Vtg expression increased when compared with the control at 2, 4, and 6 d, indicating that FoxL2 plays an important role in Vtg expression in the ovary. Our findings highlight the role of FoxL2 in banana shrimp reproduction and provide valuable information on the genes associated with the F. merguiensis reproductive system.

Biocompatibility and mineralization activity of modified glass ionomer cement in human dental pulp stem cells.

Background/purpose: Fortilin is a multi-functional protein involved in several cellular processes. It has been shown promising potential to be a bioactive molecule that can be incorporated in the dental materials. This study aimed to compare the biocompatibility and mineralization activities of modified glass ionomer cement (Bio-GIC) and Biodentine by direct and indirect method on human dental pulp stem cells (hDPSCs). Materials and methods: Conventional glass ionomer cement (GIC), Bio-GIC (GIC supplemented with chitosan, tricalcium phosphate, and recombinant fortilin from Fenneropenaeus merguiensis), and Biodentine were examined in this study. Recombinant fortilin was purified and tested for its cytotoxicity by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. Human DPSCs were treated with different material eluate for particular time intervals. At given time points, viability of hDPSCs was examined using MTT assay and calcium deposition was assessed by Alizarin red staining assay. Comparisons of the data among groups were analyzed by analysis of variance and Tukey's multiple comparisons. Results: All test materials demonstrated no cytotoxicity. In addition, Bio-GIC promoted cell proliferation at 72 h. For direct and indirect method, cells treated with Bio-GIC demonstrated significantly higher calcium deposition than other groups (P < 0.05). Conclusion: Bio-GIC and Biodentine are not cytotoxic to hDPSCs. Bio-GIC demonstrates enhanced calcium deposition comparable to Biodentine. Bio-GIC may be further developed as a bioactive material for dentin regeneration.

Validity evaluation of in-house preparation kit, vaginal pH paper test combined amine tube test, for the simple diagnosis of bacterial vaginosis.

OBJECTIVE: Determine the positive in-house preparation kit for suggested bacterial vaginosis (BV) for both elevated vaginal pH > 4.5 and positive amine test, as well as evaluate for validity of sensitivity, specificity, positive predictive value, and negative predictive value against Chandeying criteria for confirmed BV. MATERIAL AND METHOD: A cross-sectional study among the women who presented with an abnormal vaginal discharge (AVD) or asymptomatic annual cervical cytology screening was done. Each vaginal discharge was divided into two parts of investigation. The first part included the clinical criteria of confirmed BV, based on at least three out of five indicators, the vaginal pH > 4.5, homogeneous and thin discharge (milky discharge), positive sniff/amine test, clue cell > 20% of total vaginal epithelial cells, and scanty or absence lactobacilli. The second part included the in-house preparation kit of suggestive BV relied on elevated vaginal pH > 4.5 and positive amine tube test. RESULTS: Twenty-six women were enrolled. Of the complaint of AVD/asymptomatic had 2/10 of confirmed BV (12 cases), and 1/13 of confirmed non-BV (14 cases). The in-house preparation kit, compared with the clinical criteria, had sensitivity of 91%, specificity of 71%, positive predictive value of 73%, and negative predictive value of 90%. There were false negative of 1/12 cases (8.3%), and false positive of 4/14 cases (28.5%). CONCLUSION: The in-house preparation kit favorably compared with the clinical criteria and has the advantage of being simple, rapid, and easily performed in resource poor setting. Further development on sensitivity and specificity of the test is suggested.

Long-Term Effect of Modified Glass Ionomer Cement with Mimicked Biological Property of Recombinant Translationally Controlled Protein.

This study modified glass ionomer cement (GIC) by adding mimicked biological molecules to reduce cell death. GIC was modified to BIOGIC by adding chitosan and bovine serum albumin for enhancing protein release. The BIOGIC was supplemented with tricalcium phosphate (TCP) and recombinant translationally controlled tumor protein (TCTP) to improve its biological properties. Four groups of materials, GIC, BIOGIC, BIOGIC+TCP, and BIOGIC + TCP + TCTP, were examined by XRD and SEM-EDX. TCTP released from the specimens was determined by an ELISA method. Human dental pulp stem cells (hDPSCs) were harvested and analyzed by MTT assay, apoptosis, gene expression, and cell differentiation. All groups had the same crystallization characteristic peaks of La2O3. The elemental compositions composed of La, Si, and Al are the main inorganic components. The results show that BIOGIC + TCP + TCTP presented significantly higher percentages of cell viability than other groups on day 1 to day 23 (p < 0.05), but were not different after day 24 to day 41 and had reduced cell apoptosis including BAX, TPT1, BCL-2, and Caspase-3. The BIOGIC + TCP + TCTP demonstrated higher odontoblast mineralization and differentiation markers including ALP activity, DSPP, DMP-1, ALP, BMP-2, and OPN. It enhanced cell proliferation and differentiation as well as mineralization with down-regulation of genes related to apoptosis compared with other groups.

Biological Activities of Glass Ionomer Cement Supplemented with Fortilin on Human Dental Pulp Stem Cells.

This study aimed to determine the most suitable recombinant fortilin and evaluate the biological activities of glass ionomer cement (GIC) incorporated with fortilin on human dental pulp stem cells (hDPSCs). Full-length and three fragments of Penaeus merguiensis fortilin were cloned and examined for their proliferative and cytoprotective effects on hDPSCs by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Human DPSCs were cultured with GIC supplemented with fortilin, tricalcium phosphate, or a combination of tricalcium phosphate and fortilin, designated as GIC + FL, GIC + TCP, and GIC + TCP + FL, respectively (n = 4 for each group). At given time points, hDPSCs were harvested and analyzed by MTT, quantitative reverse transcription polymerase chain reaction, alkaline phosphatase activity, and Alizarin Red assays. The full-length fortilin promoted cell proliferation and significantly increased cell survival. This protein was subsequently added into the GIC along with tricalcium phosphate to investigate the biological activities. All experimental groups showed reduced cell viability after treatment with modified GICs on days 1 and 3. The GIC + TCP + FL group significantly promoted odontoblastic differentiation at particular time points. In addition, alkaline phosphatase activity and calcium phosphate deposit were markedly increased in the GIC + TCP + FL group. Among all experimental groups, the GIC incorporated with fortilin and tricalcium phosphate demonstrated the best results on odontogenic differentiation and mineral deposition in hDPSCs.

WSSV: VP26 binding protein and its biological activity.

White spot syndrome virus (WSSV) is one of the major causes of disease in the shrimp culture industry causing enormous economic losses. In this study, we displayed peptides from a cDNA library obtained from the hemolymph of shrimp infected with WSSV, on the surface of phage and screened for the peptides that interacted with the WSSV. One WSSV binding protein (WBP) gene was found to consist of 171 bp that had no matches in the NCBI database. This WBP was shown to bind to the VP26 protein of the WSSV by Western blotting. In addition, WBP reduced the binding of WSSV to shrimp haemocytes from 2.0 × 10(7)copies in the control to 6.0 × 10(2) after treatment with 80 µg of WBP. The survival rate of shrimp after WSSV were mixed with WBP at 80 µg, was 89% and the binding of WBP remained unchanged for at least 24h. Therefore, the results indicate that the WBP can bind to VP26 and inhibit the invasion of WSSV into host cells. This finding may introduce another future way to try to fight this disease in shrimp culture.