Molecular dynamics simulation and purification of chimeric L1/L2 protein from human papillomavirus type 52 expressed in Escherichia coli BL21 (DE3).

The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.

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.

Construction, heterologous expression, partial purification, and in vitro cytotoxicity of the recombinant plantaricin E produced by Lactococcus lactis against Enteropathogenic Escherichia coli K.1.1 and human cervical carcinoma (HeLa) cells.

Lactobacillus plantarum produces bacteriocin called plantaricin that can kill or inhibit other bacteria. Plantaricin E (Pln E), a recombinant bacteriocin, has been successfully constructed and produced by a GRAS host, Lactococcus lactis. A polymerase chain reaction (PCR) overlapping technique has been used to construct a ligation of signal peptide gene, Pln A and bacteriocin encoding gene, Pln E. Furthermore, the fusion fragment were cloned into pNZ8148 vector and transformed into L. lactis NZ3900. Molecular expression study shows that recombinant L. lactis NZ3900 is able to express the mature pln E at transcription level with size of 168 bp. Plantaricin E is purified by ammonium sulphate precipitation followed by gel filtration chromatography. Purified fractions were proven to be active against Enteropathogenic Escherichia coli K.1.1. The other fractions of Pln E also have antibacterial activity against several Gram positive and Gram negative bacteria. Purified recombinant plantaricin E is 3.7 kDa in size. The cytotoxicity assay shows purified Pln E inhibits 46.949 ± 3.338% of HeLa cell lines on 10 ppm dose whilst the metabolite inhibits 53.487 ± 2.957% of HeLa cell line on 100 ppm dose. The IC50 calculation of Pln E metabolite is 107.453 ppm, while the purified protein is 11.613 ppm.

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.

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.

A New Approach for Controlling Agrobacterium tumefaciens Post Transformation Using Lytic Bacteriophage.

Overgrowth of Agrobacterium tumefaciens has frequently been found in Agrobacterium-mediated plant transformation. This overgrowth can reduce transformation efficiency and even lead to explant death. Therefore, this research investigates an alternative way to mitigate or eliminate Agrobacterium after transformation using a bacteriophage. To develop this alternative method, we conducted effectiveness studies of two lytic bacteriophages (ΦK2 and ΦK4) and performed an application test to control Agrobacterium growth after transformation. According to plaque morphological characterization and molecular analysis, the two bacteriophages used in this experiment were distinct. Moreover, some stability physicochemical and growth kinetics, such as adsorption time and susceptibility test, also showed that both bacteriophages differed. On the other hand, the optimum temperature and pH of both phages were the same at 28-30 °C and pH 7. Further investigation showed that both ΦK2 and ΦK4 were able to reduce the overgrowth of A. tumefaciens post transformation. Moreover, applying the cocktail (mixture of ΦK2 and ΦK4) with antibiotic application eradicated A. tumefaciens (0% overgrowth percentage). This result indicates that the application of bacteriophage could be used as an alternative way to eradicate the overgrowth of A. tumefaciens subsequent to transformation.

Serum vitamin B12 associated with vitamin D/25(OH)D in women with recurrent aphthous stomatitis / Vitamina B12 sérica asociada con vitamina D / 25 (OH) D en mujeres con estomatitis aftosa recurrente

Vitamin B12 and Vitamin D deficiency may contribute to recurrent aphthous stomatitis (RAS). Current studies have showed vitamin B12 to be associated with vitamin D in women, however no study has assessed vitamin B12 associated with vitamin D/25(OH)D in women with RAS. Objective: To investigate the association between serum vitamin B12 and vitamin D/25(OH)D in women with RAS. Materials and Methods: Fourty one women with RAS who meet the inclusion criteria participated in this study. The inclusion criteria were women with RAS without other oral diseases. The exclusions criteria were those who have systemic diseases, taking medications or smoked. All subjects underwent venupuncture to draw blood to quantify serum vitamin B12 and vitamin D/25(OH)D. The characteristic of subjects, severity of RAS, serum Vitamin B12 and Vitamin D/25(OH)D were collected and presented descriptively. The correlation between vitamin B12 and Vitamin D/25(OH)D was analyzed using Pearson correlation test with 95% confidence interval. This study was approved by Medical and Health Ethics Committe, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Results: All RAS subjects have normal mean value of serum Vitamin B12 (453.97+154.44pg/ml) and have low mean value of serum vitamin D/25(OH)D (10.79 +3.29ng/ml) categorized as vitamin D deficiency. The Pearson correlation test showed that there was a significant positive correlation between mean serum Vitamin B12 and Vitamin D/25(OH)D (r= 0.313, p<0.05). Conclusion: There is correlation between vitamin B12 and Vitamin D, and a low level of Vitamin D may contribute in RAS in women.

Antecedentes: la deficiencia de vitamina B12 y vitamina D puede contribuir a la estomatitis aftosa recurrente (EAR). Los estudios actuales han demostrado que la vitamina B12 está asociada con la vitamina D en mujeres, sin embargo, ningún estudio ha evaluado la vitamina B12 asociada con la vitamina D/25 (OH) D en mujeres con EAR. Objetivo: investigar la asociación entre la vitamina B12 y la vitamina D / 25 (OH) D sérica en mujeres con RAS. Material y Métodos: Cuarenta y una mujeres con RAS que cumplen con los criterios de inclusión participaron en este estudio. Los criterios de inclusión fueron mujeres con RAS y sin otras enfermedades orales. Los criterios de exclusión fueron aquellos que tenían enfermedades sistémicas, tomaban medicamentos o fumaban. Todos los sujetos se sometieron a una venupuntura para extraer sangre para cuantificar la vitamina B12 y la vitamina D/25 (OH) D en suero. Las características de los sujetos, la severidad del EAR, la concentración de vitamina B12 y la vitamina D/25 (OH) D sérica fueron recolectadas y presentadas descriptivamente. La correlación entre la vitamina B12 y la vitamina D/25 (OH) D se analizó mediante la prueba de correlación de Pearson con un intervalo de confianza del 95%. Este estudio fue aprobado por el Comité de Ética Médica y de Salud, Facultad de Medicina, Universitas Gadjah Mada, Yogyakarta, Indonesia. Resultado: Todos los sujetos con EAR tienen un valor medio normal de vitamina B12 sérica (453,97pg/ml + 154,44pg/ml) y un valor medio bajo de vitamina D sérica/25 (OH) D (10,79 ng/ml + 3,29ng/ml) clasificado como deficiencia de vitamina D. La prueba de correlación de Pearson mostró que había una correlación positiva significativa entre la vitamina B12 media y la vitamina D/25 (OH) D en suero r=0.313, p<0.05). Conclusión: Existe una correlación entre la vitamina B12 y la vitamina D, y un bajo nivel de vitamina D puede contribuir al RAS en las mujeres.

Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans.

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases.