Microbiome miracles and their pioneering advances and future frontiers in cardiovascular disease.

Cardiovascular diseases (CVDs) represent a significant global health challenge, underscoring the need for innovative approaches to prevention and treatment. Recent years have seen a surge in interest in unraveling the complex relationship between the gut microbiome and cardiovascular health. This article delves into current research on the composition, diversity, and impact of the gut microbiome on CVD development. Recent advancements have elucidated the profound influence of the gut microbiome on disease progression, particularly through key mediators like Trimethylamine-N-oxide (TMAO) and other microbial metabolites. Understanding these mechanisms reveals promising therapeutic targets, including interventions aimed at modulating the gut microbiome's interaction with the immune system and its contribution to endothelial dysfunction. Harnessing this understanding, personalized medicine strategies tailored to individuals' gut microbiome profiles offer innovative avenues for reducing cardiovascular risk. As research in this field continues to evolve, there is vast potential for transformative advancements in cardiovascular medicine, paving the way for precision prevention and treatment strategies to address this global health challenge.

Nutritional profiling and in silico analysis of pharmacological activities from local rice Pulu Mandoti fermented with Pleurotus spp.

Pulu Mandoti, a local red rice (Oryza sativa L.) variety popular among Sulawesi residents, has gained recognition for its perceived health benefits, especially as a preferred dietary option for individuals with diabetes or those seeking to prevent obesity. Given the increasing consumption of mushrooms, particularly Pleurotus species, renowned for their nutritional and medicinal attributes, this study delves into the transformative effects of Pleurotus spp. fermentation on Pulu Mandoti, the indigenous rice variety. Proximate analysis disclosed elevated dry matter (91.99 ± 0.61%), crude protein (8.55 ± 0.15%), and crude fat (1.34 ± 0.05%) in Pleurotus cystidiosus fermentation compared to Pleurotus ostreatus and Pleurotus djamor. Concurrently, antioxidant and antidiabetic activities were notably improved in all Pleurotus fermentations. Pulu Mandoti fermented with P. cystidiosus outperformed other treatments, aligning with molecular docking results pinpointing 11-Eicosenoic acid, methyl ester, and butylated hydroxytoluene as optimal interactors with antioxidant receptors 5O0x and 2CKJ. Butylated hydroxytoluene demonstrated interactions with the antidiabetic receptor 2QV4, along with 9-Octadecenoic acid, methyl ester. These compounds, previously unreported in Pleurotus, displayed promising attributes as antioxidants and antidiabetic agents. Furthermore, the investigation delved into the fatty acid profiles, emphasizing the diverse range of potential bioactive compounds in fermented Pulu Mandoti. The findings of this research present a potential functional food rich in natural antioxidants and antidiabetic compounds, highlighting the yet undiscovered capabilities of Pleurotus spp. fermentation in augmenting the nutritional composition and bioactivity of indigenous rice varieties, specifically Pulu Mandoti.

Endophytic Aspergillii and Penicillii from medicinal plants: a focus on antimicrobial and multidrug resistant pathogens inhibitory activity.

The rise of multidrug resistance among microorganisms, where they develop resistance against formerly efficacious drugs, has led to increased disease prevalence and mortality rates, posing a growing challenge. Globally, antibiotic resistance has made a significant impact, causing millions of fatalities each year. Endophytic fungi have gained considerable attention in research due to their potential to produce a wide variety of secondary metabolites, including natural substances with antimicrobial capabilities. The genera Aspergillus and Penicillium stand out as the most prevalent species of endophytic fungi. Filamentous fungi, such as these are responsible for the production of 45% of known microbial metabolites. This review focuses on exploring the bioactive substances produced by endophytic fungi from these two genera, particularly in conjunction with medicinal plants. Emphasis is placed on their antimicrobial activity and their ability to inhibit multidrug-resistant pathogens. As the need for alternative treatments to combat drug-resistant infections continues to grow, endophytic fungi have the potential to provide a valuable source of bioactive molecules for medical applications.

Expression and scale-up production of recombinant human papillomavirus type 52 L1 protein in methylotrophic yeast Hansenula polymorpha.

