Progress In Microbes & Molecular Biology

Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography

Tue, 19 Mar 2024 00:00:00 +0800

The significance of immunoglobulin Y (IgY) as a particular antibody equal to mammalian IgG is well understood. However, due to a lack of reliable purification procedures, producing highly pure IgY remains problematic. In this study, we aimed to optimize the recovery of pure IgY anti-MPT64 using thiophilic adsorption chromatography. The purification of IgY anti-MPT64 was achieved by initial PEG lipid precipitation, then an optimized purification by varying the gradient concentration of elution buffer into five steps gradient (0-20, 20-40, 40-60, 60-80, and 80-100%v/v) for three injection column volume (CV) each and two steps gradient (0-50 and 50-100%v/v) in eight CV for each concentration. The obtained IgY was characterized by SDS-PAGE and dot-blot then determined its content levels using the Lowry method. The results showed that the five steps gradient purification was found to provide a better purity level of IgY than the two steps gradient. However, the IgY content obtained in the two steps gradient purification (2.2632± 0.011 mg/mL) was higher than the five steps gradient purification (1.35482 ± 0.023 mg/mL). Nevertheless, both purified IgY results can recognize MPT64 protein through a dot blot test. Therefore, it can be summarized that thiophilic adsorption chromatography with five steps gradient of purification was an efficient process to obtain a higher purity of IgY anti-MPT64, especially to be targeted as a diagnostic kit component for MPT64 detection.

Homology Modeling and Expression of Recombinant NS5-RdRp Based on the Indonesian Local Strain of Dengue Virus for Anti-Dengue Drug Development

Tue, 10 Sep 2024 00:00:00 +0800

This study aims to isolate the RNA-dependent RNA polymerase (RdRp) gene of the dengue virus (DENV) isolates from Indonesia, determine the serotype of the local DENV strain through homology modeling, and express the recombinant RdRp protein in Escherichia coli BL21 (DE3), as the target for anti-dengue drug development. We utilize reverse transcription- polymerase chain reaction (RT-PCR) and nested PCR for serotyping the DENV isolates, obtained from Hasan Sadikin Hospital, Indonesia. Then, followed by the extraction and purification of the RdRp amplicon for gene sequencing. The resulting RdRp gene sequences are translated into amino acid sequences, which are then used for protein modeling and the construction of a recombinant plasmid for RdRp protein expression. As the result, the RdRp gene, which belongs to the non-structural protein 5 (NS5) of DENV, was successfully isolated and sequenced from DENV RNA samples. Based on serotyping results and homology modeling, the serotype of the isolate DENV was identified as serotype-3. The isolated NS5-RdRp gene from the local strain was then inserted into the pET28a(+) plasmid and transformed into E. coli BL21(DE3) as the host cell. Recombinant NS5-RdRp was predominantly found in the insoluble fraction with a molecular weight of 72 kDa. Future studies on the development of an anti-dengue drug model that confirms the inhibition of the binding domain in the recombinant RdRp protein structure will be required to provide more insight and expand the development of a universal anti-dengue strategy in Indonesia.

Genomic and Resistome Analyses of Elizabethkingia anophelis Strain B2D isolated from Dental Plaque of Patient

