Multi-spectral and docking assessments to explore the combination of an antiviral drug, entecavir with bovine serum albumin.

Molecular interaction of entecavir (ETV) with the transport protein, albumin from bovine serum (BSA) was explored through multispectral and molecular docking approaches. The BSA fluorescence was appreciably quenched upon ETV binding and the quenching nature was static. The ETV-BSA complexation and the static quenching process were further reiterated using UV-visible absorption spectra. The binding constant (Ka) values of the complex were found as 1.47 × 104-4.0 × 103 M-1, which depicting a modarate binding strength in the ETV-BSA complexation. The experimental outcomes verified that the stable complexation was primarily influenced by hydrophobic interactions, hydrogen bonds and van der Waals forces. Synchronous and 3-D fluorescence spectral results demonstrated that ETV had significant impact on the hydrophobicity and polarity of the molecular environment near Tyr and Trp residues. Competitive site-markers displacement (with warfarin and ketoprofen) results discovered the suitable binding locus of ETV at site I in BSA. The molecular docking assessments also revealed that ETV formed hydrogen bonds and hydrophobic interactions with BSA, predominantly binding to site I (sub-domain IIA) of BSA.

Unveiling the molecular interaction of hepatitis B virus inhibitor, entecavir with human serum albumin through computational, microscopic and spectroscopic approaches.

Molecular docking, molecular dynamics (MD) simulation, atomic force microscopy (AFM) and multi-spectroscopic techniques were selected to unveil the molecular association between the hepatitis B virus (HBV) inhibitor, entecavir (ETR), and the major blood plasma transporter, human serum albumin (HSA). The entire docking and simulation analyses recognized ETR binding to subdomain IIA (Site I) of HSA through hydrogen bonds, hydrophobic and van der Waals forces while maintaining the complex's stability throughout the 100 ns. A gradual lessening in the Stern-Volmer quenching constant (Ksv) with rising temperatures registered ETR-induced quenching of HBV fluorescence as static quenching, thus advising complexation between ETR and HSA. The further advocation of this conclusion was seen from a larger value of the biomolecular quenching rate constant ((kq) > 1010 M-1s-1), changes in the spectra (UV-Vis absorption) of HSA following ETR inclusion and ETR-induced swelling of HSA in the AFM results. The ETR appeared to bind to HSA with moderate affinity (Ka=1.87-1.19×104 M-1) at 290, 300 and 310 K. Significant alterations in the protein's secondary and tertiary structures, including changes in the protein's Tyr/Trp microenvironment, were also detected by circular dichroism and three-dimensional fluorescence spectra when the protein was bound to ETR. The findings of the drug displacement study backed the docking results of Site I as ETR's preferred binding site in HSA.Communicated by Ramaswamy H. Sarma.

Deciphering the binding mode and structural perturbations in floxuridine-human serum albumin complexation with spectroscopic, microscopic, and computational techniques.

The binding mode of antineoplastic antimetabolite, floxuridine (FUDR), with human serum albumin (HSA), the leading carrier in blood circulation, was ascertained using multi-spectroscopic, microscopic, and computational techniques. A static fluorescence quenching was established due to decreased Ksv values with rising temperatures, suggesting FUDR-HSA complexation. UV-vis absorption spectral results also supported this conclusion. The binding constant, Ka values, were found within 9.7-7.9 × 103 M-1 at 290, 300, and 310 K, demonstrating a moderate binding affinity for the FUDR-HSA system. Thermodynamic data (ΔS = +46.35 J.mol-1.K-1 and ΔH = -8.77 kJ.mol-1) predicted the nature of stabilizing forces (hydrogen-bonds, hydrophobic, and van der Waals interactions) for the FUDR-HSA complex. Circular dichroism spectra displayed a minor disruption in the protein's 2° and 3° structures. At the same time, atomic force microscopy images proved variations in the FUDR-HSA surface morphology, confirming its complex formation. The protein's microenvironment around Trp/Tyr residues was also modified, as judged by 3-D fluorescence spectra. FUDR-bound HSA showed better resistance against thermal stress. As disclosed from ligand displacement studies, the FUDR binding site was placed in subdomain IIA (Site I). Further, the molecular docking analysis corroborated the competing displacement studies. Molecular dynamics evaluations revealed that the complex achieved equilibrium during simulations, confirming the FUDR-HSA complex's stability.

