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Construction of a Semisynthetic Human VH Single-Domain Antibody Library and Selection of Domain Antibodies against α-Crystalline of Mycobacterium tuberculosis

Hairul Bahara NH, Chin ST, Choong YS, Lim TS

J Biomol Screen, 2016 Jan;21(1):35-43.
PMID: 26423338 DOI: 10.1177/1087057115609144

The use of human variable heavy (VH) domain antibodies has been on the rise due to their small scaffold size and simple folding mechanism. A highly diverse library is largely dependent on the diversity introduced within the complementarity-determining region (CDR) cassettes. Here we introduced diversity with the use of a single framework diversifying all three CDRs using tailored codons consisting of degenerate trinucleotides (NNK). The length of the degeneracy in the CDRs was also taken into consideration based on the most frequently occurring length of CDRs and the canonical confirmation for each antibody subfamily. The semisynthetic human VH domain genes were assembled in a single pot using a temperature cascading process. The affinity selection process with Mycobacterium tuberculosis (MTb) α-crystalline was done using a semiautomated process. Enrichment of target-specific clones was observed with successful identification of monoclonal VH domain antibodies for MTb α-crystalline. In short, the semisynthetic library generated was able to select monoclonal VH domain antibodies against full MTb α-crystalline protein with complete semisynthetic CDRs displayed on a single scaffold. The library has the potential to be applied for the isolation of antibodies against other pathogenic proteins.
Rapid determination of sildenafil and its analogues in dietary supplements using gas chromatography-triple quadrupole mass spectrometry

Mokhtar SU, Chin ST, Kee CL, Low MY, Drummer OH, Marriott PJ

J Pharm Biomed Anal, 2016 Mar 20;121:188-196.
PMID: 26808068 DOI: 10.1016/j.jpba.2016.01.034

Application of gas chromatography-triple quadrupole mass spectrometry for identification, confirmation and quantification of 6 phosphodiesterase-5 (PDE-5) inhibitors (sildenafil, dimethylsildenafil, homosildenafil, thiosildenafil, thiodimethylsildenafil and thiohomosildenafil) in dietary supplements was investigated. The MS was operated in multiple reaction monitoring mode, for better sensitivity and selectivity. In this manner, the method is adequate to reduce background noise with less interference from co-eluting compounds in the samples. Two different ionisation techniques, electron ionisation (EI) and chemical ionisation (CI), were studied and compared. The chromatographic separation was performed on a short 10 m non-polar capillary column without any derivatisation step. This permitted fast analysis for all analogues with retention time less than 11 min, for both techniques. Use of backflushing can aid method retention time reduction and improves column maintenance. Evaluation of method validation included limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, precision and recovery were performed for both EI and CI techniques. The LOD obtained varied from 0.03 to 1.50 μg/g and the LLOQ ranged from 0.10 to 5.00 μg/g. Good calibration linearity was obtained for all analogues for both techniques, with correlation coefficients (r(2)) higher than 0.99. Mean recoveries of all analogues using CI show higher values (83.4-108.8%) than that of EI (61.9-91.1%). The intra- and inter-assay precisions were evaluated for all analogues at spiked concentration of 10 μg/g and the relative standard deviation was less than 15% for both methods. These methods were then successfully applied to dietary supplement samples without prior derivatisation, confirming that the samples were adulterated with sildenafil and/or its analogues.
Fulltext Measles infection causing Bacillus Calmette-Guérin reactivation: a case report

Muthuvelu S, Lim KS, Huang LY, Chin ST, Mohan A

BMC Pediatr, 2019 07 24;19(1):251.
PMID: 31340782 DOI: 10.1186/s12887-019-1635-z

BACKGROUND: Reactivation of the Bacillus Calmette-Guérin (BCG), manifesting as erythema, induration, ulceration or crust formation at a previous BCG inoculation site, is a common and highly specific feature of Kawasaki disease (KD). We report the unusual finding of BCG reactivation in an infant with laboratory-confirmed measles.
CASE PRESENTATION: A previously healthy 7-month old infant presented initially with fever, cough and coryza, and subsequently developed Koplik's spots followed by a typical morbilliform skin rash. There was significant contact history with a household relative who had recently been diagnosed with measles. On examination, a 2.5 cm area of erythema and induration was seen at the previous BCG inoculation site, in addition to the widespread maculopapular rash. No other clinical features of KD were present. Measles virus was isolated from the throat swab and measles antibodies (IgM) were present in the serum. The patient recovered completely with oral vitamin A and supportive therapy, and had normal echocardiography examination on follow up.
CONCLUSIONS: This case report highlights the rare finding of BCG reactivation in a child with confirmed measles infection, and suggests that this clinical manifestation may occasionally occur in children with infections or conditions other than KD.
Fulltext Caring for a child with cancer during COVID-19 pandemic: an assessment of the parents' perception and stress level

Nawi MA, Lau SCD, Chin ST, Teh KH, Ho LSB, Alias H

Front Public Health, 2024;12:1223362.
PMID: 38655523 DOI: 10.3389/fpubh.2024.1223362

BACKGROUND: The emergence of COVID-19 pandemic has led to heightened fear and uncertainty among parents of children with cancer. This study was conducted to evaluate the parental perceptions toward effects of COVID-19 infection to children with cancer, determine their stress level and factors contributing to high stress level during the pandemic.
METHODS: This cross-sectional study was conducted in three paediatric oncology centres in Malaysia from September 2020 until December 2022. A total of 167 parents were recruited. Parents completed a set of questionnaires to assess their perception on effect of COVID-19 infection to children with cancer and COVID Stress Scale (CSS) to assess the parents' stress level.
RESULTS: Patients' mean age at study entry was 8.75 years (SD 4.38). Ninety-one (54.5%) patients were still on active treatment. More than 80% of the parents obtained information regarding COVID-19 infection from mass media and social networking. Fear of their children contracting COVID-19 infection was high especially among patients who were still on treatment. Forty-nine (29.3%) parents were significantly affected by the pandemic leading to loss of job or monthly income. Twenty-nine (17.4%) patients required treatment modification during the pandemic. The median total score for CSS was 78.0 (IQR 25th 64.0; 75th 95.0). Ninety-one (54.5%) respondents were very/extremely stressed based on the CSS scores. Components with high scores were xenophobia (median score 18.0; IQR 25th 13.0, 75th 22.0), fear of danger (median score 17.0; IQR 25th 14.0, 75th 20.0) and contamination fears (median score 16.0; IQR 25th 12.0, 75th 19.0). Lower household income was associated with higher stress level (p = 0.006).
CONCLUSION: Our study demonstrated high awareness regarding risk of COVID-19 infection among parents of oncology children. Half of the parents had high stress level, with low household income identified as a factor associated with high stress level.
Fulltext Comparative study of IgA VH 3 gene usage in healthy TST(-) and TST(+) population exposed to tuberculosis: deep sequencing analysis

Chin ST, Ignatius J, Suraiya S, Tye GJ, Sarmiento ME, Acosta A, et al.

Immunology, 2015 Feb;144(2):302-11.
PMID: 25158076 DOI: 10.1111/imm.12372

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST(+) ) and uninfected TST(-) ) with M. tuberculosis. From a total of 22 V genes analysed, the TST(-) population preferred the VH 3-23 and Vκ1 genes. The VH 3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST(-) population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST(+) population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST(-) population. The antibodyome difference between both populations suggests a preference for antibodies with VH 3-23, D3-3, JH 4 gene usage by the TST(-) population that could be associated with resistance to infection with M. tuberculosis.
Fulltext Complete Genome Sequence of Lignin-Degrading Streptomyces sp. Strain S6, Isolated from an Oil Palm Plantation in Malaysia

Yusof N, Abdullah Tahir A, Riyadi FA, Ahmad Sabri NS, Md Akhir FN, Othman N, et al.

Microbiol Resour Announc, 2020 Jan 30;9(5).
PMID: 32001560 DOI: 10.1128/MRA.01332-19

Streptomyces spp. are bacteria that are responsible for the degradation of aromatic compounds and produce secondary metabolites. Here, we present a complete genome sequence of Streptomyces sp. strain S6, which was isolated from an oil palm plantation, with a 7.8-Mbp liner chromosome, a GC content of 72%, and 4,266 coding sequences.
Fulltext Detection of spleen abscesses facilitates diagnosis of melioidosis in Malaysian children

Mohan A, Manan K, Tan LS, Tan YC, Chin ST, Ahmad R, et al.

Int J Infect Dis, 2020 Sep;98:59-66.
PMID: 32535300 DOI: 10.1016/j.ijid.2020.06.025

OBJECTIVES: Melioidosis is associated with extremely high case fatality ratios. The aim of this study was to determine whether detection of abdominal visceral abscesses can facilitate diagnosis of melioidosis in children.
METHODS: We conducted a retrospective analysis of all children who had liver and/or spleen abscesses on abdominal ultrasonography admitted to Bintulu Hospital in Sarawak, Malaysia, from January 2014 until December 2018.
RESULTS: Fifty-three children had liver and/or spleen abscesses. Spleen abscesses were present in 48 (91%) cases; liver abscesses in 15 (28%). Melioidosis was confirmed by culture in 9 (17%) children; small occult splenic abscesses were present in all cases. In 78% of these cases, the lesions were detected before any positive culture (or serology) results were available. Four (8%) children had bacteriologically-confirmed tuberculosis. Two (4%) had Staphylococcus aureus infection. Of the remaining 38 (72%) culture-negative cases, 36 (95%) had clinical and imaging characteristics similar to that of children with culture-confirmed melioidosis and improved with empirical melioidosis antibiotic therapy.
CONCLUSIONS: A large number of children in Bintulu Hospital in Sarawak, Malaysia, were found to have spleen abscesses. Melioidosis was the most common etiology identified in these children. Abdominal ultrasonography is extremely useful in facilitating the diagnosis of pediatric melioidosis.
Fulltext Systemic Perturbations in Amine and Kynurenine Metabolism Associated with Acute SARS-CoV-2 Infection and Inflammatory Cytokine Responses

Lawler NG, Gray N, Kimhofer T, Boughton B, Gay M, Yang R, et al.

J Proteome Res, 2021 May 07;20(5):2796-2811.
PMID: 33724837 DOI: 10.1021/acs.jproteome.1c00052

We performed quantitative metabolic phenotyping of blood plasma in parallel with cytokine/chemokine analysis from participants who were either SARS-CoV-2 (+) (n = 10) or SARS-CoV-2 (-) (n = 49). SARS-CoV-2 positivity was associated with a unique metabolic phenotype and demonstrated a complex systemic response to infection, including severe perturbations in amino acid and kynurenine metabolic pathways. Nine metabolites were elevated in plasma and strongly associated with infection (quinolinic acid, glutamic acid, nicotinic acid, aspartic acid, neopterin, kynurenine, phenylalanine, 3-hydroxykynurenine, and taurine; p < 0.05), while four metabolites were lower in infection (tryptophan, histidine, indole-3-acetic acid, and citrulline; p < 0.05). This signature supports a systemic metabolic phenoconversion following infection, indicating possible neurotoxicity and neurological disruption (elevations of 3-hydroxykynurenine and quinolinic acid) and liver dysfunction (reduction in Fischer's ratio and elevation of taurine). Finally, we report correlations between the key metabolite changes observed in the disease with concentrations of proinflammatory cytokines and chemokines showing strong immunometabolic disorder in response to SARS-CoV-2 infection.
Fulltext Incomplete Systemic Recovery and Metabolic Phenoreversion in Post-Acute-Phase Nonhospitalized COVID-19 Patients: Implications for Assessment of Post-Acute COVID-19 Syndrome

Holmes E, Wist J, Masuda R, Lodge S, Nitschke P, Kimhofer T, et al.

J Proteome Res, 2021 Jun 04;20(6):3315-3329.
PMID: 34009992 DOI: 10.1021/acs.jproteome.1c00224