Wed, 17 Apr 2024 00:00:00 +0800

In this study, strain B2D isolated from a dental plaque sample of a human patient was studied for its general characteristics, taxonomic identification, genome features, and resistome profile. The bacterium exhibited antibiotic resistance to all beta-lactam antibiotics, nitrofuran, and sulfonamides, with high minimum inhibitory concentrations. It was only sensitive to the fluoroquinolone ciprofloxacin and intermediately susceptible to aminoglycoside tobramycin. A preliminary identification through 16S rRNA gene sequences revealed that it shared the highest sequence identity with Elizabethkingia anophelis subsp. endophytica JM-87^T (100%) and Elizabethkingia anophelis subsp. anophelis R26^T (99.31%). The draft genome of strain B2D was approximately 3.9 Mbp with 50 contigs and 35.5% GC content. A 16S rRNA gene and core genes-based phylogenetic analyses revealed a close phylogenetic relationship between strain B2D and the other Elizabethkingia type strains. An above species level threshold average nucleotide identity value confirmed its taxonomic identity as Elizabethkingia anophelis. Furthermore, we conducted a resistome analysis of strain B2D and Elizabethkingia type strains, revealing the presence of widespread antibiotic resistance genes, including beta-lactamases and genes associated with cationic antiseptic resistance and glycopeptide resistance. Overall, the multidrug resistant profile of strain B2D as elucidated and confirmed through whole genome analysis indicated its potential as a reservoir of beta-lactamase genes. Moreover, its presence within dental plaque in the human oral cavity prompts speculation regarding its role as an opportunistic pathogen capable of causing infections, particularly in immunocompromised individuals.

Environmental Metagenomic Analysis of "ESKAPE" Pathogens in the Pediatric Intensive Care Unit of General Hospital Yogyakarta Indonesia

Ludhang Pradipta Rizki, Indah Kartika Murni, Abu Tholib Aman, Titik Nuryastuti — Wed, 07 Feb 2024 00:00:00 +0800

Healthcare-associated infections (HAIs) are infections that occur while receiving health care, develop in a hospital or other healthcare facility, and first appear 48 hours or more after hospital admission, or within 30 days after having received health care. HAIs are linked to high mortality rates, prolonged stays, increased hospital overhead costs, and financial burdens on patients. Bacterial transmission from medical personnel or the environment, or patient-to-patient contact are all potential causes of these infections. A molecular epidemiology approach is needed to examine the contribution of risk factors and the distribution of "ESKAPE" pathogens within the hospital environment. In this study, we conducted a comprehensive analysis of the distribution of ESKAPE bacterial pathogens in the environment of pediatric intensive care units over a 30-day time interval using shotgun metagenomics. We collected samples from handwashing sinks, the floor around patients, and ventilator screens and tubes in the pediatric intensive care unit (PICU) of General Hospital, Yogyakarta, Indonesia in March 2022. We determine taxonomic profiles and also detect resistome, and virulome distribution of ESKAPE pathogens on various environmental surfaces through shotgun metagenomic sequencing. The microbiomes of the floor, sink, and mechanical ventilator exhibit a diverse composition of microbial communities, featuring significant species richness based on Shannon and Simpson’s index. These microbiomes encompass a wide array of microbial species, including ESKAPE bacterial pathogens, as well as profiles related to resistome and virulome. ESKAPE pathogens, especially Acinetobacter baumannii, predominated in the PICU environment. Most virulome have been associated with metabolism/nutrition and adhesion. Noteworthy findings include resistome genes characterized by mechanisms like efflux pumps (MDR) and alterations in antibiotic targets.

Streptomyces sp. MUM 195J: A Promising Probiotic for Controlling Vibrio parahaemolyticus Infection in Aquaculture

Wed, 12 Jun 2024 00:00:00 +0800

Aquaculture is gaining prominence in meeting the increasing global food demand. However, persistent episodes of pathogenic infections have greatly affected production and incurred substantial financial losses to the industry. Regrettably, there is a lack of effective contemporary therapeutic measures to control infectious diseases in aquaculture. The repercussions of antimicrobial resistance have underscored the drawbacks of the contemporary practice of relying solely on antibiotics in disease control. The aquaculture industry needs a safer, environmentally viable, and economically efficient means for disease management. In this regard, this study aims to investigate the effectiveness of mangrove-derived Streptomyces sp. probiotics in controlling Vibrio parahaemolyticus infections. In vitro screenings were undertaken to evaluate the inhibitory activity of five Streptomyces sp. isolates. Subsequently, a series of in vivo trials was conducted, with the Malaysian giant freshwater prawn, Macrobrachium rosenbergii larvae as the animal model. Following that, molecular analyses were employed to examine the changes in gene expression. In essence, Streptomyces sp. MUM 195J emerges as a promising probiotic strain that demonstrates a strong inhibitory effect against V. parahaemolyticus. Its application as a feed additive elevates the survival rate of M. rosenbergii threefold, thus demonstrating efficacy at par with florfenicol antibiotic when challenged with the V. parahaemolyticus pathogen. Besides, Streptomyces sp. MUM 195J elevated the growth rate of M. rosenbergii by 17%. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed that probiotic supplementation elevated the immune function of the animal. Additionally, Streptomyces sp. MUM 195J demonstrates the potential to ameliorate the quality of the rearing water.

