Investigating D-Amino Acid Oxidase Expression and Interaction Network Analyses in Pathways Associated With Cellular Stress: Implications in the Biology of Aging.

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids by oxidative deamination, producing hydrogen peroxide (H2O2) as a by-product. The generation of intracellular H2O2 may alter the redox-homeostasis mechanism of cells and increase the oxidative stress levels in tissues, associated with the pathogenesis of age-related diseases and organ decline. This study investigates the effect of DAO knockdown using clustered regularly interspaced short palindromic repeats (CRISPR) through an in silico approach on its protein-protein interactions (PPIs) and their potential roles in the process of aging. The target sequence and guide RNA of DAO were designed using the CCTop database, PPI analysis using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, Reactome biological pathway, protein docking using GalaxyTongDock database, and structure analysis. The translated target sequence of DAO lies between amino acids 43 to 50. The 10 proteins that were predicted to interact with DAO are involved in peroxisome pathways such as acyl-coenzyme A oxidase 1 (ACOX1), alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT), catalase (CAT), carnitine O-acetyltransferase (CRAT), glyceronephosphate O-acyltransferase (GNPAT), hydroxyacid oxidase 1 (HAO1), hydroxyacid oxidase 2 (HAO2), trans-L-3-hydroxyproline dehydratase (L3HYPDH), polyamine oxidase (PAOX), and pipecolic acid and sarcosine oxidase (PIPOX). In summary, DAO mutation would most likely reduce activity with its interacting proteins that generate H2O2. However, DAO mutation may result in peroxisomal disorders, and thus, alternative techniques should be considered for an in vivo approach.

Advancing Precision Medicine with Gene and Cell Therapy in Malaysia: Ethical, Legal, and Social Implications.

A new era of gene and cell therapy for treating human diseases has been envisioned for several decades. However, given that the technology can alter any DNA/cell in human beings, it poses specific ethical, legal, and social difficulties in its application. In Malaysia, current bioethics and medical ethics guidelines tackle clinical trials and biomedical research, medical genetic services, and stem cell research/therapy. However, no comprehensive framework and policy is available to cater to ethical gene and cell therapy in the country. Incorporating ethical, legal, and social implications (ELSI) would be crucial to guide the appropriate use of human gene and cell therapy in conjunction with precision medicine. Policy experts, scientists, bioethicists, and public members must debate the associated ELSI and the professional code of conduct while preserving human rights.

HLA-B*57:01 typing in a Malaysian cohort: implications of abacavir hypersensitivity in people living with HIV.

Background: Abacavir (ABC) in combination with other antiretroviral drugs, is used to treat people living with HIV (PLWH). However, it is linked to a fatal hypersensitivity reaction in susceptible individuals, and is strongly associated with the HLA-B*57:01 allele. Materials & methods: A total of 152 patients, 50 PLWH and 102 HIV-1 negative patients, were assessed for the HLA-B*57:01 allele through a sequence-specific primer PCR. Results: All PLWH tested negative for the HLA-B*57:01 allele, but two HIV-negative patients were found to have HLA-B*57, with one of them expressing the HLA-B*57:01 allele. Conclusion: Given the low prevalence of this risk allele in the population, testing for the presence of HLA-B*57:01 in PLWH may not provide significant benefit for the reported population.

Playing God? Religious Perspectives on Manipulating the Genome.

The Human Genome Project (HGP) is a remarkable medical science breakthrough that enables the understanding of genetics and the intervention of human health. An individual's health is influenced by physical, emotional, social, intellectual, and religious factors. Among these, religious beliefs shape our thinking on cloning, stem cells, and gene editing, affecting healthcare decisions and the motivation for seeking treatment. Is the human genome sacred? Does editing it violate the idea that we're made in God's image or allow us to "play God"? Understanding the perspectives behind the fundamental religious doctrines of Islam, Christian, Hindu, and Buddhist on gene editing/therapy in somatic and germline cells would ensure a right balance between geneticists and theologians in providing the best healthcare while catering to individual beliefs.

A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system.

Gene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 µg/mL and 7 µg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Is Malaysia Ready for Human Gene Editing: A Regulatory, Biosafety and Biosecurity Perspective.