We present a multivariate metabotyping approach to assess the functional recovery of nonhospitalized COVID-19 patients and the possible biochemical sequelae of "Post-Acute COVID-19 Syndrome", colloquially known as long-COVID. Blood samples were taken from patients ca. 3 months after acute COVID-19 infection with further assessment of symptoms at 6 months. Some 57% of the patients had one or more persistent symptoms including respiratory-related symptoms like cough, dyspnea, and rhinorrhea or other nonrespiratory symptoms including chronic fatigue, anosmia, myalgia, or joint pain. Plasma samples were quantitatively analyzed for lipoproteins, glycoproteins, amino acids, biogenic amines, and tryptophan pathway intermediates using Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Metabolic data for the follow-up patients (n = 27) were compared with controls (n = 41) and hospitalized severe acute respiratory syndrome SARS-CoV-2 positive patients (n = 18, with multiple time-points). Univariate and multivariate statistics revealed variable patterns of functional recovery with many patients exhibiting residual COVID-19 biomarker signatures. Several parameters were persistently perturbed, e.g., elevated taurine (p = 3.6 × 10-3 versus controls) and reduced glutamine/glutamate ratio (p = 6.95 × 10-8 versus controls), indicative of possible liver and muscle damage and a high energy demand linked to more generalized tissue repair or immune function. Some parameters showed near-complete normalization, e.g., the plasma apolipoprotein B100/A1 ratio was similar to that of healthy controls but significantly lower (p = 4.2 × 10-3) than post-acute COVID-19 patients, reflecting partial reversion of the metabolic phenotype (phenoreversion) toward the healthy metabolic state. Plasma neopterin was normalized in all follow-up patients, indicative of a reduction in the adaptive immune activity that has been previously detected in active SARS-CoV-2 infection. Other systemic inflammatory biomarkers such as GlycA and the kynurenine/tryptophan ratio remained elevated in some, but not all, patients. Correlation analysis, principal component analysis (PCA), and orthogonal-partial least-squares discriminant analysis (O-PLS-DA) showed that the follow-up patients were, as a group, metabolically distinct from controls and partially comapped with the acute-phase patients. Significant systematic metabolic differences between asymptomatic and symptomatic follow-up patients were also observed for multiple metabolites. The overall metabolic variance of the symptomatic patients was significantly greater than that of nonsymptomatic patients for multiple parameters (χ2p = 0.014). Thus, asymptomatic follow-up patients including those with post-acute COVID-19 Syndrome displayed a spectrum of multiple persistent biochemical pathophysiology, suggesting that the metabolic phenotyping approach may be deployed for multisystem functional assessment of individual post-acute COVID-19 patients.
Fulltext JAK-STAT and G-protein-coupled receptor signaling pathways are frequently altered in epitheliotropic intestinal T-cell lymphoma

Nairismägi ML, Tan J, Lim JQ, Nagarajan S, Ng CC, Rajasegaran V, et al.

Leukemia, 2016 06;30(6):1311-9.
PMID: 26854024 DOI: 10.1038/leu.2016.13

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available.
Fulltext Genetic risk of extranodal natural killer T-cell lymphoma: a genome-wide association study

Li Z, Xia Y, Feng LN, Chen JR, Li HM, Cui J, et al.

Lancet Oncol, 2016 Sep;17(9):1240-7.
PMID: 27470079 DOI: 10.1016/S1470-2045(16)30148-6

BACKGROUND: Extranodal natural killer T-cell lymphoma (NKTCL), nasal type, is a rare and aggressive malignancy that occurs predominantly in Asian and Latin American populations. Although Epstein-Barr virus infection is a known risk factor, other risk factors and the pathogenesis of NKTCL are not well understood. We aimed to identify common genetic variants affecting individual risk of NKTCL.
METHODS: We did a genome-wide association study of 189 patients with extranodal NKTCL, nasal type (WHO classification criteria; cases) and 957 controls from Guangdong province, southern China. We validated our findings in four independent case-control series, including 75 cases from Guangdong province and 296 controls from Hong Kong, 65 cases and 983 controls from Guangdong province, 125 cases and 1110 controls from Beijing (northern China), and 60 cases and 2476 controls from Singapore. We used imputation and conditional logistic regression analyses to fine-map the associations. We also did a meta-analysis of the replication series and of the entire dataset.
FINDINGS: Associations exceeding the genome-wide significance threshold (p<5 × 10(-8)) were seen at 51 single-nucleotide polymorphisms (SNPs) mapping to the class II MHC region on chromosome 6, with rs9277378 (located in HLA-DPB1) having the strongest association with NKTCL susceptibility (p=4·21 × 10(-19), odds ratio [OR] 1·84 [95% CI 1·61-2·11] in meta-analysis of entire dataset). Imputation-based fine-mapping across the class II MHC region suggests that four aminoacid residues (Gly84-Gly85-Pro86-Met87) in near-complete linkage disequilibrium at the edge of the peptide-binding groove of HLA-DPB1 could account for most of the association between the rs9277378*A risk allele and NKTCL susceptibility (OR 2·38, p value for haplotype 2·32 × 10(-14)). This association is distinct from MHC associations with Epstein-Barr virus infection.
INTERPRETATION: To our knowledge, this is the first time that a genetic variant conferring an NKTCL risk is noted at genome-wide significance. This finding underlines the importance of HLA-DP antigen presentation in the pathogenesis of NKTCL.
FUNDING: Top-Notch Young Talents Program of China, Special Support Program of Guangdong, Specialized Research Fund for the Doctoral Program of Higher Education (20110171120099), Program for New Century Excellent Talents in University (NCET-11-0529), National Medical Research Council of Singapore (TCR12DEC005), Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore National Cancer Centre Research Fund, and the US National Institutes of Health (1R01AR062886, 5U01GM092691-04, and 1R01AR063759-01A1).

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