Subverting the Canon: Novel Cancer-Promoting Functions and Mechanisms for snoRNAs.

Small nucleolar RNAs (snoRNAs) constitute a class of intron-derived non-coding RNAs ranging from 60 to 300 nucleotides. Canonically localized in the nucleolus, snoRNAs play a pivotal role in RNA modifications and pre-ribosomal RNA processing. Based on the types of modifications they involve, such as methylation and pseudouridylation, they are classified into two main families-box C/D and H/ACA snoRNAs. Recent investigations have revealed the unconventional synthesis and biogenesis strategies of snoRNAs, indicating their more profound roles in pathogenesis than previously envisioned. This review consolidates recent discoveries surrounding snoRNAs and provides insights into their mechanistic roles in cancer. It explores the intricate interactions of snoRNAs within signaling pathways and speculates on potential therapeutic solutions emerging from snoRNA research. In addition, it presents recent findings on the long non-coding small nucleolar RNA host gene (lncSNHG), a subset of long non-coding RNAs (lncRNAs), which are the transcripts of parental SNHGs that generate snoRNA. The nucleolus, the functional epicenter of snoRNAs, is also discussed. Through a deconstruction of the pathways driving snoRNA-induced oncogenesis, this review aims to serve as a roadmap to guide future research in the nuanced field of snoRNA-cancer interactions and inspire potential snoRNA-related cancer therapies.

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

Association of two BRM promoter polymorphisms and smoking status with malignant pleural mesothelioma risk and prognosis.

Brahma (BRM), of the SWI/SNF complex, has two 6 to 7 bp insertion promoter polymorphisms (BRM-741/BRM-1321) that cause epigenetic BRM suppression, and are associated with risk of multiple cancers. BRM polymorphisms were genotyped in malignant pleural mesothelioma (MPM) cases and asbestos-exposed controls. Multivariable logistic regression (risk) and Cox regression (prognosis) were performed, including stratified analyses by smoking status to investigate the effect of polymorphisms on MPM risk and prognosis. Although there was no significant association overall between BRM-741/BRM-1321 and risk in patients with MPM, a differential effect by smoking status was observed (P-interaction < .001), where homozygous variants were protective (aOR of 0.18-0.28) in ever smokers, while never smokers had increased risk when carrying homozygous variants (aOR of 2.7-4.4). While there was no association between BRM polymorphisms and OS in ever-smokers, the aHR of carrying homozygous-variants of BRM-741, BRM-1321 or both were 4.0 to 8.6 in never-smokers when compared to wild-type carriers. Mechanistically, lower mRNA expression of BRM was associated with poorer general cancer prognosis. Electrophoretic mobility shift assays and chromatin immunoprecipitation experiments (ChIP) revealed high BRM insertion variant homology to MEF2 regulatory binding sites. ChIP experimentation confirmed MEF2 binding only occurs in the presence of insertion variants. DNA-affinity purification assays revealed YWHA scaffold proteins as vital to BRM mRNA expression. Never-smokers who carry BRM homozygous variants have an increased chance of developing MPM, which results in worse prognosis. In contrast, in ever-smokers, there may be a protective effect, with no difference in overall survival. Mechanisms for the interaction between BRM and smoking require further study.

A Rare Embryonic Arterial Anomaly: Thrombosis of Persistent Sciatic Artery.

Persistent sciatic artery (PSA) is an exceptionally rare congenital vascular anomaly with profound clinical implications. This condition occurs when the primitive sciatic artery, responsible for fetal lower limb blood supply, fails to regress during embryonic development. PSA persists into adulthood, representing an intriguing vascular variation that can present as gluteal aneurism and thrombosis. We present the case of a 72-year-old female patient admitted with abdominal pain and blackening of her right foot. Clinical examination revealed dry gangrene affecting the toes, limb edema, and absent peripheral pulses in the right lower limb. Septic shock and electrolyte imbalances prompted immediate resuscitation and antibiotic therapy. Diagnostic investigations, including Doppler ultrasonography, CT angiography, and 2D echocardiography, identified a right-sided PSA. With limb ischemia being irreversible, a below-knee amputation was performed. This case highlights the clinical presentation, diagnostic workup, and management of a rare PSA, emphasizing the importance of prompt recognition and intervention in complex vascular anomalies.

Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast.

Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends replication. However, their function remains elusive. Here, we have analyzed genome-wide replication and show that ST does not affect genome-wide replication but is crucial for the efficient replication of a subtelomeric region called STE3-2. We further show that, when ST function is compromised, a homologous recombination (HR)-based fork restart mechanism becomes necessary for STE3-2 stability. While both Taz1 and Stn1 bind to STE3-2, we find that the STE3-2 replication function of ST is independent of Taz1 but relies on its association with the shelterin proteins Pot1-Tpz1-Poz1. Finally, we demonstrate that the firing of an origin normally inhibited by Rif1 can circumvent the replication defect of subtelomeres when ST function is compromised. Our results help illuminate why fission yeast telomeres are terminal fragile sites.