Clinical features and mutational analysis of X-linked agammaglobulinemia patients in Malaysia.

Background: Bruton's tyrosine kinase (BTK) is a cytoplasmic protein involved in the B cell development. X-linked agammaglobulinemia (XLA) is caused by mutation in the BTK gene, which results in very low or absent B cells. Affected males have markedly reduced immunoglobulin levels, which render them susceptible to recurrent and severe bacterial infections. Methods: Patients suspected with X-linked agammaglobulinemia were enrolled during the period of 2010-2018. Clinical summary, and immunological profiles of these patients were recorded. Peripheral blood samples were collected for monocyte BTK protein expression detection and BTK genetic analysis. The medical records between January 2020 and June 2023 were reviewed to investigate COVID-19 in XLA. Results: Twenty-two patients (from 16 unrelated families) were molecularly diagnosed as XLA. Genetic testing revealed fifteen distinct mutations, including four splicing mutations, four missense mutations, three nonsense mutations, three short deletions, and one large indel mutation. These mutations scattered throughout the BTK gene and mostly affected the kinase domain. All mutations including five novel mutations were predicted to be pathogenic or deleterious by in silico prediction tools. Genetic testing confirmed that eleven mothers and seven sisters were carriers for the disease, while three mutations were de novo. Flow cytometric analysis showed that thirteen patients had minimal BTK expression (0-15%) while eight patients had reduced BTK expression (16-64%). One patient was not tested for monocyte BTK expression due to insufficient sample. Pneumonia (n=13) was the most common manifestation, while Pseudomonas aeruginosa was the most frequently isolated pathogen from the patients (n=4). Mild or asymptomatic COVID-19 was reported in four patients. Conclusion: This report provides the first overview of demographic, clinical, immunological and genetic data of XLA in Malaysia. The combination of flow cytometric assessment and BTK genetic analysis provides a definitive diagnosis for XLA patients, especially with atypical clinical presentation. In addition, it may also allow carrier detection and assist in genetic counselling and prenatal diagnosis.

Comprehensive views toward the biomolecular recognition of an anticancer drug, leflunomide with human serum albumin.

Biomolecular association of an anticancer drug, leflunomide (LEF) with human serum albumin (HSA), the leading ligands carrier in human circulation was characterized using biophysical (i.e., fluorescence, absorption and voltammetric) methods and computational (i.e., molecular docking and molecular dynamics simulation) techniques. Evaluations of fluorescence, absorption and voltammetric findings endorsed the complex formation between LEF and HSA. An inverse relationship of Stern-Volmer constant-temperature and hyperchromic shift of the protein's absorption signal with addition of LEF confirmed the LEF quenched the HSA fluorescence through static process. Moderate nature of binding strength (binding constant = 2.76-4.77 × 104 M-1) was detected towards the LEF-HSA complexation, while the association process was naturally driven via hydrophobic interactions, van der Waals interactions and hydrogen bonds, as evident from changes in entropy (ΔS= + 19.91 J mol-1 K-1) and enthalpy (ΔH = - 20.09 kJ mol-1), and molecular docking assessments. Spectral analyses of synchronous and three-dimensional fluorescence validated microenvironmental fluctuations near Trp and Tyr residues upon LEF binding to the protein. LEF association with HSA significantly defended temperature-induced destabilization of the protein. Although LEF was found to attach to HSA at Sudlow's sites I and II, but exhibited greater preference toward its site I, as detected by the investigations of competitive site-marker displacement. Molecular dynamics simulation assessment revealed that the complex attained equilibrium throughout simulations, showing the LEF-HSA complex constancy.Communicated by Ramaswamy H. Sarma.

Gut microbiota of healthy Asians and their discriminative features revealed by metagenomics approach.

