Diagnosis of abdominal tuberculosis by multi-targeted (mpt64 and IS6110) loop-mediated isothermal amplification assay.

BACKGROUND AND AIM: Diagnosis of abdominal TB is an exigent task due to variable anatomical sites and non-specific clinical manifestations that closely resemble other diseases. Most of the available diagnostic modalities yield low sensitivities and need expertise to handle the specialized equipment. Hence, there is an urgent need to develop a rapid and reliable diagnostic test, so as to reduce the unnecessary morbidity. Therefore, we designed a multi-targeted loop-mediated isothermal amplification (MT-LAMP) for diagnosing abdominal TB. METHODS: We evaluated an MT-LAMP (using mpt64 and IS6110) to diagnose abdominal TB within ascitic fluids and intestinal/peritoneal biopsies and compared these results with multiplex-PCR (M-PCR) using the same targets. MT-LAMP products were analyzed by gel electrophoresis and visual detection methods, that is, hydroxy naphthol blue and SYBR Green I reaction. RESULTS: Sensitivities of 80.9% and 84.6% were obtained in suspected (n = 42) and total abdominal TB (n = 52) cases, respectively by gel-based MT-LAMP, with 97.3% (n = 37) specificity in non-TB controls. Notably, sensitivities attained by gel-based/SYBR Green I MT-LAMP in both clinically suspected and total abdominal TB cases were significantly higher (P < 0.05) than M-PCR. Furthermore, sensitivity obtained with SYBR Green I was equivalent to that of gel-based MT-LAMP, while somewhat lesser specificity (94.6%) was attained with SYBR Green I, compared with gel-based MT-LAMP. CONCLUSION: Both gel-based and SYBR Green MT-LAMP exhibited equivalent sensitivities to diagnose abdominal TB. Because SYBR Green LAMP is easier to perform than a gel-based assay, we are currently focused on improving the specificity of this assay so as to develop a diagnostic kit.

Kinetic study and optimization of recombinant human tumor necrosis factor-alpha (rhTNF-α) production in Escherichia coli.

Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays a major role in immune regulation, homeostatic function, and cellular organization. The present study was undertaken to overproduce recombinant human TNF-α (rhTNF-α) in Escherichia coli (E.coli) in high cell density culture. The use of a codon-optimized gene and strong promoter-based (T7) expression system, choice of Terrific Broth (TB) as medium, and subsequent optimization of culture conditions in shake flasks resulted in production of 0.95 g/L insoluble rhTNF-α comprising upto 50% of total cellular protein (TCP) The protein yield further increased upto 1.26 g/L in 1 L TB medium batch culture in bioreactor with the controlled temperature, pH, and dissolved oxygen. In a series of chemostats operated at dilution rates of 0.2 h-1, 0.3 h-1, 0.4 h-1 and 0.5 h-1 the specific growth rate (µ) positively correlated with specific yield (Yp/x) and a maximum yield of 164 mg/g DCW was obtained at µ = 0.4 h-1 within 4 h post-induction. A fed-batch cultivation in TB with an exponential feeding profile (µ = ∼0.4 h-1) of concentrated feed resulted in an accumulation of 5.5 g/L of rhTNF-α within 14 h of cultivation which accounted for ∼29% of TCP.

Natural and grafted cyclotides in cancer therapy: An insight.

Cyclotides is a rapidly growing class of plant-derived cyclic peptides exhibiting several bioactivities with potential applications in the agricultural and pharmaceutical sectors. Both natural and grafted cyclotides have shown promise in cancer therapy. Approximately 70 natural cyclotides belonging to three plant families (Fabaceae, Rubiaceae, and Violaceae) have shown cytotoxicity against several cancer cell lines. Cyclotides exhibit considerable stability against thermal and enzymatic proteolysis, owing to their unique structure with knotted topology and head to tail cyclization. Further, their small size, high stability, oral bioavailability, and tolerance to amino acid substitution in structural loops make them an ideal platform for designing peptide-based drugs for cancer. Thus, cyclotides provide ideal scaffolds for bioactive epitope grafting and facilitating drug delivery in cancer treatment. Many anticancer linear peptides have been grafted in cysteine knotted cyclic framework of cyclotide for enhancing their cell permeability across cellular membranes, thereby improving their delivery and pharmacokinetics. The present review comprehensively discusses the distribution, toxicity, and anticancer bioactivity of natural cyclotides. Further, it systematically elaborates on the role and action of epitopes' into grafted cyclotides in targeting cancer. The review also encompasses related patents landscape study and future challenges in peptide-based cancer therapy.

Microbial enzymes for deprivation of amino acid metabolism in malignant cells: biological strategy for cancer treatment.

Amino acid deprivation therapy (AADT) is emerging as a promising strategy for the development of novel therapeutics against cancer. This biological therapy relies upon the differences in the metabolism of cancer and normal cells. The rapid growth of tumors results in decreased expression of certain enzymes leading to auxotrophy for some specific amino acids. These auxotrophic tumors are targeted by amino acid-depleting enzymes. The depletion of amino acid selectively inhibits tumor growth as the normal cells can synthesize amino acids by their usual machinery. The enzymes used in AADT are mostly obtained from microbes for their easy availability. Microbial L-asparaginase is already approved by FDA for the treatment of acute lymphoblastic leukemia. Arginine deiminase and methionase are under clinical trials and the therapeutic potential of lysine oxidase, glutaminase and phenylalanine ammonia lyase is also being explored. The present review provides an overview of microbial amino acid depriving enzymes. Various attributes of these enzymes like structure, mode of action, production, formulations, and targeted cancers are discussed. The challenges faced and the combat strategies to establish AADT in standard cancer armamentarium are also reviewed.Key Points • Amino acid deprivation therapy is a potential therapy for auxotrophic tumors. • Microbial enzymes are used due to their ease of manipulation and high productivity. • Enzyme properties are improved by PEGylation, encapsulation, and genetic engineering. • AADT can be employed as combinational therapy for better containment of cancer.

Genotypic diversity in multi-drug-resistant E. coli isolated from animal feces and Yamuna River water, India, using rep-PCR fingerprinting.

Genotypic diversity among multi-drug-resistant (MDR) aquatic E. coli isolated from different sites of Yamuna River was analyzed using repetitive element PCR (rep-PCR) methods viz. ERIC-PCR and (GTG)5-PCR and compared with the MDR animal fecal isolates. The 97 E. coli isolates belonging to different serotypes, phylogroups, and multi-drug resistance patterns were analyzed. High genetic diversity was observed by both the methods; however, (GTG)5 typing showed higher discriminating potential. Combination of ERIC types (E1-E32) and (GTG)5 types (G1-G46) generated 77 genotypes. The frequency of genotypes ranged from 0.013 to 0.065. The genotype composition of E. coli isolates was highly diverse at all the sampling sites across Yamuna River except at its entry site in Delhi. The sampling sites under the influence of high anthropogenic activities showed an increase in number of unique genotype isolates. These sites also exhibited high multiple antibiotic resistance (MAR) indexes (above 0.25) suggesting high risk of contamination. Principal coordinate analysis (PCoA) showed limited clustering of genotypes based on the sampling sites. The most frequent genotypes were grouped in the positive zone of both the principal coordinates (PC1 and PC2). The genotypes of most of the animal fecal isolates were unique and occupied a common space in the negative PC1 area forming a separate cluster. High genotypic diversity among the aquatic E. coli and the drain isolates, discharging the untreated municipal waste in the river, was observed, suggesting that the sewage effluents contribute substantially to contamination of this river system than animal feces. The presence of such a high diversity among the MDR E. coli isolates in the natural river systems is of great public health significance and highlights the need of an efficient surveillance system for better management of Indian natural water bodies.

Characterization of a two component system, Bas1213-1214, important for oxidative stress in Bacillus anthracis.