Decoding the Methylation Patterns of ABC Transporters in Colorectal Cancer

Fri, 18 Oct 2024 00:00:00 +0800

Colorectal cancer (CRC) is a significant global health concern, posing a major threat to morbidity and mortality rates. The molecular mechanisms that drive CRC, particularly the DNA methylation patterns in ATP-binding cassette (ABC) genes, have attracted considerable attention because of their potential roles in CRC drug resistance, initiation, and progression. This study aimed to investigate the methylation patterns of ABC genes in CRC using a high-throughput microarray technology. Microarray methylation data from CRC and adjacent normal tissues were subjected to preprocessing, differential methylation analysis, and correlation analysis using the MethSurv tool for a detailed study of methylation patterns. The study revealed global hypomethylation of 43 ABC transporters and two pseudogenes in CRC compared to normal tissues. Receiver operating characteristic curve analysis identified 369 CpG sites as potential biomarkers for CRC diagnosis, with area under the curve values ranging from acceptable to outstanding. Survival analysis demonstrated the correlation between DNA methylation of individual CpG sites and patient survival probability in colon and rectal adenocarcinoma datasets from The Cancer Genome Atlas. The study provides insights into the epigenetic landscape of ABC genes in CRC, highlighting their potential roles in drug resistance, disease initiation, and progression. The findings offer opportunities for developing innovative therapeutic approaches and improving patient outcomes in the fight against CRC. Further research is needed to validate these results and investigate the functional implications of ABC gene methylation in CRC pathogenesis and treatment response.

Impact of Cyclosporine A on Cognitive Functions and Neuronal Oxidative Stress, Apoptosis and Inflammatory Markers in Rats

Tue, 16 Jul 2024 00:00:00 +0800

Cyclosporine A (CYP-A), a potent immunosuppressive agent, significantly impacts organ transplantation and the treatment of autoimmune disorders, and its ability to control autoimmune reactions and prevent organ rejection has made it indispensable in contemporary medicine. However, its negative effects, such as nephrotoxicity, neurotoxicity, and hepatotoxicity, limit its usefulness. The study investigated the effects of CYP-A on cognitive and neurological functions in rats over a fifteen-day period. Four groups of six rats each were treated with normal saline (control) or varying doses of CYP-A (20, 50, and 100 mg/kg). Behavioural assessments included the elevated plus maze (EPM) and novel object recognition (NOR) tests used to evaluate memory capabilities and recognition memory respectively, and the Y-maze assessed the exploration of a new environment and navigational skills. Post-experiment, brain tissues were analyzed for apoptosis, neuroinflammation, and oxidative stress markers to assess potential neurotoxicity. Histopathological analysis was also performed to provide further insights into CYP-A's effects on brain tissue. Compared to the control rats, there were significant changes in the memory parameters with two higher doses (50 and 100 mg/kg). The findings indicated that MDA, TNF-α, NF-κB, PGE2, and Caspase-3 levels increased significantly in the CYP-A 100 treatment compared to the control animals. However, a significant drop was observed in CAT, and GSH when comparing the CYP-A 100 group to the control rats. Conversely, comparable alterations were also noted in MDA, TNF-α, PGE2, Bcl-2, Bax, and Caspase-3 levels between the CYP-A 100 and other treatment groups (20 or 50 mg/kg). Moreover, the histopathological analysis revealed that as compared to the control rats, CYP-A at three different dose levels (20, 50, or 100 mg/kg, p.o.) exhibited various changes. It was noticed that as the dose levels of CYP-A increased, there was a corresponding increase in brain tissue architectural changes. Precisely, the group treated with 100 mg/kg exhibited severe haemorrhages, inflammation, necrosis, and congestion compared to the control animals. In conclusion, CYP-A induces neurotoxicity, leading to oxidative stress, neuroinflammation, apoptosis, and cognitive deficits. Understanding the underlying mechanisms of this neurotoxicity is crucial to mitigating the adverse effects of CYP-A and improving patient outcomes. Further research is needed to develop targeted therapies and enhance the safe use of CYP-A.