BACKGROUND: Human papillomavirus (HPV) vaccination is one of the crucial national vaccination programs aimed at reducing the prevalence of the diseases associated with HPV infections, which continue to pose a global health concern. However, a significant disparity exists in the distribution of HPV vaccine, particularly in low-middle income countries where the cost of HPV vaccine becomes a major obstacle. Thus, it is essential to ensure the availability of an economically feasible HPV vaccine, necessitating immediate efforts to enhance the cost-effectiveness of vaccine production. This study aimed to develop an efficient production system for the recombinant HPV type 52 L1 protein as HPV vaccine material using methylotrophic yeast Hansenula polymorpha expression system. RESULTS: This study presents an in-depth examination of the expression and scale-up production of HPV type 52 L1 protein using DASGIP® parallel bioreactor system. The pHIPX4 plasmid, which is regulated by the MOX promoter, generates stable clones that express the target protein. Cultivation employing the synthetic medium SYN6(10) with controlled parameters (e.g. temperature, pH, feeding strategy, and aeration) produces 0.15 µg/mL of HPV type 52 L1 protein, suggesting a possibility for scaling up to a higher production level. CONCLUSION: The scale-up production of HPV type 52 L1 protein using Hansenula polymorpha expression system described in this study provides an opportunity for an economical manufacturing platform for the development of the HPV vaccine.

The Formulation and Characterization of Wound Dressing Releasing S-Nitrosoglutathione from Polyvinyl Alcohol/Borax Reinforced Carboxymethyl Chitosan Self-Healing Hydrogel.

Self-healing hydrogels often lack mechanical properties, limiting their wound-dressing applications. This study introduced S-Nitrosoglutathione (GSNO) to self-healing hydrogel-based wound dressings. Self-healing hydrogel mechanical properties were improved via polymer blends. Applying this hydrogel to the wound site allows it to self-heal and reattach after mechanical damage. This work evaluated polyvinyl alcohol (PVA)-based self-healing hydrogels with borax as a crosslinking agent and carboxymethyl chitosan as a mechanical property enhancer. Three formulations (F1, F4, and F7) developed self-healing hydrogels. These formulations had borax concentrations of 0.8%, 1.2%, and 1.6%. An FTIR study shows that borate ester crosslinking and hydrogen bonding between polymers generate a self-healing hydrogel. F4 has a highly uniform and regular pore structure, as shown by the scanning electron microscope image. F1 exhibited faster self-healing, taking 13.95 ± 1.45 min compared to other formulations. All preparations had pH values close to neutrality, making them suitable wound dressings. Formula F7 has a high drug content (97.34 ± 1.21%). Good mechanical qualities included high tensile stress-strain intensity and Young's modulus. After 28 h of storage at -20 °C, 5 °C, and 25 °C, the self-healing hydrogel's drug content dropped significantly. The Korsmeyer-Peppas release model showed that the release profile of GSNO followed Fickian diffusion. Thus, varying the concentration of crosslinking agent and adding a polymer affects self-healing hydrogels' physicochemical properties.

Molecular Recognition of Moringa oleifera Active Compounds for Stunted Growth Prevention Using Network Pharmacology and Molecular Modeling Approach.

In this study, network pharmacology was used to analyze the active compounds of Moringa oleifera as food supplements for stunted growth prevention. Thirty-eight important proteins were discovered that may be strongly related to stunting. Those proteins were uploaded to several online tool platforms in order to determine the shared genes' pathways. Six pathways were identified that may be correlated with human growth. Furthermore, ligands for molecular docking analysis were retrieved from the top 5 active substances discovered through experimental investigation. In the meantime, the first-degree rank based on the protein-protein interaction (PPI) topological analysis was utilized to choose albumin protein (ALB) as a receptor. Our docking results showed that every ligand binds to the receptors, indicating that they can bind to the binding site of the ALB protein to form a complex formation. Further, MD simulation was used to verify the stability of the ligand in complex with the protein in the TIP3P water model. Based on the validation parameters, our results suggested that all models achieved a stable phase along the simulation. Additionally, the MM-GBSA method was used to calculate the binding energies of all models. Ligands 2 and 4 have strong binding to the binding pocket of ALB, followed by ligands 3, 5, and 2, suggesting that those ligands could be promising food supplements that can be utilized for the prevention of stunted growth in children.

Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115.

BACKGROUND: Cervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein. RESULT: The recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 µg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP. CONCLUSION: The recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.

Production of codon-optimized Factor C fragment from Tachypleus gigas in the Pichia pastoris GS115 expression system for endotoxin detection.

BACKGROUND: Factor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C-type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. METHODS AND RESULTS: The FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. CONCLUSIONS: Protein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating them for 72 h. These optimized conditions are well-suited for upscaling the production of recombinant FC fragments using a bioreactor.

Whole-genome sequence analysis and probiotic characteristics of Lactococcus lactis Subsp. lactis strain Lac3 isolated from traditional fermented buffalo milk (Dadih).

BACKGROUND: Probiotics are live microorganisms that provide beneficial effects on the host's health when exploited in adequate amounts. This study aimed at carrying out whole-genome sequence analysis and in vitro potential probiotic characteristics of Lactococcus lactis subsp. lactis strain Lac3 isolated from the spontaneously fermented buffalo milk named Dadih. RESULTS: The results from de novo assembly indicated that the assembled genome consisted of 55 contigs with a genome size of 2,441,808 bp ~ (2.44 Mb), and GC % content of 34.85%. The evolution history result showed that the strain Lac3 was closely related to Lactococcus lactis species deposited in NCBI with a sequence similarity ≥ 99.93%. L. lactis subsp. lactis Lac3 was non-pathogenic with a probability of 0.21 out of 1 and had a pathogenicity score of zero (0), and neither harbored virulence factors nor acquired antibiotic resistance phenotypes. L. lactis subsp. lactis Lac3 exhibited the potential probiotic characteristics to tolerate acid at pH (2.0 and 5.0), salinity (1-5% NaCl), bile salt of (0.3-1.0%) and had auto-aggregation capacity increased from 6.0 to 13.1%. CONCLUSION: This study described a novel strain of Lactococcus lactis subsp. lactis called Lac3, which exhibits probiotic properties that could be beneficial in the development of probiotics.

Optimization, characterization, comparison of self-assembly VLP of capsid protein L1 in yeast and reverse vaccinology design against human papillomavirus type 52.

BACKGROUND: Vaccination is the one of the agendas of many countries to reduce cervical cancer caused by the Human papillomavirus. Currently, VLP-based vaccine is the most potent vaccine against HPV, which could be produced by a variety of expression systems. Our study focuses on a comparison of recombinant protein expression L1 HPV52 using two common yeasts, Pichia pastoris and Hansenula polymorpha that have been used for vaccine production on an industrial scale. We also applied bioinformatics approach using reverse vaccinology to design alternative multi-epitope vaccines in recombinant protein and mRNA types. RESULTS: Our study found that P. pastoris relatively provided higher level of L1 protein expression and production efficiency compared to H. polymorpha in a batch system. However, both hosts showed self-assembly VLP formation and stable integration during protein induction. The vaccine we have designed exhibited high immune activation and safe in computational prediction. It is also potentially suitable for production in a variety of expression systems. CONCLUSION: By monitoring the overall optimization parameter assessment, this study can be used as the basis reference for large-scale production of the HPV52 vaccine.

Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas).

BACKGROUND: Horseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural horseshoe crab's factor C and prevent its excessive harvest from nature. In response to these concerns, this study aimed to characterize the amebocyte lysates and factor C protein modeling of T. gigas originated from Banyuasin South Sumatra Estuary. METHODS AND RESULTS: Sampling of T. gigas was carried out in Banyuasin South Sumatra Estuary, Indonesia. The endotoxin test or TAL (Tachypleus amebocyte lysates) assay was performed using gel coagulation method. Protein characterization of protease enzyme was conducted by protease activity, SDS-PAGE, and zymogram analysis. The cDNA of mitochondrial COI gene was amplified for molecular identification followed by cDNA cloning of factor C. Protein modeling was investigated by molecular docking and molecular dynamic (MD) simulation. Endotoxin test results showed that TAL-35 had endotoxin sensitivity in a range of 0.0156-1 EU/ml, while TAL 36 had a sensitivity between 00,625 and 1 EU/ml. T. gigas amebocytes have protease activity in molecular mass sizes less than 60 kDa, with 367 U/ml for TAL 35 and 430 U/ml for TAL 36. The molecular identification revealed 98.68% identity similarity to T. gigas. The docking results suggested three ligands; i.e., diphosphoryl lipid A, core lipid A, and Kdo2 lipid A can be activators of the factor C protein by binding to the region of the receptor to form a ligand-receptor complex. CONCLUSIONS: Endotoxins can be detected using horseshoe crab amebocytes. The presence of proteases is considered responsible for this ability, as evidenced by casein zymogram results. According to docking and MD analysis, we found that lipopolysaccharides (LPS) participate to the binding site of factor C.