This study is conducted to identify the microbial architecture and its functional capacity in the Asian population via the whole metagenomics approach. A brief comparison of the Asian countries namely Malaysia, India, China, and Thailand, was conducted, giving a total of 916 taxa under observation. Results show a close representation of the taxonomic diversity in the gut microbiota of Malaysia, India, and China, where Bacteroidetes, Firmicutes, and Actinobacteria were more predominant compared to other phyla. Mainly due to the multi-racial population in Malaysia, which also consists of Malays, Indian, and Chinese, the population tend to share similar dietary preferences, culture, and lifestyle, which are major influences that shapes the structure of the gut microbiota. Moreover, Thailand showed a more distinct diversity in the gut microbiota which was highly dominated by Firmicutes. Meanwhile, functional profiles show 1034 gene families that are common between the four countries. The Malaysia samples are having the most unique gene families with a total of 67,517 gene families, and 51 unique KEGG Orthologs, mainly dominated by the metabolic pathways, followed by microbial metabolism in diverse environments. In conclusion, this study provides some general overview on the structure of the Asian gut microbiota, with some additional highlights on the Malaysian population. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03671-3.

In vitro interactions of two pesticides, propazine and quinoxyfen with bovine serum albumin: Spectrofluorometric and molecular docking investigations.

Binding mechanisms of two selected pesticides, propazine (PRO) and quinoxyfen (QUI) with bovine serum albumin (BSA) was examined using fluorescence, absorption and molecular docking methods. Intrinsic fluorescence of BSA was quenched in the presence of both PRO and QUI. The quenching was ascertained to be conversely linked to temperature, which suggested the contribution of static quenching process in the PRO-BSA and QUI-BSA complex formations. This results were validated by the enhancement in absorption spectrum of BSA upon binding with PRO and QUI. Binding constant values (Kf = 9.55-0.60 × 10-3 M-1 for PRO-BSA system; Kf = 7.08-5.01 × 102 M-1 for QUI-BSA system) and number of binding site (n) values for the PRO-BSA and QUI-BSA systems at different temperatures affirmed a weak binding strength with a set of equivalent binding sites on BSA. Thermodynamic data obtained for both the PRO-BSA and QUI-BSA interactions predicted that the association process was spontaneous and non-covalent contacts such as hydrophobic interactions, van der Waals forces and hydrogen bonds participated in the binding reactions. This result was further supported by the molecular docking assessments. Three-dimensional spectral results revealed the microenvironmental alterations near tryptophan (Trp) and tyrosine (Tyr) residues in BSA by the addition of PRO and QUI. The docking analysis demonstrated the binding pattern for the PRO-BSA and QUI-BSA systems and disclosed the preferred binding site of both PRO and QUI as site I (subdomain IIA) of BSA.

The role and influence of bacteria on the growth of Blastocystis sp. subtype 3.

Blastocystis sp. is an enteric protistan parasite that affects individuals worldwide with gastrointestinal symptoms such as abdominal discomfort, diarrhea, and flatulence. However, its pathogenicity is controversial due to its presence among asymptomatic individuals. Blastocystis sp. subtype 3 (ST3) is the most prevalent subtype among humans that have been associated with irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, and colorectal cancer. Axenization of the parasite has been shown to impede its growth thus revealing the importance of accompanying bacteria in ensuring Blastocystis sp. survival. This study aims to identify the influence of accompanying bacteria on the growth of Blastocystis sp. ST3. Blastocystis sp. cultures were treated with Meropenem, Vancomycin, and Amoxicillin-Clavulanic acid (Augmentin). Bacteria-containing supernatant of antibiotic-treated and control cultures were isolated and identified through 16 s rRNA sequencing. Morphological changes of antibiotic-treated Blastocystis sp. ST3 were also observed. The cultures treated with meropenem and augmentin exhibited opposing effects with reduced growth of isolates from symptomatic patients and a significant increase in asymptomatic isolates. Whereas, vancomycin-treated cultures had no difference in the growth of Blastocystis sp. ST3 isolates from symptomatic and asymptomatic patients. Isolates from symptomatic and asymtomatic patients had 6 and 2 distinct bacterial species identified with Proteus mirabilis as the common bacteria among both types of isolates. Morphologically, Blastocystis sp. ST3 cultures exposed to meropenem and augmentin demonstrated an increase in pre-cystic forms. These findings demonstrate the effects of accompanying bacteria on the growth of Blastocystis sp. ST3 that could translate into clinical manifestations observed among Blastocystis sp.-infected patients.

Ensemble-based molecular docking and spectrofluorometric analysis of interaction between cytotoxin and tumor necrosis factor receptor 1.