Serosurveillance of Health Care Workers in a COVID Hospital: Immune Response, and Its Longevity.

OBJECTIVE: We aimed to study the seroprevalence of coronavirus disease 2019 (COVID-19) and sustainability of the immune response in health care workers (HCWs). A cross-sectional study was conducted between October 7 and November 30, 2020, in a multi-specialty hospital in Eastern India designated as COVID hospital during this pandemic. Study participants included 2,110 HCWs, including those who have recovered from COVID infection. METHOD: HCWs were required to complete a questionnaire and give written consent to participate in the study. Their venous blood sample was collected for serum analysis of IgG antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by chemiluminescent immunoassay. RESULTS: Positive IgG antibodies were seen in 924 participants with a point prevalence of 43.79%. Slightly higher reactivity was seen in males. History of COVID-19 infection was noted in 10.9%, with the highest antibody response in 81% cases. A maximum of 87.9% reactivity was seen in the first two months, and a significant fall was noted in the fourth month, with reactivity seen in only 50% of the study participants. CONCLUSION: SARS-CoV-2 infection is associated with a variable immune response in the infected population. The declining trend of the antibodies correlates with short-lived protective immunity and the possibility of re-infection. Further studies are needed to explore the probable reasons for varied seroprevalence.

Results of Serosurveillance and Forecasting the Third Wave of COVID-19 in an Industrial District in India.

Prevalence of immunoglobulin G (IgG) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in the industrial district of East Singhbhum (Jharkhand, India) from July, August, November, and December 2020 and January 2021 after the first wave and in July 2021 after the second wave of coronavirus disease 2021 (COVID-19) infections may be utilized to find the possibility of a third wave of COVID-19 infections. Based on the trend of the loss of protective IgG antibodies after the first wave and the seropositivity of 75% in the district in July 2021, simple forecasting and proportional estimates of the seropositivity in the next eight months and the estimated maximum number of the cases was done. We also considered the seropositivity without vaccination in July 2021 (63%). Additionally, the trend of the weekly RT-PCR and rapid antigen testing for SARS-CoV-2 may also preemptively predict an imminent wave. Based on the East Singhbhum population and the vaccination coverage with at least one dose till July 2021 (Covishield or Covaxin), it is estimated that a 4-5% monthly vaccination coverage rate of new individuals will not allow the seropositivity to fall below 50% and hold at bay a major wave. Vaccination coverage of 3% or less would allow a continuous drop in acquired immunity in the district and can potentially cause a rise in cases, making the community susceptible to a future surge of infections. A 3-5% vaccination rate of new individuals is unlikely to see a drop in the community seropositivity below 50% and the number of new cases of COVID-19 infections going above 478 to 712 per month at least till March 2022. The assumptions are based on presuming that there will be no new mutant of SARS-CoV-2 that escapes the immunity provided by previous infection or vaccination over the next eight months. However, currently, there is no evidence to speculate on any new variant of concern causing a major wave globally. The B.1.617.2 (delta) variant was first identified in October 2020 and there was a lag of six months to the second surge of COVID-19 infections in East Singhbhum, primarily caused by this variant. Additionally, 3% and above, with a rising weekly trend of reverse transcription-polymerase chain reaction (RT-PCR) positivity for SARS-CoV-2 can provide at least four to eight weeks advance warning before the peak of the wave if an imminent future wave is impending.

Prolonged Oxygen Therapy Post COVID-19 Infection: Factors Leading to the Risk of Poor Outcome.