Gene editing platforms have revolutionized the field of genetics with a direct impact on the public health system. Although there are apparent benefits, it is often accompanied by public debates over its uncertainties and risks. In the Malaysian context, modern biotechnology has raised questions about how to best govern gene editing in regulations, biosafety, and biosecurity. Even though standards and guidelines on stem cell and cell-based therapies have been developed, there are no appropriate legal frameworks available for gene editing yet. Nevertheless, biosafety regulations were established to balance promoting biotechnology and protecting against their potential environmental and human health risks. There is also a need to address the potential of genetically modified organisms (GMOs) as bioweapons. Numerous frameworks from several international organizations may provide valuable input in formulating documents on gene editing. By establishing comprehensive guidelines, legal policies, and standards to tackle the challenges and risks associated with gene editing, Malaysia can successfully apply this modern technology in this country.

Lessons Learned From Failures and Success Stories of HIV Breakthroughs: Are We Getting Closer to an HIV Cure?

There is a continuous search for an HIV cure as the success of ART in blocking HIV replication and the role of CD4+ T cells in HIV pathogenesis and immunity do not entirely eradicate HIV. The Berlin patient, who is virus-free, serves as the best model for a 'sterilizing cure' and many experts are trying to mimic this approach in other patients. Although failures were reported among Boston and Essen patients, the setbacks have provided valuable lessons to strengthen cure strategies. Following the Berlin patient, two more patients known as London and Düsseldorf patients might be the second and third person to be cured of HIV. In all the cases, the patients underwent chemotherapy regimen due to malignancy and hematopoietic stem cell transplantation (HSCT) which required matching donors for CCR5Δ32 mutation - an approach that may not always be feasible. The emergence of newer technologies, such as long-acting slow-effective release ART (LASER ART) and CRISPR/Cas9 could potentially overcome the barriers due to HIV latency and persistency and eliminate the need for CCR5Δ32 mutation donor. Appreciating the failure and success stories learned from these HIV breakthroughs would provide some insight for future HIV eradication and cure strategies.

Iron and Virulence in Stenotrophomonas Maltophilia: All We Know So Far.

Stenotrophomonas maltophilia is a multi-drug-resistant global opportunistic nosocomial pathogen, which possesses a huge number of virulence factors and antibiotics resistance characteristics. Iron has a crucial contribution toward growth and development, cell growth and proliferation, and pathogenicity. The bacterium found to acquire iron for its cellular process through the expression of two iron acquisition systems. Two distinct pathways for iron acquisition are encoded by the S. maltophilia genome-a siderophore-and heme-mediated iron uptake system. The entAFDBEC operon directs the production of the enterobactin siderophore of catecholate in nature, while heme uptake relies on hgbBC and potentially hmuRSTUV operon. Fur and sigma factors are regulators of S. maltophilia under iron-limited condition. Iron potentially act as a signal which plays an important role in biofilm formation, extracellular polymeric substances (EPS), extracellular enzymes production, oxidative stress response, diffusible signal factor (DSF) and siderophore production in S. maltophilia. This review summarizes the current knowledge of iron acquisition in S. maltophilia and the critical role of iron in relation to its pathogenicity.

Putative Iron Acquisition Systems in Stenotrophomonas maltophilia.

Iron has been shown to regulate biofilm formation, oxidative stress response and several pathogenic mechanisms in Stenotrophomonas maltophilia. Thus, the present study is aimed at identifying various iron acquisition systems and iron sources utilized during iron starvation in S. maltophilia. The annotations of the complete genome of strains K279a, R551-3, D457 and JV3 through Rapid Annotations using Subsystems Technology (RAST) revealed two putative subsystems to be involved in iron acquisition: the iron siderophore sensor and receptor system and the heme, hemin uptake and utilization systems/hemin transport system. Screening for these acquisition systems in S. maltophilia showed the presence of all tested functional genes in clinical isolates, but only a few in environmental isolates. NanoString nCounter Elements technology, applied to determine the expression pattern of the genes under iron-depleted condition, showed significant expression for FeSR (6.15-fold), HmuT (12.21-fold), Hup (5.46-fold), ETFb (2.28-fold), TonB (2.03-fold) and Fur (3.30-fold). The isolates, when further screened for the production and chemical nature of siderophores using CAS agar diffusion (CASAD) and Arnows's colorimetric assay, revealed S. maltophilia to produce catechol-type siderophore. Siderophore production was also tested through liquid CAS assay and was found to be greater in the clinical isolate (30.8%) compared to environmental isolates (4%). Both clinical and environmental isolates utilized hemoglobin, hemin, transferrin and lactoferrin as iron sources. All data put together indicates that S. maltophilia utilizes siderophore-mediated and heme-mediated systems for iron acquisition during iron starvation. These data need to be further confirmed through several knockout studies.