Cytotoxin (CTX) is a three-finger toxin presents predominantly in cobra venom. The functional site of the toxin is located at its three hydrophobic loop tips. Its actual mechanism of cytotoxicity remains inconclusive as few conflicting hypotheses have been proposed in addition to direct cytolytic effects. The present work investigated the interaction between CTX and death receptor families via ensemble-based molecular docking and fluorescence titration analysis. Multiple sequence alignments of different CTX isoforms obtained a conserved CTX sequence. The three-dimensional structure of the conserved CTX was later determined using homology modelling, and its quality was validated. Ensemble-based molecular docking of CTX was performed with different death receptors, such as Fas-ligand and tumor necrosis factor receptor families. Our results showed that tumor necrosis factor receptor 1 (TNFR1) was the best receptor interacting with CTX attributed to the interaction of all three functional loops and evinced with low HADDOCK, Z-score and RMSD value. The interaction between CTX and TNFR1 was also supported by a concentration-dependent reduction of fluorescence intensity with increasing binding affinity. The possible intermolecular interactions between CTX and TNFR1 were Van der Waals forces and hydrogen bonding. Our findings suggest a possibility that CTX triggers apoptosis cell death through non-covalent interactions with TNFR1.Communicated by Ramaswamy H. Sarma.

Characterization of Climbazole-Bovine serum albumin interaction by experimental and in silico approaches.

Interactive association of an antifungal drug, climbazole (CBZ) with the carrier protein in bovine circulation, bovine serum albumin (BSA) was explored by fluorescence and absorption spectroscopy along with in silico techniques. The fluorescence and absorption spectral alterations of the protein upon addition of CBZ affirmed the complex foration between CBZ and BSA. The inverse temperature dependence behaviour of the KSV values as well as the hyperchromic result of the protein's absorption signals characterized CBZ-triggered quenching of BSA fluorescence as the static quenching. A weak binding affinity (Ka = 3.12-1.90-× 103 M-1) was reported towards the CBZ-BSA association process. Interpretation of thermodynamic data (entropy change = +14.68 J mol-1 K-1 and enthalpy change = -15.07 kJ mol-1) and in silico analyses anticipated that hydrophobic forces, van der Waals forces and hydrogen bonds were the key intermolecular forces in the complex stabilization. Inclusion of CBZ to BSA produced microenvironmental perturbations around Tyr and Trp residues, and also significantly defended temperature-induced destabilization of BSA. The binding locus of CBZ was detected in the proximity of Sudlow's sites I (subdomain IIA) and II (subdomain IIIA) of BSA, exhibiting greater preference towards site II, as revealed by competitive site-marker displacement investigations and in silico analysis. The stability of the CBZ-BSA complex was further validated by the molecular dynamics simulation assessments.

Spectrofluorometric and computational approaches for the interaction studies of aclonifen and bifenox with human serum albumin.

Interaction of two broadly used herbicides, aclonifen (ACF) and bifenox (BIF) with the major transporter in human circulation, human serum albumin (HSA) were examined using fluorescence and absorption spectral measurements combined with in silico analyses. Assessment of the fluorescence and absorption spectral results affirmed the complexation between ACF/BIF and HSA. Increase in the KSV value with temperature characterized the ACF/BIF-induced quenching of the protein fluorescence as dynamic quenching. The moderate binding affinities (Kf = 1.74×104 - 1.95×106 M-1 for ACF-HSA complex; Kf = 2.00×103 - 1.02×106 M-1 for BIF-HSA complex) were pointed out between ACF/BIF and HSA, showing a relatively higher binding constant values with increasing temperatures. Quantitative evaluation of thermodynamic data (ΔS = +0.86 kJ mol-1 K-1 and ΔH = +225.43 kJ mol-1 for ACF-HSA complex; ΔS = +1.11 kJ mol-1 K-1 and ΔH = +304.63 kJ mol-1 for BIF-HSA complex) predicted the contribution of hydrophobic interactions in the ACF-HSA and BIF-HSA association processes, which were well supported by our molecular docking results. In silico analyses were made to acquire insight details into the ACF and BIF binding to HSA at the binding sites and suggested the locations of ACF and BIF binding sites as both subdomain IIA (site I) and subdomain IIIA (site II) of HSA, showing more preference toward site I.