Microbial colonization is an outcome of appropriate sensing and regulation of its gene expression. Bacillus anthracis adapts and thrives in its environment through complex regulatory mechanisms, among them, the two component systems (TCS). Many bacteria respond to the oxygen fluctuations via TCS. In the present work, a previously uncharacterized TCS, Bas1213-1214, of B. anthracis with a probable role in oxygen sensing has been characterized as a functional TCS. A substantial increase in the expression of Bas1213 was observed during the stationary growth phase, in presence of bicarbonate ions, and under oxidative stress thereby speculating the role of Bas1213 in toxin production and adaptive responses. Electrophoretic mobility shift assay (EMSA) and ANS assay highlighted autoregulation of the system. Identification of Bas1213 regulon further suggested its regulatory function in metabolism and adaptive responses. A marked reduction in sporulation was observed on overexpression of Bas1213 in B. anthracis which can be correlated with the augmented expression of sporulation kinase D. Additionally, Bas1213 was shown to regulate catalase, and ABC transporter (mntH) further implicating its essential role during oxidative stress. Finally, crucial residues involved in the DNA binding activity of Bas1213 were also identified. This study reports that the role of Bas1213-1214 in the regulation of metabolism and adaptive responses during oxidative stress. Both sporulation and response to environmental oxygen are important for the maintenance of B. anthracis lifecycle, therefore, characterization of Bas1213-1214 provides a step closer toward understanding the regulatory network governing in B. anthracis.

Laccase grafted membranes for advanced water filtration systems: a green approach to water purification technology.

PURPOSE: Conventional wastewater treatment technologies are not good enough to completely remove all endocrine disrupting compounds (EDCs) from the water. Membrane separation systems have emerged as an attractive alternative to conventional clarification processes for waste and drinking water. Coupling of a membrane separation process with an enzymatic reaction has opened up new avenues to further enhance the quality of water. This review article deliberates the feasibility of implementing enzymatic membrane reactors has been deliberated. MATERIALS AND METHODS: A comprehensive study of conventional water treatment technologies was carried out and their shortcomings were pointed out. Research findings from the leading groups working on enzyme grafted membrane based water purification were summarized. This review also comprehends the patent documents pertinent to the technology of enzyme grafted membranes for water purification. RESULTS: Immobilization of an enzyme on a membrane improves the performance of membrane filtration, and processes for the treatment of polluted water. Research has started exploring the potential for laccase enzymes because it can catalyze the oxidation of a wide range of substrates, structurally comparable to EDCs, by a radical-catalyzed reaction mechanism, with corresponding reduction of oxygen to water in an electron transfer process. Further, in the presence of certain mediators, the substrate range of laccases can be further enhanced to non-aromatic substrates. CONCLUSIONS: Removal of EDCs by laccase cross-linked enzyme aggregates in fixed-bed reactors or fluidized-bed reactors and laccase immobilized ultrafiltration (LIUF) membranes are proving their worth in water purification technology. The major operational issues with the use of LIUF membranes are enzyme instability in real wastewater and membrane fouling. In view of the above-stated characteristics, laccases are considered as the most promising enzyme for a greener and less expensive water purification technology.

Recent trends in targeted delivery of smart nanocarrier-based microbial enzymes for therapeutic applications.

Smart carrier-based immobilization has widened the use of enzymes for the treatment of several disorders. Large surface areas, tunable morphology, and surface modification ability aid the targeted and controlled release of therapeutic enzymes from such formulations. Smart nanocarriers, such as polymeric carriers, liposomes, and silica have also increased the stability, half-life, and permeability of these enzymes. In this review, summarize recent advances in the smart immobilization of microbial enzymes and their development as precision nanomedicine for the treatment of cancer, thrombosis, phenylketonuria (PKU), and wound healing. We also discuss the challenges and measures to be adopted for the successful clinical translation of these formulations.

Microbial arginine deiminase: A multifaceted green catalyst in biomedical sciences.

Arginine deiminase is a well-recognized guanidino-modifying hydrolase that catalyzes the conversion of L-arginine to citrulline and ammonia. Their biopotential to regress tumors via amino acid deprivation therapy (AADT) has been well established. PEGylated formulation of recombinant Mycoplasma ADI is in the last-phase clinical trials against various arginine-auxotrophic cancers like hepatocellular carcinoma, melanoma, and mesothelioma. Recently, ADIs have attained immense importance in several other biomedical applications, namely treatment of Alzheimer's, as an antiviral drug, bioproduction of nutraceutical L-citrulline and bio-analytics involving L-arginine detection. Considering the wide applications of this biodrug, the demand for ADI is expected to escalate several-fold in the coming years. However, the sustainable production aspects of the enzyme with improved pharmacokinetics is still limited, creating bottlenecks for effective biopharmaceutical development. To circumvent the lacunae in enzyme production with appropriate paradigms of 'quality-by-design' an explicit overview of its properties with 'biobetter' formulations strategies are required. Present review provides an insight into all the potential biomedical applications of ADI along with the improvements required for its reach to clinics. Recent research advances with special emphasis on the development of ADI as a 'biobetter' enzyme have also been comprehensively elaborated.

Recent updates in diagnosis of abdominal tuberculosis with emphasis on nucleic acid amplification tests.

INTRODUCTION: Abdominal tuberculosis (TB) is a common epitome of extrapulmonary TB (EPTB), wherein peritoneal and intestinal TB are the most prevalent forms. Diagnosis of abdominal TB is a daunting challenge owing to variable anatomical locations, paucibacillary nature of specimens and atypical clinical presentations that mimic other abdominal diseases, such as Crohn's disease and malignancies. In this review, we made a comprehensive study on the diagnosis of abdominal TB. AREA COVERED: Various modalities employed for abdominal TB diagnosis include clinical features, imaging, bacteriological tests (smear/culture), histopathological/cytological observations, interferon-gamma release assays and nucleic acid amplification tests (NAATs). Among NAATs, loop-mediated isothermal amplification assay, PCR, multiplex-PCR, nested PCR, real-time PCR and GeneXpert® MTB/RIF were discussed. Identification of circulating Mycobacterium tuberculosis cell-free DNA by real-time PCR within ascitic fluids is another useful approach. EXPERT OPINION: Several novel molecular/immunological methods, such as GeneXpert Ultra, aptamer-linked immobilized sorbent assay, immuno-PCR (I-PCR) and nanoparticle-based I-PCR have recently been developed for detecting pulmonary TB and several EPTB types, which may also be explored for abdominal TB diagnosis. Precise and prompt diagnosis of abdominal TB may initiate an early therapy so as to reduce the complications, i.e. abdominal pain, ascites, abdominal distension, intestinal obstruction/perforation, etc., and avoid surgical involvement.Plain Language SummaryAbdominal tuberculosis (TB) is a manifestation of extrapulmonary TB (EPTB), where peritoneal and intestinal TB are two major forms. Diagnosis of abdominal TB is difficult owing to low bacterial load present in clinical samples and non-specific clinical presentations as it mimics other diseases such as inflammatory bowel diseases, abdominal malignancies, etc. Bacteriological tests (smear/culture) almost fail owing to poor sensitivities and it is not always possible to get representative tissue samples for histopathological and cytological observations. In recent years, molecular tests i.e. nucleic acid amplification tests (NAATs), such as PCR/multiplex-PCR (M-PCR), nested PCR and GeneXpert are widely employed. Markedly, PCR/M-PCR and nested PCR exhibited reasonable good sensitivities/specificities, while GeneXpert revealed low sensitivity in most of the studies but high specificity, thus it could assist in differential diagnosis of intestinal TB and Crohn's disease. Further, novel molecular/immunological tests employed for pulmonary TB and other EPTB types were described and those tests can also be utilized to diagnose abdominal TB. Reliable and rapid diagnosis of abdominal TB would initiate an early start of anti-tubercular therapy and reduce the severe complications.

In silico and in vitro analysis of arginine deiminase from Pseudomonas furukawaii as a potential anticancer enzyme.