Lutein Suppresses the Maturation and Function of Bone Marrow-Derived Dendritic Cells

Seulah Choi, Tae Sung Kim, Hui Xuan Lim — Thu, 18 Apr 2024 00:00:00 +0800

Lutein, nonivamide, and baicalein, organic compounds found in a variety of plants, are known to exert anti-inflammatory effects in animal cells. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) and link the innate and adaptive immune systems. DCs can be directed into fully mature APCs by exposure to bacterial or viral components, resulting in inflammatory situations. The manipulation of DC maturation provides a strategy for the treatment of allergic and inflammatory diseases. In this study, we evaluated the effects of lutein, nonivamide, and baicalein on the maturation and activation of DCs. Compared to nonivamide and baicalein, lutein significantly and dose-dependently reduced the levels of maturation-associated cell surface markers, including CD40, co-stimulatory molecule CD86, and major histocompatibility complex class II (I-A^b) molecule in lipopolysaccharide (LPS)-stimulated DCs. Lutein also decreased IL-12p40 and IL-6 gene expression and secretion in LPS-stimulated DCs. Furthermore, lutein significantly enhanced the endocytic ability of LPS-stimulated DCs. These results demonstrated that lutein may exhibit immunosuppressive activity by inhibiting the phenotypic and functional maturation of DCs, and provide new evidence for the value of lutein in the search for novel therapeutic agents in the treatment of inflammatory diseases.

A Review on Colorectal Cancer and the Role of Traditional Chinese Herbal Medicine as Complementary Therapy

Tue, 05 Nov 2024 00:00:00 +0800

Colorectal cancer (CRC), encompassing cancers of the colon and rectum area, is the third most frequently diagnosed cancer and the second leading cause of cancer-related deaths globally, posing a significant health challenge. In 2020, CRC contributed to 9.4% of all cancer fatalities. With the significant rise in cases among the elderly, it is projected that the figure will double by 2035 globally, particularly in less developed countries. For many years, surgery and chemotherapy have been the mainstay treatments for cancer, but patients with metastatic disease have generally faced poor outcomes. Currently, there is a renewal of interest to explore the potential of natural products to treat cancers. Nature provides many effective treatments for severe diseases, with around 75-80% of the global population relying on conventional medical practices due to limited access to healthcare and concerns about synthetic medicine safety. Natural products are crucial for treating infectious diseases, cancer, and neurological disorders, and references in religious texts have spurred scientific validation of traditional claims. These products are excellent sources for CRC treatments, with nearly 50% of current cancer therapies derived from natural ingredients. This review focuses on the discussion of colorectal cancer and the potential of Chinese traditional herbs used in treating colorectal cancers. Furthermore, various signalling pathways of CRC and the possible mechanism of TCM intervening in these pathways are also discussed.

