Plasmodium knowlesi circumsporozoite protein: genetic characterisation and predicted antigenicity of the central repeat region

@#Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif “AGQPQAQGDGANAGQPQAQGDGAN” has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.

Cloning, expression and purification of Plasmodium knowlesi circumsporozoite protein and immunoblot analysis with P. knowlesi strain A1H1 protein extract

@#Circumsporozoite protein (CSP) is a sporozoite major surface protein of Plasmodium species. The protein showed promising protection level as a vaccine candidate against Plasmodium falciparum infection. There is a lack of studies on P. knowlesi CSP (PkCSP) as a vaccine candidate due to the high polymorphic characteristic of central repeat region. Recent studies showed the protein has a relatively conserved region at the C-terminal, which consists of T- and B-cell epitopes. This could be the target region for vaccine development against the pre-erythrocytic stage of the parasite. In this study, recombinant PkCSP was expressed using Escherichia coli system. Recombinant PkCSP was immunized in animal models and the antiserum was evaluated using immunoblot analysis. Results showed that PkCSP can be successfully expressed using the bacterial system. Endpoint titre of the antiserum were ranged up to 1:819200. Immunoblot analysis showed the antiserum recognized recombinant PkCSP but not total protein extract from P. knowlesi erythrocytic stage. In conclusion, PkCSP could elicit strong immune response in animal models. However, serum antibodies could not recognize protein from the parasite’s erythrocytic stage extract indicating it is not expressed at the erythrocytic stage. Therefore, PkCSP remains as a potential pre-erythrocytic vaccine candidate against P. knowlesi infection.

Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

@#Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Complications of Sub-microscopic Plasmodium vivax Malaria among Orang Asli in Pos Lenjang, Kuala Lipis

@#In recent years, increasing cases of Plasmodium vivax complications had been reported all over the world. This former benign Plasmodium species is now recognized to be one of the human malaria parasites that can produce severe disease. In this article, we report two cases of sub-microscopic P. vivax malaria confirmed by PCR. Both patients were asymptomatic before treatment. They showed unusual presentations few days after initiation of antimalarial treatment. Both patients had subsequently completed antimalarial treatment and recovered completely.

Pancreatic ß-cell function is inhibited by miR-3666 in type 2 diabetes mellitus by targeting adiponectin

Type 2 diabetes mellitus (T2D) is a common endocrine and metabolic disorder, and poses threats to human health worldwide. Recently, microRNAs (miRNAs) have been suggested to play important roles in the pathophysiology of T2D. In this study, we explored the role of miR-3666 in T2D. miR-3666 was significantly down-regulated in the serum of T2D patients when compared to that of healthy volunteers, and miR-3666 expression level was negatively correlated with blood glucose levels of T2D patients. Overexpression of miR-3666 inhibited cell proliferation, reduced insulin secretion, and promoted cell apoptosis of pancreatic β-cell line (INS-1 cells). On the other hand, knockdown of miR-3666 had the opposite effects in INS-1 cells. The bio-informatics analysis using TargetScan revealed that adiponectin (ADIPOQ) was a downstream target of miR-3666, and the interaction between miR-3666 and ADIPOQ was validated by luciferase reporter assay. In addition, miR-3666 negatively regulated the mRNA and protein expression of ADIPOQ. Overexpression of ADIPOQ promoted insulin secretion after glucose stimulation, promoted cell proliferation, inhibited cell apoptosis, and partially abolished the effects of miR-3666 overexpression on insulin secretion, cell proliferation, and cell apoptosis of INS-1 cells. In conclusion, our results revealed that miR-3666 inhibited pancreatic cell proliferation, reduced insulin sensitivity, and promoted apoptosis by targeting ADIPOQ.

Inhibition of STAT3 by RNA interference suppresses angiogenesis in colorectal carcinoma