Visual inspection reveals a novel pathogenic mutation in PKD1 missed by the variant caller in whole­exome sequencing.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common type of inherited cystic kidney disease. The feasibility of whole­exome sequencing (WES) to obtain molecular diagnosis of ADPKD is still in question as previous studies showed conflicting results. Utilizing WES on a patient with ADPKD, standard bioinformatics pipeline demonstrated no pathogenic variant in the genes of interest. By visualizing read alignments using the Integrative Genomics Viewer, a region with atypical alignment of numerous soft­clipped reads at exon 45 of polycystin 1, transient receptor potential channel interacting (PKD1) gene was demonstrated. A total of four visual inspection steps were outlined to assess the origin of these soft­clipped reads as strand bias during capture, poor mapping, sequencing error or DNA template contamination. Following assessment, the atypical alignment at PKD1 was hypothesized to be caused by an insertion/deletion mutation. Sanger sequencing confirmed the presence of a novel 20­bp insertion in PKD1 (NM_001009944.3; c.12143_12144insTCC​CCG​CAG​TCT​TCC​CCG​CA; p.Val4048LeufsTer157), which introduced a premature stop codon and was predicted to be pathogenic. The present study demonstrated that WES could be utilized as a molecular diagnostic tool for ADPKD. Furthermore, visual inspection of read alignments was key in identifying the pathogenic variant. The proposed visual inspection steps may be incorporated into a typical WES data analysis workflow to improve the diagnostic yield.

A Novel De Novo NFKBIA Missense Mutation Associated to Ectodermal Dysplasia with Dysgammaglobulinemia.

Background: Inborn errors of immunity (IEIs) are comprised of heterogeneous groups of genetic disorders affecting immune function. In this report, a 17-month-old Malay patient suspected of having Hyper IgM syndrome, a type of IEIs, was described. However, the diagnosis of Hyper IgM syndrome was excluded by the normal functional studies and the mild features of ectodermal dysplasia observed from a further clinical phenotype inspection. Methods: Whole-exome sequencing (WES) was performed to unravel the causative mutation in this patient. Results: The variant analysis demonstrated a novel missense mutation in NFKBIA (NM_020529:c.94A > T,NP_065390:p.Ser32Cys) and was predicted as damaging by in silico prediction tools. The NFKBIA gene encodes for IκBα, a member of nuclear factor kappa B (NF-κB) inhibitors, playing an important role in regulating NF-κB activity. The mutation occurred at the six degrons (Asp31-Ser36) in IκBα which were evolutionarily conserved across several species. Prediction analysis suggested that the substitution of Ser32Cys may cause a loss of the phosphorylation site at residue 32 and a gain of the sumoylation site at residue 38, resulting in the alteration of post-translational modifications of IκBα required for NF-κB activation. Conclusion: Our analysis hints that the post-translational modification in the NFKBIA Ser32Cys mutant would alter the signaling pathway of NF-κB. Our findings support the usefulness of WES in diagnosing IEIs and suggest the role of post-translational modification of IκBα.

Potential Acetylcholinesterase Inhibitor Acting on the Pesticide Resistant and Susceptible Cotton Pests.

Gossypium spp., produces economically important cotton fiber, and its yield is highly affected due to pest attacks. Insecticidal target site mutation is one of the reasons behind insecticide resistance to a wide range of pesticides. Acetylcholinesterase (AChE) protein sequences from major pests of cotton were analyzed to assess various physicochemical properties, presence of motifs, and understand evolutionary relationship. The impact of three mutant AChE1, A. lucorum A216S, B. tabaci F392W, and A. gossypii A302S, on the strucutral stability was assessed, and F392W_AChE1 was selected based on 100 ns molecular dynamics simulation. Virtual screening of the zinc database and high-throughput virtual screening, standard precision, and extra precision docking resulted in the identification of six compounds. The six identified compounds and six known commercial pesticdes were docked with three mutant and three wild type AChE1, and one (C1) was selected based on Tice criteria. The conformational and interaction stability of the AChE1-C1 and F392W_AChE1-C1 complexes were monitored at 100 ns Gromacs simulation and were found to be thermodynamically favorable. Therefore, C1 may have the potential to bind to the resistant and susceptible strains of cotton pest, and the resistance developed by insects could be arrested. Furthermore, synthesis and field study of C1 will lead us to a better understanding of the efficacy of the identified compound.