Epigenetic Drug Interventions in Breast Cancer: A Narrative Review of Current Research and Future Directions

Mon, 02 Sep 2024 00:00:00 +0800

Breast cancer is a life-threatening disease known for its extensive molecular heterogeneity. The study of the breast cancer epigenome has revealed potential avenues for improving breast cancer treatment risk stratification, early detection, and treatment. With renewed interest in epigenetic-modifying pharmaceutical agents, namely DNA methyltransferase inhibitors (DNMTi), histone deacetylase inhibitors (HDACi), bromodomain and extra-terminal inhibitors (BETi), and enhancer of zeste homolog 2 inhibitors (EZH2i), there have been extensive preclinical and clinical studies to evaluate the safety and efficacy of these agents as potential treatments for breast cancer. In this review, we summarise and present the preclinical and clinical evidence for epigenetic drugs in treating breast cancer. We review the challenges associated with the translation of these findings into improved patient outcomes, namely the optimisation of dosage and treatment regimens, and the emergence of resistance. These challenges have been widely recognised in the field and are of utmost importance for the successful implementation of personalised medicine. While there is strong evidence that epigenetic alterations, consisting of changes in DNA methylation, histone modifications, and non-coding RNAs, play a crucial role in breast cancer initiation and development, additional research is warranted to elucidate the safety profile of long-term interventions involving epigenetic drugs and to validate the role of epigenetic markers in disease diagnosis, prognosis, and personalised treatment.

The Impact of Antidepressants on Gut Microbiome and Depression Management

Mon, 22 Jul 2024 00:00:00 +0800

Depression is a widespread psychiatric disorder that significantly impacts an individual’s quality of life. It affects mental and emotional well-being and has far-reaching consequences on their physical health, relationships, and overall ability to function in daily life. Recent advances in psychiatric research have revealed a connection between the gut-brain axis, a bidirectional communication system linking the brain's emotional and cognitive centers with intestinal functions. Antidepressants, including selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), and monoamine oxidase inhibitors (MAOIs), are commonly prescribed to alleviate symptoms of depression. However, their influence extends beyond neurotransmitter modulation in the brain to significant effects on gastrointestinal physiology. Antidepressants can alter gut motility, secretion, and microbiota composition, which in turn can influence mood and mental health. This review aims to provide insights into the impact of antidepressants on gut health and their implications for depression treatment. Additionally, it explores the potential of probiotics as an adjunct treatment to enhance the efficacy of antidepressants and mitigate gastrointestinal side effects. Restoring healthy gut microbiota can improve gastrointestinal and mental health outcomes, suggesting a promising avenue for integrated therapeutic approaches.

Tackling Microbial Resistance and Emerging Pathogens with Next-Generation Antibiotics

Thu, 22 Aug 2024 00:00:00 +0800

In the 19^th century, the discovery of penicillin revolutionized medicine, saving millions from infectious diseases. People believed that they had won the war against infections. However, the misuse and abuse of antimicrobial agents are accompanied by major ramifications like antimicrobial resistance, creating drug-resistant superbugs. This issue is concerning worldwide, as dwindling effective antibiotics lead to rising healthcare costs, re-hospitalization, and disease severity. Consequently, multiple initiatives have been undertaken to address these phenomena, including the development of antimicrobials with novel modes of action. Without novel discoveries of newer antimicrobial agents, we may face the risk of entering a post-antibiotic era where uncomplicated infections become untreatable. Ultimately, the morbidity and mortality rate would rise higher than in the pre-antibiotic era. This study highlights the recent developments in antimicrobials over the past five years and explores the strategies employed by the new generation of drugs to act against resistance. For example, we discuss the treatment of Carbapenem-resistant Enterobacteriaceae, such as Klebsiella pneumoniae Carbapenamase-producing Gram-negative bacteria, by using meropenem-vaborbactam. Plazomicin, lacking a hydroxyl group, effectively combats metallo-beta-lactamase, which meropenem-vaborbactam is unable to address. It is also preferred over tobramycin and gentamicin due to its hydroxyethyl group. Furthermore, we explore the conjugation of nanoparticles with antibiotics, which demonstrated synergistic effects and positive outcomes on different bacterial resistance. Mechanisms include increased drug adhesion to bacterial cell walls, generating oxidative stress, and causing mistranslation by detaching ribosomes from tRNA. Additionally, the IspH inhibitors like 4’-flurouridine targeting the MEP pathway which is also included in the discussion. This report thoroughly examines newer generations and classes of antibiotics, highlighting the improvements made by scientists to combat bacterial resistance effectively.