Gold nanoparticles (AuNP)-based aptasensor for enteropathogenic Escherichia coli detection.

BACKGROUND: Diarrhea is a major cause of severe gastrointestinal illness in the infant especially in many developing countries. Although this molecular technique has been accepted as standard technique to detect Diarrhea-causing EPEC, the practical aspect of this technique for in-site rapid screening purposes is still facing a major challenge. In this study, we characterized EPEC specific aptamers and applied it as an AuNP-based aptasensor for point of care (POC) diagnosis purpose. METHODS: As many as six selected DNA aptamers was screened using target bacteria and the bound aptamer was measured by qPCR technique. Moreover, Kd value for each optimal bound aptamer was measured by using the same technique. Colorimetry assay was applied to test specificity and LOD of AuNP-based aptasensor. RESULTS: Two DNA aptamers have been successfully obtained to detect Enteropathogenic Escherichia coli K.1.1. DNA aptamer S8-7 exhibited constant dissociation (Kd) value of 17.08 nM, while DNA aptamer S10-5 exhibited Kd value of 34.14 nM. AuNP-based aptasensor showed high selectivity and specificity for EPEC K.1.1 with a limit of detection (LOD) value of 105 CFU/mL. Truncation study on DNA aptamer S8-7 showed that elimination of primer binding sequence only slightly increased both performance of detection and LOD value of AuNP-based aptasensor. CONCLUSION: Further study is necessary to improve AuNP-aptasensor performance such as through mutagenesis approach on targeted DNA aptamers before AuNP-based aptasensor can be applied as a biosensor in point of care (POC) diagnosis.

A sequential toggle cell-SELEX DNA aptamer for targeting Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli bacteria.

BACKGROUND: Mastitis is an inflammation of the mammary glands caused by a microbial infection. The common bacteria causing this infection in dairy farms are Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. The aptamer is a new biosensor platform for detecting pathogens; however, its use for simultaneous detection of S. aureus, S. agalactiae, and E. coli bacteria has not been reported. This study's objective is to isolate and characterize polyclonal DNA aptamer with broad reactivity to the mastitis bacteria S. aureus, S. agalactiae, and E. coli using a sequential toggle cell-SELEX. METHODS AND RESULTS: The DNA aptamer pool from SELEX 15 was inserted into the pGEM-T easy plasmid. Furthermore, the transformant clones were selected by PCR colony, plasmid isolation, and sequencing. Six DNA aptamers, consisting of S15K3, S15K4, S15K6, S15K13, S15K15, and S15K20 with a constant region and the right size of 81 bp were derived from the sequencing analysis. The secondary structure of the DNA was predicted using Mfold software. The DNA was analyzed with binding characteristics, including binding capacity and affinity (Kd), using qPCR. The results indicated aptamer S15K15 has the highest binding ability into S. agalactiae, while S15K13 performed binding capacity most to E. coli EPEC 4, and S15K3 has the highest capacity of binding to S. aureus BPA-12. CONCLUSION: Aptamer S15K3 has the best binding characteristics on all three bacterial targets.

Dissection of Capsid Protein HPV 52 to Rationalize Vaccine Designs Using Computational Approaches Immunoinformatics and Molecular Docking.

BACKGROUND: Human Papillomavirus type 52 (HPV 52) is considered one of the threatening HPV types inducing cervical cancer worldwide. This study was conducted to address strategies of an effective vaccine against cervical cancer using computational approaches immuno-informatics and molecular docking. METHODS: Major capsid protein L1 and L2 HPV 52 (L1 and L2 HPV 52) sequences were investigated by multiple analyses including B and T cell epitope, toxicity, allergenicity, Immunogenicity, epitope conservancy, population coverage, and molecular docking. RESULTS: L1 and L2 HPV 52 showed a conserved sequence among amino acid levels. Q307K, S383D/N, and D473E are found as major mutations in L1, while mutations in L2 are S122T, Q247H, L247S, and E365D. Multiple epitopes were identified and elicited strong immune responses against cross types of HPV in various HLA populations. To enhance vaccine effectiveness that allows having cross-protection over HPV types, N terminus HPV L2 was analyzed suggesting multi-candidates chimeric L1/L2 vaccine design. CONCLUSION: This study shed a light on a useful pipeline with robust analysis for effective vaccine production.

Spontaneous expression of the gene of KI67 and P53 in cynomolgus monkeys infected with papillomavirus.

Background and Aim: Cynomolgus monkeys (Macaca fascicularis) develop spontaneous infection of Papillomavirus (PV); thus, potentially beneficial for modeling human PV (HPV) infection study. Contrary to human origin, infection in cynomolgus monkeys does not always show evident clinical symptoms of cervical cancer. The absence of cervical cancer clinical symptoms leads us to investigate the molecular mechanism of the HPV infection in cynomolgus monkeys. This study aimed to investigate the messenger ribonucleic acid (mRNA) expression levels of KI67 and P53 genes, majorly known as biomarker oncogenesis of PV infection. Materials and Methods: The polymerase chain reaction (PCR) technique was used with MY11/MY09 primer to screen PV in cynomolgus monkey, further grouped as positive-PV and negative-PV infection groups. Real-time quantitative PCR was also applied to quantify the mRNA expression levels of KI67 and P53 genes in animals. Results: Increased expression of mRNA level of KI67 genes was significantly higher in Positive- PV group than negative-PV group. In contrast, the P53 mRNA expression level increased markedly higher in the negative-PV group than in the positive-PV group. Conclusion: Our study describes the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection-type. However, we used a limited number of cancer genetic markers. So, further study of other genetic markers is required to prove that cervical cancer could be developed naturally in cynomolgus monkeys.

Construction, expression, and in vitro assembly of virus-like particles of L1 protein of human papillomavirus type 52 in Escherichia coli BL21 DE3.

BACKGROUND: A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein. RESULTS: The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoRI. Digestion by EcoRI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs. CONCLUSION: Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.

Isolation and enzyme bioprospection of bacteria associated to Bruguiera cylindrica, a mangrove plant of North Sumatra, Indonesia.

Mangrove-associated bacteria are of industrial interest due to their diverse and versatile enzyme properties. This study investigates the culturable bacteria from a wide range of habitat in a Bruguiera cylindrica mangrove ecosystem in North Sumatra. Screening of extracellular hydrolytic enzymes showed multiple potential traits in amylase, cellulase, chitinase, phosphatase, protease, and urease production by bacterial isolates. Molecular identification based on 16S rDNA region of a potential strain, Vibrio alginolyticus Jme3-20 is then reported as a newly proteolytic agent. The strain also showed a stable growth under salinity (NaCl) stress with considerable phosphate solubilization activities. Protease activity was enhanced by optimizing the 0.5 % (w/v) sucrose and soy peptone in the fermentation medium. SDS-PAGE and zymogram analysis showed the presence of a 35-kDa MW protease. Hence, our study revealed important insights into the bacterial diversity and activity in mangrove ecosystems, evidencing the importance of microbial exploration in this ecosystem.

Inhibition of enteropathogenic Escherichia coli biofilm formation by DNA aptamer.