Arginine deiminase (ADI), a promising anticancer enzyme from Mycoplasma hominis, is currently in phase III of clinical trials for the treatment of arginine auxotrophic tumors. However, it has been associated with several drawbacks in terms of low stability at human physiological conditions, high immunogenicity, hypersensitivity and systemic toxicity. In our previous work, Pseudomonas furukawaii 24 was identified as a potent producer of ADI with optimum activity under physiological conditions. In the present study, phylogenetic analysis of microbial ADIs indicated P. furukawaii ADI (PfADI) to be closely related to experimentally characterized ADIs of Pseudomonas sp. with proven anticancer activity. Immunoinformatics analysis was performed indicating lower immunogenicity of PfADI than MhADI (M. hominis ADI) both in terms of number of linear and conformational B-cell epitopes and T-cell epitope density. Overall antigenicity and allergenicity of PfADI was also lower as compared to MhADI, suggesting the applicability of PfADI as an alternative anticancer biotherapeutic. Hence, in vitro experiments were performed in which the ADI coding arcA gene of P. furukawaii was cloned and expressed in E. coli BL21. Recombinant ADI of P. furukawaii was purified, characterized and its anticancer activity was assessed. The enzyme was stable at human physiological conditions (pH 7 and 37 °C) with Km of 1.90 mM. PfADI was found to effectively inhibit the HepG2 cells with an IC50 value of 0.1950 IU/ml. Therefore, the current in silico and in vitro studies establish PfADI as a potential anticancer drug candidate with improved efficacy and low immunogenicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03292-2.

Diagnosis of peritoneal tuberculosis by real-time immuno-PCR assay based on detection of a cocktail of Mycobacterium tuberculosis CFP-10 and HspX proteins.

BACKGROUND: Diagnosis of peritoneal TB is difficult owing to unusual clinical manifestations and low sensitivities obtained with most of the available diagnostic modalities. Hence, there is an urgent need to design a reliable diagnostic test so that an early therapy is initiated. RESEARCH DESIGN AND METHODS: We designed a quantitative real-time immuno-PCR (RT-I-PCR) assay to detect a cocktail of Mycobacterium tuberculosis CFP-10 (Rv3874) and HspX (Rv2031c) proteins in clinical samples (ascitic fluids and peritoneal biopsies) of peritoneal TB patients, and results were compared with I-PCR/ELISA. RESULTS: A wide range of CFP-10+ HspX (0.6 pg/mL to 9.9 ng/mL) was detected in clinical samples of peritoneal TB patients by RT-I-PCR, whereas ELISA exhibited a narrow range (3 ng/mL to 11.5 ng/mL). Sensitivities of 81.5% and 65.7% and specificities of 92.5% and 90% were obtained in a total of 78 cases (comprising 38 peritoneal TB and 40 non-TB controls) by RT-I-PCR and I-PCR, respectively. Markedly, sensitivity obtained by RT-I-PCR was significantly higher than I-PCR (p = 0.0143) and ELISA (p = 0.0005). CONCLUSIONS: Our RT-I-PCR revealed good accuracy for the rapid diagnosis of peritoneal TB cases. After further improving the specificity and reducing the cost, this assay may develop into a diagnostic kit.

An insight into biological activities of native cyclotides for potential applications in agriculture and pharmaceutics.

Cyclotides are plant-derived mini-proteins of 28 - 37 amino acids. They have a characteristic head-to-tail cyclic backbone and three disulfide cross-linkages formed by six highly conserved cysteine residues, creating a unique knotted ring structure, known as a cyclic cystine knot (CCK) motif. The CCK topology confers immense stability to cyclotides with resistance to thermal and enzymatic degradation. Native cyclotides are of interest due to their multiple biological activities with several potential applications in agricultural (e.g. biopesticides, antifungal) and pharmaceutical (e.g. anti-HIV, cytotoxic to tumor cells) sectors. The most recent application of insecticidal activity of cyclotides is the commercially available biopesticidal spray known as 'Sero X' for cotton crops. Cyclotides have a general mode of action and their potency of bioactivity is determined through their binding ability, pore formation and disruption of the target biological membranes. Keeping in view the important potential applications of biological activities of cyclotides and the lack of an extensive and analytical compilation of bioactive cyclotides, the present review systematically describes eight major biological activities of the native cyclotides from four angiosperm families viz. Fabaceae, Poaceae, Rubiaceae, Violaceae. The bioactivities of 94 cytotoxic, 57 antibacterial, 44 hemolytic, 25 antifungal, 21 anti-HIV, 20 nematocidal, 10 insecticidal and 5 molluscicidal cyclotides have been comprehensively elaborated. Further, their distribution in angiosperm families, mode of action and future prospects have also been discussed.

Microbial Enzymes used in Prodrug Activation for Cancer Therapy: Insights and Future Perspectives.