In order to investigate signal transduction and activation of transcription 3 (STAT3) signaling on angiogenesis in colorectal carcinoma (CRC) after inhibiting STAT3 expression, we constructed the HT-29-shSTAT3 cell line by lentivirus-mediated RNAi. Cell growth was assessed with MTT and the cell cycle distribution by flow cytometry. CRC nude mouse models were established and tumor growth was monitored periodically. On day 30, all mice were killed and tumor tissues were removed. Microvessel density (MVD) was determined according to CD34-positive staining. The expression of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-2 (MMP2) and basic fibroblast growth factor (FGF2) was monitored by quantitative real-time PCR and Western blot analysis. Knockdown of STAT3 expression significantly inhibited cell growth in HT-29 cells, with a significantly higher proportion of cells at G0/G1 (P < 0.01). Consistently, in vivo data also demonstrated that tumor growth was significantly inhibited in mice injected with HT-29-shSTAT3 cells. MVD was 9.80 ± 3.02 in the HT-29-shSTAT3 group, significantly less than that of the control group (P < 0.01). mRNA and protein levels of VEGFA and MMP2 in the HT-29-shSTAT3 group were significantly lower than in the control group (P < 0.05), but no significant difference was observed in the mRNA or protein level of FGF2 (P > 0.05). Taken together, these results demonstrate that STAT3 signaling is important to the growth of CRC and promotes angiogenesis by regulating VEGFA and MMP2 expression.

Common allergens in contact dermatitis as determined by patch testing: a 4-year experience at Jose R. Reyes Memorial Medical Center (JRRMMC) - preliminary report

Objective: This study sought to determine through patch testing the ten most common allergens among patients with suspected allergic contact dermatitis (ACD) and thus, provide dermatologists with a useful guide in patient evaluation and counseling regarding the avoidance of triggering factors that perpetuate their allergic skin problemsDesign: An observational desriptive studySetting: Tertiary government hospitalPatients: A total of 119 patients who presented with skin lesions at the outpatient section of the JRRMMC Dermatology Department from July 1991-June 1995 were patch tested. From these, 80 patients diagnosed to have ACD were given emphasis in the final analysis and evaluationResults: Of the total 119 patients patch tested during this 4-year time period, 80 patients (67.22%) were clinically diagnosed to have allergic contact dermatitis. The sites of dermatitis commonly affected in this group were the feet, the hands and the arms. the ten most common skin sensitizers identified among these patients were as folows: fragrance mix, nickel sulfate, potassium dichromate and thiurum mix-both at third, p-phenylenediamine, cinnamic aldehyde, balsam of Peru, epoxy resin and paraben mix-both at seventh, carba rubber mix, bronopol and 2-mercaptobenzothiazole both at ninth, wool alcohol and mercapto-mix sharing the tenth place. It is interesting to note that the top five allergens in this group share similar ranking to that which figured prominently among patients who exhibited various kinds of dermatitidesConclusions: As the result of rapid industrialization, the incidence of ACD has risen to the leaping proportion in the last two decades. Definite cure is obtained primarily by avoidance of the specific allergen(s). These are best indentified through patch testing. By undertaking this study, the authors hope to provide some local statistics on the most common skin sensitizers causing the ACD and therefore, place physicians and dermatologists in particular, in a better position to give their patients sound advice regarding the avoidance of tigerring factors that can readily perpetuate their skin problems.(Author)

Miliary tuberculosis in Filipino infants and children: a study of twenty-seven cases in an urban hospital from 1970 to 1980

This study determined the age distribution, the number of miliary tuberculosis as accidental findings on autopsy cases not recognized by radiographic examination of the chest. A retrospective study of 27 cases admitted at National Childrens Hospital from 1970-1980 was done. Sex incidence was noted and found to be more common in females. The age distribution showed a high incidence of miliary tuberculosis in infants two years old and below with the youngest patient aged three months old and the oldest fourteen years of age. Fever and cough with anorexia, weight loss, anemia and hepotomegaly dominated the clinical picture of these patients. Malnutrition and poor living condition and the development of measles were significant contributing factors to the development miliary tuberculosis. Because of the young age of the patient, presence of other diseases and poor host defense mechanism, miliary tuberculosis has a high mortality rate. (Auth)

The spectrum of beta-thalassemia mutations in Malays in Singapore and Kelantan.