Enteropathogenic Escherichia coli (EPEC) is a bioagent that causes diarrhea through the formation of biofilm. The recalcitrant of EPEC to the current conventional antibiotic treatment has grown a big concern in a way to find effective alternative inhibitors. Aptamers have been demonstrated to show the ability to kill the pathogenic bacteria through inhibition of biofilm formation. Therefore, this study aimed to investigate antibiofilm activities of six types of aptamers against EPEC K1.1 which was isolated from patients with diarrhea. Environmental conditions such as temperatures and pH which impacted on biofilm formation of EPEC K1.1 and also biofilm inhibition of aptamer on EPEC K1.1 were performed by counting the crystal violet formation in 96-well polystyrene microplates at OD570. The motility examination combined with qPCR were applied to prove the mechanism of aptamers inhibition on biofilm by targeting essential genes that involve biofilm formation. The result showed that by applying cut off value at 0.399, aptamer SELEX 10 Colony 5 exhibited the highest biofilm inhibition against EPEC K1.1 with an absorbance value of 0.126. Further analysis showed that this aptamer also was able to reduce the motility diameter of EPEC K1.1. The effect of this aptamer on EPEC K1.1 motility was confirmed by qPCR where the mRNA level of motB, csgA and lsrA gene reduced significantly compared to the untreated group. Aptamer SELEX 10 Colony 5 was able to inhibit biofilm formation through interfering the motility ability of EPEC K1.1 and also by reducing the mRNA level of biofilm formation-related genes. This study provides evidences that aptamer is effective and promising for both antibiofilm of EPEC K1.1 and alternative treatment of diarrhea.

Efficacy, toxicity study and antioxidant properties of plantaricin E and F recombinants against enteropathogenic Escherichia coli K1.1 (EPEC K1.1).

Enteropathogenic Escherichia coli (EPEC) is one of the resistance bacteria towards antibiotics and have been raising problem during treatments. Therefore, a new antibiotic candidate is required. Plantaricin E and F recombinant have been successfully produced by a GRAS host Lactococcus lactis. This study was aimed to evaluate the efficacy and toxicity of plantaricin E and F recombinant against EPEC K1.1 infection by in vivo assay. The production of plantaricin E and F recombinants from Lactococcus lactis was conducted and encapsulated. The in vivo study was carried out by inoculating the mice perorally with EPEC K1.1 for 7 days then treated with 100, 250, and 500 mg/kg body weight/day of recombinant plantaricin E and F for another 7 days. The toxicity assay were observed in ddY mice using various concentrations of treatment (50, 100, 1000, and 5000 mg/kg/body weight) doses perorally for 48 h. The result showed that the plantaricin E and F recombinant were successfully produced in Lactococcus lactis expression host with 3.7 kDa and 3.8 kDa in size. The efficacy study revealed the optimal doses of plantaricin E and F recombinant against EPEC K1.1 infection was 250 mg/kgBW for plantaricin E and 500 mg/kgBW for plantaricin F. The plantarisin E and F recombinant treatment showed improvement in leukocyte, hematocrit, and hemoglobin levels as well in decreasing malondialdehyde (MDA) level. Observation of the intestine histopathology showed small amounts of mononuclear inflammatory cell infiltration than the other groups of treatment. The acute toxicity assay showed that there was no mortality observed during the assay, even after 5000 mg/kg body weight of plantarisin E and F recombinant treatment (LD50 > 5000 mg/KgBW). The hematological and biochemical observations showed normal levels in leukocytes, erythrocytes, hematocrit, hemoglobin, platelets, urea, creatinine, and alanine transaminase aspartate transaminase (SGOT and SGPT) while histopathological observation shows a picture of normal liver and kidney cells. This study confirmed the application of bacteriocin for further academic and industrial purposes as a non-toxic substance for food preservative and antibiotic candidate.

The proportion-ratio on dipeptidyl aminopeptidase-4 (DP-4) inhibition by gelatin compared to synthetic sitagliptin.

The current study aims to determine the inhibition activity gelatin against dipeptidyl aminopeptidase 4 (DP-4). Two commercial gelatins, i.e., bovine and fish skin gelatin and one extracted (in our laboratory) gelatin, i.e., fish bone gelatin were selected for analysis. Each gelatin have same protein pattern (75-245 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis with mean of protein concentration of 1.72 mg/mL. The inhibition activity was measured on the capacity to inhibit DP-4 by using Gly-Pro-p-nitroanilide as their substrate. The sitagliptin was used as standard comparison. Based on the percent inhibition, gelatin has been shown to be the prospective DP-4 inhibitor.