Carica papaya Leaf Juice for Dengue: A Scoping Review.

The potential therapeutic effect of Carica papaya leaf juice has attracted wide interest from the public and scientists in relieving dengue related manifestations. Currently, there is a lack of evaluated evidence on its juice form. Therefore, this scoping review aims to critically appraise the available scientific evidence related to the efficacy of C. papaya leaf juice in dengue. A systematic search was performed using predetermined keywords on two electronic databases (PubMed and Google Scholar). Searched results were identified, screened and appraised to establish the association between C. papaya and alleviating dengue associated conditions. A total of 28 articles (ethnobotanical information: three, in vitro studies: three, ex vivo studies: one, in vivo study: 13, clinical studies: 10) were included for descriptive analysis, which covered study characteristics, juice preparation/formulations, study outcomes, and toxicity findings. Other than larvicidal activity, this review also reveals two medicinal potentials of C. papaya leaf juice on dengue infection, namely anti-thrombocytopenic and immunomodulatory effects. C. papaya leaf juice has the potential to be a new drug candidate against dengue disease safely and effectively.

Ensemble-based high-throughput virtual screening of natural ligands using the Super Natural-II database against cell-wall protein dTDP-4-dehydrorhamnose reductase (RmlD) in Mycobacterium tuberculosis.

The disease Tuberculosis (TB) is caused by a bacterium called Mycobacterium tuberculosis (Mtb). The bacterial cell-wall consists of peptidoglycan layer maintains the cellular integrity and cell viability. The main problem resides in the cell cycle of Mycobacterium tuberculosis in its quiescent form which is not targeted by any drugs hence there is an immediate need for new antibiotics to target the cell wall. The current study deals with the dTDP-4-dehydrorahmnose reductase (RmlD) which is the final enzyme in the series of cell-wall proteins of Mtb. The RmlD is a part of Carbohydrate biosynthesis has been considered as a good drug target for the novel class of antibiotics. Our study begins with the protein structure prediction, Homology studies were conducted using the Phyre2 web server. The structure is then refined and subjected to molecular dynamics simulations for 50 ns using GROMACS. The clustering analysis has been carried out and generated 41 clusters with 2 Å as the cut-off. Blind docking virtual screening was performed against RmlD protein using the Super Natural-II database with AutoDock4.0. its results helped to screen top ligands based on best binding energies. In both dockings, there are some common residues in which the ligands are interacting and forming the Hydrogen bonds such as Asp-105, Val-158, Thr-160, Gly-161, Arg-224, Arg-256. The ligand-567 giving the best results by being in the top-3 of all the clusters in both blind docking as well as the active-site docking. Hence ligand-567 can be a potential inhibitor of RmlD which can further inhibit the cell-wall synthesis of Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.

Biomolecular interaction mechanism of an anticancer drug, pazopanib with human serum albumin: a multi-spectroscopic and computational approach.

Pazopanib (PZP) is a multi-targeting tyrosine kinase inhibitor and is currently approved by FDA for the treatment of soft tissue sarcoma and renal cancer. Molecular interaction mechanism of PZP with human serum albumin (HSA) was explored under simulated physiological conditions (pH = 7.4), using fluorescence and UV absorption spectroscopy along with computational methods. Based on the inverse correlation between the Stern-Volmer constant (Ksv) and temperature, it was concluded that PZP quenched the protein fluorescence through static quenching mechanism. This was also confirmed from the UV-vis absorption spectral results. Moderate binding affinity between PZP and HSA was evident from the Ka values (5.51 - 1.05 × 105 M-1) while PZP-HSA complex formation was driven by hydrophobic and van der Waals interactions as well as hydrogen bonds, as revealed by positive entropy change (ΔS = +98.37 J mol-1 K-1) and negative enthalpy change (ΔH = -60.31 kJ mol-1). Three-dimensional fluorescence spectral results disclosed microenvironmental perturbations around Trp and Tyr residues of the protein upon PZP binding. Interestingly, the addition of PZP to HSA significantly protected the protein against thermal stress. Competitive drug displacement results obtained with warfarin, phenylbutazone and diazepam elucidated Sudlow's Site I, positioned in subdomain IIA of HSA, as the preferred binding site of PZP which was well supported by molecular docking analysis, while molecular dynamics simulation results suggested the stability of the PZP-HSA complex.Communicated by Vsevolod Makeev.

