An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.
CHRM3 codes for the M3 muscarinic acetylcholine receptor that is located on the surface of smooth muscle cells of the detrusor, the muscle that effects urinary voiding. Previously, we reported brothers in a family affected by a congenital prune belly-like syndrome with mydriasis due to homozygous CHRM3 frameshift variants. In this study, we describe two sisters with bladders that failed to empty completely and pupils that failed to constrict fully in response to light, who are homozygous for the missense CHRM3 variant c.352G > A; p.(Gly118Arg). Samples were not available for genotyping from their brother, who had a history of multiple urinary tract infections and underwent surgical bladder draining in the first year of life. He died at the age of 6 years. This is the first independent report of biallelic variants in CHRM3 in a family with a rare serious bladder disorder associated with mydriasis and provides important evidence of this association.
Between 2005 and 2013 six severe pneumonia cases (all requiring mechanical ventilation, two fatal outcomes) caused by human adenovirus type 21 (HAdV-B21) were observed in Germany. So far, HAdV-B21 was mainly associated with non-severe upper and lower respiratory tract infections. However, a few highly virulent HAdV types, e.g. HAdV-B14p1, were previously associated with severe, fatal pneumonia. Complete genomic sequences of the German HAdV-B21 pneumonia isolates formed a single phylogenetic cluster with very high sequence identity (≥ 99.897%). Compared to the HAdV-B21 prototype (only 99.319% identity), all isolates had a unique 15 amino acid deletion and a 2 amino acid insertion in the RGD loop of the penton base which may affect binding to the secondary receptor on the host cells. Moreover, a recombinant E4 gene region derived of HAdV-B3 was identified by bootscan analysis. Thus, the highly virulent, pneumotropic HAdV-B21 was denominated as subtype 21a. Surprisingly, there was 99.963% identity with agent Y/SIBU97 (only 13.4 kb available in GenBank of the 35.4 kb genome) which was associated with 10 fatalities due to cardiopulmonary failure in Sarawak, Malaysia, in 1997. In conclusion, a HAdV-B21 subtype (21a) associated with severe pneumonia in Germany was phylogenetically linked to an adenovirus isolated in Malaysia.
This paper presents a study on gene knockout strategies to identify candidate genes to be knocked out for improving the production of succinic acid in Escherichia coli. Succinic acid is widely used as a precursor for many chemicals, for example production of antibiotics, therapeutic proteins and food. However, the chemical syntheses of succinic acid using the traditional methods usually result in the production that is far below their theoretical maximums. In silico gene knockout strategies are commonly implemented to delete the gene in E. coli to overcome this problem. In this paper, a hybrid of Ant Colony Optimization (ACO) and Minimization of Metabolic Adjustment (MoMA) is proposed to identify gene knockout strategies to improve the production of succinic acid in E. coli. As a result, the hybrid algorithm generated a list of knockout genes, succinic acid production rate and growth rate for E. coli after gene knockout. The results of the hybrid algorithm were compared with the previous methods, OptKnock and MOMAKnock. It was found that the hybrid algorithm performed better than OptKnock and MOMAKnock in terms of the production rate. The information from the results produced from the hybrid algorithm can be used in wet laboratory experiments to increase the production of succinic acid in E. coli.
Microbial strain optimization focuses on improving technological properties of the strain of microorganisms. However, the complexities of the metabolic networks, which lead to data ambiguity, often cause genetic modification on the desirable phenotypes difficult to predict. Furthermore, vast number of reactions in cellular metabolism lead to the combinatorial problem in obtaining optimal gene deletion strategy. Consequently, the computation time increases exponentially with the increase in the size of the problem. Hence, we propose an extension of a hybrid of Bees Algorithm and Flux Balance Analysis (BAFBA) by integrating OptKnock into BAFBA to validate the result. This paper presents a number of computational experiments to test on the performance and capability of BAFBA. Escherichia coli, Bacillus subtilis and Clostridium thermocellum are the model organisms in this paper. Also included is the identification of potential reactions to improve the production of succinic acid, lactic acid and ethanol, plus the discussion on the changes in the flux distribution of the predicted mutants. BAFBA shows potential in suggesting the non-intuitive gene knockout strategies and a low variability among the several runs. The results show that BAFBA is suitable, reliable and applicable in predicting optimal gene knockout strategy.