Enzyme prodrug therapy has gained momentum in recent years due to its ability to improve therapeutic index (benefits versus toxic side-effects) and efficacy of chemotherapy in cancer treatment. Inactive prodrugs used in this system are converted into active anti-cancerous drugs by enzymes, specifically within the tumor cells. This therapy involves three components namely prodrug, enzyme and gene delivery vector. Past reports have clearly indicated that the choice of enzyme used is the major determinant for the success of this therapy. Generally, enzymes from nonhuman sources are employed to avoid off-target toxicity. Exogenous enzymes also give better control to the clinician regarding the calibration of treatment by site-specific initiation. Amongst these exo-enzymes, microbial enzymes are preferred due to their high productivity, stability and ease of manipulation. The present review focuses on the commonly used microbial enzymes, particularly cytosine deaminase, nitroreductase, carboxypeptidase, purine nucleoside phosphorylase in prodrug activation therapy. Various aspects viz. source of the enzymes, types of cancer targeted, mode of action and efficacy of the enzyme/prodrug system, efficient vectors used and recent research developments of each of these enzymes are comprehensively elaborated. Further, the results of the clinical trials and various strategies to improve their clinical applicability are also discussed.

A Novel miR-146a-POU3F2/SMARCA5 Pathway Regulates Stemness and Therapeutic Response in Glioblastoma.

Rapid tumor growth, widespread brain-invasion, and therapeutic resistance critically contribute to glioblastoma (GBM) recurrence and dismal patient outcomes. Although GBM stem cells (GSC) are shown to play key roles in these processes, the molecular pathways governing the GSC phenotype (GBM-stemness) remain poorly defined. Here, we show that epigenetic silencing of miR-146a significantly correlated with worse patient outcome and importantly, miR-146a level was significantly lower in recurrent tumors compared with primary ones. Further, miR-146a overexpression significantly inhibited the proliferation and invasion of GBM patient-derived primary cells and increased their response to temozolomide (TMZ), both in vitro and in vivo. Mechanistically, miR-146a directly silenced POU3F2 and SMARCA5, two transcription factors that mutually regulated each other, significantly compromising GBM-stemness and increasing TMZ response. Collectively, our data show that miR-146a-POU3F2/SMARCA5 pathway plays a critical role in suppressing GBM-stemness and increasing TMZ-response, suggesting that POU3F2 and SMARCA5 may serve as novel therapeutic targets in GBM. IMPLICATIONS: miR-146a predicts favorable prognosis and the miR-146a-POU3F2/SMARCA5 pathway is important for the suppression of stemness in GBM.

An overview of acyclotides: Past, present and future.

Acyclotides are plant-based, acyclic miniproteins with cystine knot motif formed by three conserved disulfide linkages and lack head to tail ligation. Acyclotides may not necessarily be less stable, even though they lack cyclic backbone, as the conserved cystine knot feature provides the required stability. Violacin A was the first acyclotide, isolated from Viola odorata in 2006. Until now, acyclotides have been reported from five dicot families (Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae, Fabaceae) and one monocot family (Poaceae). In Poaceae, only acyclotides have been found whereas in dicot families both cyclotides and acyclotides have been isolated. In last 15 years, several acyclotides with antimicrobial, cytotoxic and hemolytic bioactivities have been discovered. Thus, although many naturally expressed acyclotides do exhibit bioactivities, the linearization of the cyclic peptides may result in loss of bioactivities. Although, bioactivities of acyclotides are comparable to their cyclic counterparts, the numbers of isolated acyclotides are still few. Further, those discovered, have the scope to be screened for agriculturally important activities (insecticidal, anti-helminthic, molluscicidal) and pharmaceutical properties (anticancer, anti-HIV, immuno-stimulant). The feasibility of application of acyclotides is because of their relatively less complex biological synthesis compared to cyclotides, as the cyclization step is not needed. This attribute facilitates the production of transgenic crops and/or its expression in heterologous organisms, lacking cyclization machinery. Keeping in view the bioactivities and the wide array of emerging potential applications of acyclotides, the present review discusses their distribution in plants, gene and protein structure, biosynthesis, bioactivities and mechanism of action. Further, their potential applications and future perspectives to exploit them in agriculture and pharmaceutical industries have been highlighted.