Revealing Chronic Granulomatous Disease in a Patient With Williams-Beuren Syndrome Using Whole Exome Sequencing.

Blended phenotypes exhibited by a patient may present a challenge to the establishment of diagnosis. In this study, we report a seven-year-old Murut girl with unusual features of Williams-Beuren syndrome (WBS), including recurrent infections and skin abscesses. Considering the possibility of a second genetic disorder, a mutation screening for genes associated with inborn errors of immunity (IEI) was conducted using whole exome sequencing (WES). Analysis of copy number variations (CNVs) from the exome data revealed a 1.53Mb heterozygous deletion on chromosome 7q11.23, corresponding to the known WBS. We also identified a biallelic loss of NCF1, which indicated autosomal recessive chronic granulomatous disease (CGD). Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Coamplification of NCF1 and its pseudogenes identified a GT-deletion (ΔGT) at the start of exon 2 in NCF1 (NM_000265.7: c.75_76delGT: p.Tyr26Hisfs*26). Estimation of NCF1-to-NCF1 pseudogenes ratio using ΔGT and 20-bp gene scans affirmed nil copies of NCF1 in the patient. While the father had a normal ratio of 2:4, the mother had a ratio of 1:5, implicating the carrier of ΔGT-containing NCF1. Discovery of a 7q11.23 deletion involving one NCF1 allele and a ΔGT in the second NCF1 allele explained the coexistence of WBS and CGD in our patient. This study highlights the capability of WES to establish a molecular diagnosis for a case with blended phenotypes, enabling the provision of appropriate prophylactic treatment.

A single-center pilot study in Malaysia on the clinical utility of whole-exome sequencing for inborn errors of immunity.

Primary immunodeficiency diseases refer to inborn errors of immunity (IEI) that affect the normal development and function of the immune system. The phenotypical and genetic heterogeneity of IEI have made their diagnosis challenging. Hence, whole-exome sequencing (WES) was employed in this pilot study to identify the genetic etiology of 30 pediatric patients clinically diagnosed with IEI. The potential causative variants identified by WES were validated using Sanger sequencing. Genetic diagnosis was attained in 46.7% (14 of 30) of the patients and categorized into autoinflammatory disorders (n = 3), diseases of immune dysregulation (n = 3), defects in intrinsic and innate immunity (n = 3), predominantly antibody deficiencies (n = 2), combined immunodeficiencies with associated and syndromic features (n = 2) and immunodeficiencies affecting cellular and humoral immunity (n = 1). Of the 15 genetic variants identified, two were novel variants. Genetic findings differed from the provisional clinical diagnoses in seven cases (50.0%). This study showed that WES enhances the capacity to diagnose IEI, allowing more patients to receive appropriate therapy and disease management.

Atypical Presentation of Severe Fungal Necrotizing Fasciitis in a Patient with X-Linked Agammaglobulinemia.

X-linked agammaglobulinemia is a rare primary immunodeficiency due to a BTK mutation. The patients are characteristically deficient in peripheral B cells and serum immunoglobulins. While they are susceptible to infections caused by bacteria, enteroviruses, and parasites, fungal infections are uncommon in XLA patients. Here, we report a boy of Malay ethnicity who suffered from recurrent upper respiratory tract infections and severe progressive necrotizing fasciitis caused by Saksenaea erythrospora. Immunological tests showed a B cell deficiency and hypogammaglobulinemia. Whole-exome sequencing identified a dinucleotide deletion (c.1580_1581del) in BTK, confirmed by Sanger sequencing and predicted to be disease causing by in silico functional prediction tools (Varsome and MutationTaster2) but was absent in the gnomAD database. This mutation resulted in a frameshift and premature termination (p.C527fs), which disrupted the protein structure. The mother was heterozygous at the mutation site, confirming her carrier status. Flow cytometric analysis of monocyte BTK expression showed it to be absent in the patient and bimodal in the mother. This study describes a novel BTK mutation in a defined hotspot and an atypical fungal phenotype in XLA. Further studies are required to understand the pathogenesis of fungal infection in XLA.