About a fifth of malaria cases in 1999 for the Kapit division of Malaysian Borneo had routinely been identified by microscopy as Plasmodium malariae, although these infections appeared atypical and a nested PCR assay failed to identify P malariae DNA. We aimed to investigate whether such infections could be attributable to a variant form of P malariae or a newly emergent Plasmodium species.
The long non-coding RNAs (lncRNAs) are the most prevalent and functionally diverse member of the non-coding RNA (ncRNA). The lncRNA has previously been considered to be a form of transcriptional "noise" but recent studies have found that the lncRNA to be associated with various disease conditions. It has also been found to play important roles in various physiological processes such as haemopoiesis, where lncRNA is reported to act as a fine-tuner of this very important process. To date, the effects of dysregulated lncRNA in thalassaemia has not been fully explored. This review article focuses on the possible roles of dysregulated lncRNAs in the pathogenesis of thalassaemia.
Matched MeSH terms: Thalassemia/genetics*; RNA, Long Noncoding/genetics*
Urinary bladder tuberculosis (UB-TB) is one of the gravest public health issues of renal TB, and it is diagnosed with <50% of urogenital TB. Unsatisfactory and delayed diagnosis with imprudent medications for bladder TB frequently resulted in several urinary and complications, including contraction of the UB. The objectives of this research were to build awareness among medical professionals and subsequently minimize the sufferings of patients. This was a case report-based study regarding UB-TB. All routine tests for cystitis were conducted. In addition, 24-h urine sample for TB identification, including a polymerase chain reaction test, was performed. Twenty-four hours of urine sample revealed confirmatory findings of TB. The patient had responded well with the national TB guideline-designated medication. Recurrent cystitis had a higher possibility of tuberculous origin. Medical doctors must rethink when a patient visited multiple times for cystitis for the etiology of the disease.
Chemokines influence the migration of leukocytes to secondary lymphoid tissue and sites of inflammation. In HIV patients, they are implicated in inflammatory complications of antiretroviral therapy (ART), notably Immune Reconstitution Disease (IRD) and Sensory Neuropathy (SN). However most chemokines have not been monitored as patients begin ART or correlated with IRD and SN.
This study was conducted to determine the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in Malaysian hospitals. A total of 264 MRSA isolates from eight hospitals were subjected to typing by pulsed-field gel electrophoresis (PFGE) of SmaI restricted DNA. Antibiotic disk susceptibility testing was also carried out to determine their resistance patterns. Thirty-one PFGE pattern types were identified. Three major pattern types A, ZC and K were found with type A the predominant profile in c. 80% of strains and present in all hospitals. Unlike type A, other DNA pattern types were unique to the hospitals in which they were isolated. PFGE type A also consisted of strains that were multiply antibiotic resistant. The presence of a single predominant PFGE type in Malaysian hospitals is an important finding which suggests that inter-hospital spread of MRSA had occurred frequently and regularly.
The co-occurrence of JAK2 V617F mutation with BCR-ABL reciprocal translocation is uncommon. We report a 60-year-old man who initially presented with phenotype of polycythemia vera (PV), which evolved into chronic myeloid leukemia and back to PV once treatment with imatinib was commenced. JAK2 V617F mutation and BCR-ABL fusion transcripts were detected in the initial sample. However, JAK2 V617F alleles diminished when BCR-ABL mRNA burden increased and reappeared once the patient was commenced on imatinib. The dynamic interaction between JAK2 V617F and BCR-ABL implies that two independent clones exist with the JAK2 V617F clone only achieving clonal dominance when BCR-ABL positive clones are suppressed by imatinib.