Biomedical applications of microbial phenylalanine ammonia lyase: Current status and future prospects.

Phenylalanine ammonia lyase (PAL) has recently emerged as an important therapeutic enzyme with several biomedical applications. The enzyme catabolizes l-phenylalanine to trans-cinnamate and ammonia. PAL is widely distributed in higher plants, some algae, ferns, and microorganisms, but absent in animals. Although microbial PAL has been extensively exploited in the past for producing industrially important metabolites, its high substrate specificity and catalytic efficacy lately spurred interest in its biomedical applications. PEG-PAL drug named Palynziq™, isolated from Anabaena variabilis has been recently approved for the treatment of adult phenylketonuria (PKU) patients. Further, it has exhibited high potency in regressing tumors and treating tyrosine related metabolic abnormalities like tyrosinemia. Several therapeutically valuable metabolites have been biosynthesized via its catalytic action including dietary supplements, antimicrobial peptides, aspartame, amino-acids, and their derivatives. This review focuses on all the prospective biomedical applications of PAL. It also provides an overview of the structure, production parameters, and various strategies to improve the therapeutic potential of this enzyme. Engineered PAL with improved pharmacodynamic and pharmacokinetic properties will further establish this enzyme as a highly efficient biological drug.

Evaluation of genetic diversity among aquatic and fecal isolates of Escherichia coli using multilocus variable number of tandem repeat analysis.

In developing countries like India, fecal pollution of surface waters is a major threat to public and environmental health. The aim of the study was to assess serological, phylogenetic and molecular diversity among aquatic Escherichia coli isolates from Yamuna river and their comparison with the animal fecal isolates. A total of 97 E. coli isolates from Yamuna river and domesticated animals were characterized by multilocus variable number tandem repeat analysis (MLVA) using four VNTR loci. The pathogenicity of these strains by serological and phylogenetic analysis was also determined. E. coli strains were differentiated into 53 distinct MLVA types with high discriminatory power, Simpson's index of 0.95 (95% CI 0.923-0.978). Cluster analysis and population modeling using minimum spanning tree suggested a possible epidemiological linkage among aquatic and fecal isolates. The study also reported the presence of highly diverse and pathogenic serotypes belonging to STEC and EPEC strains, particularly O157 and high prevalence of pathogenic phylogroups (phylogroup, B2 and D). The presence of such a high molecular heterogeneity among aquatic and fecal E. coli isolates emphasizes upon the need to develop proper fecal pollution abatement strategies for Indian natural bodies.

High Prevalence of Antibiotic Resistance and Integrons in Escherichia coli Isolated from Urban River Water, India.

The contamination of surface waters with multidrug resistant (MDR) coliforms is a major public health concern in developing countries. This study was aimed to evaluate the occurrence of antibiotic resistance and role of integrons in the spread of resistance genes in Escherichia coli isolated from urban waters of river Yamuna. One hundred and forty-one strains of E. coli were isolated and assessed for antibiotic resistance wherein high resistance was observed for Cefazolin. Integrons (class 1 and class 2) were detected in 32% of the isolates. Variable region of class 1 integron carried different gene cassettes, namely. dfrA17-aadA5, dfrA12-orfF-aadA2, blaOXA-1-aadA1, and unusual phage tail tape measure protein. These integron-positive isolates were further characterized by phylogrouping, serotyping, and BOX-PCR typing. Phylogroup B2 was found to be the most prevalent. Pathogenic E. coli O157 was also reported. Majority of the isolates (54%; 7/13) carrying "dfrA17-aadA5" gene cassette were clustered predominantly into a single BOX-PCR type (B8), suggesting a genetic relatedness among the isolates. This study thus depicts very high incidence of multidrug resistance and class 1 integrons in surface water of India. The prevalence of integrons in aquatic E. coli correlated well with resistance to increasing number of antibiotic classes and multiple antibiotic resistance (MAR) index at various sites. Integron-positive MDR E. coli isolates were found to be serologically and genetically diverse suggesting major role of integrons in the emergence and dissemination of resistance traits in waterborne E. coli.