Role of SarA in Staphylococcus aureus: A Virulence Target For Therapeutic Strategies

Fri, 14 Jun 2024 00:00:00 +0800

Methicillin-resistant Staphylococcus aureus (MRSA) infection gives rise to significant morbidity and carries a grave prognosis, resulting in the demise of approximately 21.8% of afflicted individuals on a yearly basis Staphylococcus aureus has the capability to induce a myriad of diverse diseases, a phenomenon attributed to its extensive array of virulence factors and formation of biofilms. The regulation of key virulence determinants, crucial for pathogenicity, is intricately controlled by the staphylococcal accessory regulatory (sarA) system. SarA plays a crucial role in the pathogenic mechanisms of S. aureus and the development of biofilms, while simultaneously modulating the synthesis of multiple virulence factors and influencing the expression of specific colonization determinants, and mutations in sarA partially limit the extent of S. aureus biofilms formation. In this review, we present an overview of the current understanding of the molecular mechanisms underlying the regulation of sarA gene expression, with a particular emphasis on its relevance in the development and sustenance of antimicrobial resistance, along with in the processes of biofilm formation and activation of virulence genes in MRSA. This review demonstrated that suppressing the expression of sarA gene exerts a notable impact on both biofilm development and the pathogenicity of MRSA strains, thereby offering a hopeful approach to the efficient management and treatment of MRSA infections.

Gut Microbiota in Autism Spectrum Disorder: A Systematic Review

Wed, 15 May 2024 00:00:00 +0800

Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition with stereotyped behavior and deficits in communication and social interaction. There is increasing evidence of the implication of gut microbiota in ASD. We conducted a systematic review to summarize previously published data to compare the profile of gut microbiota between autistic and neurotypical subjects. The outcomes of interventions such as prebiotics, probiotics, and microbiota transplantation therapy to overcome the symptomatology of ASD were also discussed. The current review allows us to associate gut microbiota dysbiosis and ASD. To date, there is still little consensus on which bacterial species are consistently altered in individuals with autism. Further studies are required to obtain stronger evidence of the relationship between gut microbiota and the severity of ASD conjointly with the effectiveness of dietary/probiotic interventions in reducing autistic behaviors compared to their healthy siblings.

Complete Whole-Genome Sequence of Streptomyces sp. MUM 178J, a Potential Anti-Vibrio Agent

Fri, 16 Feb 2024 00:00:00 +0800

Streptomyces sp. is a group of filamentous, Gram-positive bacteria notoriously known for their capabilities in producing bioactive compounds that have been used as novel drugs and lead in drug development. The Streptomyces sp. MUM 178J was isolated from a mangrove forest in Malaysia. This isolate was found to harbor anti-Vibrio properties as its crude extract inhibited the growth of multidrug-resistant Vibrio parahaemolyticus. Therefore, the strain was subjected to whole genome sequencing to unearth its genomic potential. The genome of Streptomyces sp. MUM 178J consists of 6,699,249 bp with a G+C content of 71.3%. 66 tRNA genes and 18 rRNA genes were also predicted to be present within the genome. Further analysis with the bioinformatics tool, antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), detected nine biosynthetic gene clusters displaying more than 70% similarity to known gene clusters, including one associated with melanin production. Melanin has demonstrated antagonistic activity against the growth of members of the Vibrio family, including V. parahaemolyticus. This indicates the potential correlation between the production of melanin and the anti-Vibrio properties of MUM 178J. The availability of the whole genome sequence of Streptomyces sp. MUM 178J allows for future in-depth investigation and potential exploitation of MUM 178J to harvest useful bioactive compounds.