The spectrum of beta-thalassemia mutations in Malays in Singapore and Kelantan (Northeast Malaysia) was studied. Allele specific priming was used to determine the mutations in beta-carriers at -28, Codon 17, IVSI #1, IVSI #5, Codon 41-42 and IVSII #654 along the beta-globin gene. The most common structural hemoglobin variant in Southeast Asia, Hb E, was detected by DNA amplification with restriction enzyme (Mnl1) analysis. Direct genomic sequencing was carried out to detect the beta-mutations uncharacterized by allele-specific priming. The most prevalent beta-mutations in Singaporean Malays were IVSI #5 (45.83%) followed by Hb E (20.83%), codon 15 (12.5%) and IVSI #1 and IVSII #654 at 4.17% each. In contrast, the distribution of the beta-mutations in Kelantan Malays differed, with Hb E as the most common mutation (39.29%) followed by IVSI #5 (17.86%), codon 41-42 (14.29%), codon 19 (10.71%) and codon 17 (3.57%). The beta-mutations in Kelantan Malays follow closely the distribution of beta-mutations in Thais and Malays of Southern Thailand and Malays of West Malaysia. The AAC-->AGC base substitution in codon 19 has been detected only in these populations. The spectrum of beta-mutations in the Singaporean Malays is more similar to those reported in Indonesia with the beta-mutation at codon 15 (TGG-->TAG) present in both populations. The characterization of beta-mutations in Singaporean and Kelantan Malays will facilitate the establishment of effective prenatal diagnosis programs for beta-thalassemia major in this ethnic group.

Detection of Mycobacterium tuberculosis in sputum, pleural and bronchoalveolar lavage fluid using DNA amplification of the MPB 64 protein coding gene and IS6110 insertion element.

Two gene sequences specific for Mycobacterium tuberculosis were evaluated for the diagnosis of pulmonary tuberculous (PTB) in pleural fluid (PF), bronchoalveolar lavage fluid (BAL) and sputum (Sp). The 240 bp sequence (nts 460-700) coding for the MPB 64 protein coding gene and the 123 bp IS6110 insertion element present in multiple copies in the mycobacterial genome were amplified using the polymerase chain reaction. Fifty-nine clinical specimens were studied. The diagnosis of PTB was confirmed by positive M. tuberculosis cultures in 14 specimens, and by the presence of characteristic histological features of granuloma and Langerhan's giant cells on pleural biopsy in 3 PF specimens through cultures for M. tuberculosis were negative. The remaining 42 specimens were obtained from patient's with non-tuberculosis pulmonary infections or malignancy, and these served as negative controls. Our results showed that the IS6110 insertion element and MPB 64 gene sequence were detected in all 14 culture positive PTB cases, although detection of the latter sequence required both DNA amplification and oligonucleotide hybridization. There was however one false positive specimen with the MPB 64 detection protocol. More importantly, both the MPB 64 sequence and IS6110 insertion element protocols were unable to detect M. tuberculosis DNA in the 3 PF samples diagnosed by histological characteristics on pleural biopsy and culture negative. We conclude that DNA amplification for M. tuberculosis-specific sequences is a useful method for rapid diagnosis of PTB in culture positive specimens. However, the false negative results with TB culture negative cases of tuberculosis pleurisy, limits its usefulness for the diagnosis of tuberculous pleurisy.

Molecular analysis of Hb Q-H disease and Hb Q-Hb E in a Singaporean family.

Hb Q (alpha 74Asp-His) results from a mutation in the alpha-gene such that abnormal alpha Q-chains are synthesized. The alpha Q-chains combine with the normal Beta A-chains to form abnormal Hb alpha 2Q beta 2A (Hb Q). Hb Q-H disease is rare, and has been reported only in the Chinese. We report here a Chinese family, were the mother diagnosed with Hb Q-H disease and the father with Hb E heterozygosity and a child with Hb Q-E-thalassemia. Thalassemia screening of the mother's blood revealed a Hb level of 6.8g/dl with low MCV and MCH. Her blood film was indicative of thalassemia. Cellulose acetate electrophoresis showed Hb H and Hb Q with the absence of Hb A. Globin chain biosynthesis was carried out and alpha Q- and beta-chains were detected. Normal alpha- chains were absent. Digestion of the mother's DNA with Bam HI and Bgl II followed by hybridization with the 1.5 kb alpha-Pst probe showed a two alpha-gene deletion on one chromosome and the -alpha Q chain mutant with the -alpha 4.2 defect on the other chromosome. DNA amplification studies indicated the two-gene deletion to be of the -SEA/ defect. The patient was concluded to possess Hb Q-H disease (--SEA/-alpha 4.2Q). Cellulose acetate electrophoresis of the father's blood showed the presence of Hb A, F and E. Molecular analysis of the father's DNA confirmed an intact set of alpha-genes (alpha alpha/alpha alpha). Globin chain biosynthesis of fetal blood of their child showed gamma, beta A, beta E, alpha A and alpha Q-chains. Molecular analysis of the child's DNA showed one alpha-gene deletion, thus giving a genotype of alpha alpha/-alpha 4.2Q beta beta E.