The 'Checkmate' for Iron Between Human Host and Invading Bacteria: Chess Game Analogy.

Iron is an essential nutrient for all living organisms with critical roles in many biological processes. The mammalian host maintains the iron requirements by dietary intake, while the invading pathogenic bacteria compete with the host to obtain those absorbed irons. In order to limit the iron uptake by the bacteria, the human host employs numerous iron binding proteins and withholding defense mechanisms that capture iron from the microbial invaders. To counteract, the bacteria cope with the iron limitation imposed by the host by expressing various iron acquisition systems, allowing them to achieve effective iron homeostasis. The armamentarium used by the human host and invading bacteria, leads to the dilemma of who wins the ultimate war for iron.

Examining the effect of ions and proteins on the heat dissipation of iron oxide nanocrystals.

In this paper, the effect and contribution of physiological components like ions and proteins under an applied alternating magnetic field (AMF) towards heat dissipation of superparamagnetic iron oxide nanoparticles (SPIONs) are discussed. Our results have shown that under an applied AMF, magnetic hyperthermia efficiency could be significantly enhanced if SPIONs were suspended in 1× phosphate buffered saline (PBS) compared to a suspension in de-ionized (DI) water. However, no heat enhancement was found when SPIONs were suspended in blood which is an amalgamation of physiological ions and proteins. Closer investigations have revealed that the presence of physiological ions can contribute positively to heating efficiency, and the heating efficiency increases with concentration of ions, ionic mass and solubility. However, the heating efficiency of ions can be suppressed to an insignificant level (comparable with measurement error), in the presence of physiological proteins in 1×PBS. Our electrochemical studies also showed that ionic mobility can be reduced significantly if proteins were present in the solution, thus retarding the heating efficiency.

Congenital segmental dilatation of the duodenum--differential diagnosis for an antenatally-diagnosed intra-abdominal cyst.

We report the third case of segmental dilatation of the duodenum and the first to be diagnosed antenatally. The lesion presented as an intra-abdominal cyst on an antenatal scan at 17 weeks and was followed up with more specific investigations after birth. The final diagnosis was obtained only at laparotomy where the dilated segment was resected. The patient is well at eighteen month follow-up. Segmental dilatation of the gastrointestinal tract, although rare, should be considered in the differential diagnosis of antenatally discovered intra-abdominal cysts.

Rare pancreatic tumours in children (other than nesidioblastosis).

BACKGROUND: The rarity of pancreatic tumours other than nesidioblastosis in children is such that the experience of any one surgeon or institution is small. As a consequence, there is limited information on the appropriate management and outcome of these tumours. For this reason a review was conducted of the experience of a large paediatric surgical institution. METHODS: During a 23-year period, six patients with pancreatic tumours other than nesidioblastosis were treated at the Royal Children's Hospital, Melbourne. RESULTS: Despite the varied modes of presentation and surgery undertaken, all six patients with surgery alone, with follow-up from 1 to 18 years. Two of the tumours were malignant; the remaining four being islet cell adenomata. No form of adjuvant treatment (chemotherapy or radiotherapy) was used. CONCLUSION: These rare paediatric tumours appear to have a good prognosis, even when malignant, and respond well to radical surgical ablation. Adjuvant therapy appears to be unnecessary.

The Dandy-Walker syndrome--a 10-year experience of its management and outcome.

Hydroephalus of childhood may be associated with intracranial haemorrhage, infection, aqueductal stenosis, Arnold-Chiari malformation (spina bifida), tumours or more uncommonly with the Dandy-Walker syndrome (DWS). We present our experience with the management of this condition over a 10-year period. Twelve children with a definite diagnosis of DWS are reviewed with regard to the age at diagnosis, investigations, treatment, associated problems and overall outcome. Two patients died; there was a high incidence of mental retardation (65%) and associated problems. All but one child had ventriculo-peritoneal shunting and two had cysto-peritoneal shunts in addition to their V-P shunts. There were no familial cases; one child had a chromosomal abnormality. Although the long-term outcome is dismal in most cases, about 35% of the children have a reasonable outcome and hence the condition warrants energetic treatment.