Outbreaks of foodborne diseases have become a global health concern; hence, many improvements and developments have been made to reduce the risk of food contamination. We developed a centrifugal microfluidic automatic wireless endpoint detection system integrated with loop mediated isothermal amplification (LAMP) for monoplex pathogen detection. Six identical sets were designed on the microfluidic compact disc (CD) to perform 30 genetic analyses of three different species of foodborne pathogens. The consecutive loading, mixing, and aliquoting of the LAMP primers/reagents and DNA sample solutions were accomplished using an optimized square-wave microchannel, metering chambers and revulsion per minute (RPM) control. We tested 24 strains of pathogenic bacteria (Escherichia coli, Salmonella spp and Vibrio cholerae), with 8 strains of each bacterium, and performed DNA amplification on the microfluidic CD for 60min. Then, the amplicons of the LAMP reaction were detected using the calcein colorimetric method and further analysed via the developed electronic system interfaced with Bluetooth wireless technology to transmit the results to a smartphone. The system showed a limit of detection (LOD) of 3 × 10-5ngμL-1 DNA by analysing the colour change when tested with chicken meat spiked with the three pathogenic bacteria. Since the entire process was performed in a fully automated way and was easy to use, our microdevice is suitable for point-of-care (POC) testing with high simplicity, providing affordability and accessibility even to poor, resource-limited settings.
Bioremediation of heavy metals and dyes is one of the emerging techniques globally as it is evident from the numerous publications made by various research groups. Biofilm-assisted bioremediation is one of the trending approaches as it facilitates negatively charged extracellular polymeric substances which makes the bacteria resistant to the toxic chemicals. Genetic engineering of microbes will make them unique in the bioremediation process. This mini-review concentrates on source and toxic effects of heavy metals and dyes on aqueous and living beings. Further, the genetic improvement strategies for effective bioremediation are described. However, the gap between practicability and real-time applicability needs to test with real-time wastewater in the industrial scale.
Gene expression data are expected to be of significant help in the development of efficient cancer diagnoses and classification platforms. In order to select a small subset of informative genes from the data for cancer classification, recently, many researchers are analyzing gene expression data using various computational intelligence methods. However, due to the small number of samples compared to the huge number of genes (high dimension), irrelevant genes, and noisy genes, many of the computational methods face difficulties to select the small subset. Thus, we propose an improved (modified) binary particle swarm optimization to select the small subset of informative genes that is relevant for the cancer classification. In this proposed method, we introduce particles' speed for giving the rate at which a particle changes its position, and we propose a rule for updating particle's positions. By performing experiments on ten different gene expression datasets, we have found that the performance of the proposed method is superior to other previous related works, including the conventional version of binary particle swarm optimization (BPSO) in terms of classification accuracy and the number of selected genes. The proposed method also produces lower running times compared to BPSO.
In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.
Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.
Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.
The crystallins are a family of monomeric proteins present in the mammalian lens and mutations in these proteins cause various forms of cataracts. The aim of our current study is to emphasize the structural characterization of aggregation propensity of mutation R58H on γD crystallin using molecular dynamics (MD) approach. MD result revealed that difference in the sequence level display a wide variation in the backbone atomic position, and thus exhibits rigid conformational dynamics. Changes in the flexibility of residues favoured to increase the number of intra-molecular hydrogen bonds in mutant R58H. Moreover, notable changes in the hydrogen bonding interaction resulted to cause the misfolding of mutant R58H by introducing α-helix. Principal component analysis (PCA) result suggested that mutant R58H showed unusual conformational dynamics along the two principal components when compared to the wild-type (WT)-γD crystallin. In a nutshell, the increased surface hydrophobicity could be the cause of self-aggregation of mutant R58H leading to aculeiform cataract.