BACKGROUND: Coronavirus disease 2019 (COVID-19) infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded ribonucleic acid (RNA) ß-coronavirus. Prolonged duration of symptoms, ill health, disability, and need for hospitalisation are all well-known features of severe COVID-19 disease. OBJECTIVE: To describe the epidemiological, clinical and imaging characteristics of hospitalised patients of COVID-19 who required prolonged oxygen therapy after testing negative for SARS-CoV-2 and attempt to determine the associated factors leading to delayed recovery, failure to wean, and mortality. MATERIAL AND METHOD: Prospective observational study from 9th September to 6th November 2020 in a tertiary care COVID hospital of Jharkhand. Included COVID-19-infected patients requiring oxygen to maintain a saturation of ≥95% after testing reverse transcription polymerase chain reaction (RT-PCR) negative. Patients were classified as Group I, those who could be weaned off oxygen, and Group II, those who could not be weaned off oxygen during their stay in the isolation ward. A detailed assessment for outcome in these two groups related to age, gender, presence or absence of co-morbidities, nature of co-morbidities and findings of high-resolution CT (HRCT) thorax was done to ascertain risk factors for failure to wean and adverse outcomes. RESULTS: During the study period, 93 patients suffering from moderate to severe COVID-19 infection, could not be discharged from the hospital and were admitted to the post-COVID isolation ward after testing RT-PCR negative, due to breathlessness and need for oxygen therapy, with a male predominance, M:F ratio of 2.2:1. Of these 93 patients, 51 could be weaned off oxygen in the isolation ward. The mean and median age of patients who could be successfully weaned was 58.5±14.3 years and 60 years respectively, compared to a mean age of 64±12.4 years and a median age of 67 years for patients who could not be weaned off oxygen during the isolation period. Patients aged ≥60 years were at risk for prolonged requirement of oxygen compared to those <50 years of age, relative risk (RR) 1.43 (95%CI 0.9-2, p=0.051). Failure to wean in <50 years was noted in presence of co-morbidities, RR 4 (95%CI 1.5-10.6, p=0.005). Multivariable logistic regression analysis calculated an odds ratio (OR) of 12.22 (95%CI 2.4-61.5, p<0.002) in patients of coronary artery disease (CAD), and 3.34 (95%CI 1.01-10.9, p<0.046) in patients of diabetes, for failure to wean with delayed recovery in patients aged 50 years and more, having multiple co-morbidities. Presence of ≥three co-morbid conditions was associated with increased risk of critical care unit (CCU) admissions (RR 2.1, p=0.02), failure to wean (RR 1.79, p<0.006), and death (p=0.02). Elderly male patients (mean age of 81.3±7.2years) with CAD and multiple comorbidities were at a high risk of mortality (p=0.01). CONCLUSION:  Patients ≥50 years of age having ≥three co-morbidities are at increased risk of prolonged hospitalisation and oxygen therapy in moderate to severe COVID-19 infection, precluding their discharge even after they test negative for SARS-CoV-2. Elderly male patients of COVID-19 with CAD and multiple comorbidities are at a high risk of mortality.

Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II.

An alkaline-protease-producing bacterial strain (AS-S24-II) isolated from a soil sample in Assam is a Gram-stain-positive, catalase-positive, endospore-forming rod and grows at temperatures ranging from 30 degrees C to 60 degrees C and salinity ranging from 0% to 7% (w/v) NaCl. Phenotypic characterisation, chemotaxonomic properties, presence of Paenibacillus-specific signature sequences, and ribotyping data suggested that the strain AS-S24-II represents a novel species of the genus Paenibacillus, for which the name Paenibacillus tezpurensis sp. nov. (MTCC 8959) is proposed. Phylogenetic analysis revealed that P. lentimorbus strain DNG-14 and P. lentimorbus strain DNG-16 represent the closest phylogenetic neighbour of this novel strain. Alkaline protease production (598 x 10(3) U l(-1)) by P. tezpurensis sp. nov. in SmF was optimised by response surface method. A laundry-detergent-stable, Ca(2+)-independent, 43-kDa molecular weight alkaline serine protease from this strain was purified with a 1.7-fold increase in specific activity. The purified protease displayed optimum activity at pH 9.5 and 45-50 degrees C temperature range and exhibited a significant stability and compatibility with surfactants and most of the tested commercial laundry detergents at room temperature. Further, the protease improved the wash performance of detergents, thus demonstrating its feasibility for inclusion in laundry detergent formulations.

A Prospective Study on Rapidly Declining SARS-CoV-2 IgG Antibodies Within One to Three Months of Testing IgG Positive: Can It Lead to Potential Reinfections?

Background COVID-19 immunoglobulin G (IgG) antibodies have been considered to provide protective immunity and its immunoassays have been widely used for serosurveillance. In our serosurveillance on an industrial workforce of randomly selected 3296 subjects, COVID-19 IgG antibody positivity was reported in 7.37% (243) subjects. However, when 30 days later, eight of the 243 COVID-19 IgG antibody-positive individuals complained of symptoms suggestive of COVID-19 infection and were confirmed as COVID-19 infection by reverse transcription-polymerase chain reaction (RT-PCR), their COVID-19 IgG antibodies were retested. Seven of the eight previously IgG positive individuals had lost their protective antibodies. Methods Subsequently, a prospective clinical trial was planned by repeating the test for IgG antibodies on the remaining earlier positive 235 individuals at 45-65 days after their initial test. Only 201 of the 235 individuals consented and participated in the non-randomized single-arm observational trial. Results Only 28.36% (57/201) retained their IgG antibodies and 70.15% (141/201) had lost their IgG antibodies. Three cases reported equivocal results on retesting. Conclusions Our findings show that the protective COVID-19 IgG antibodies rapidly decline over one to three months. Further studies are needed with a quantitative assay over a period with neutralizing antibodies to establish if its decay can potentially lead to reinfections. Rapidly decaying protective IgG antibodies would impact herd immunity and vaccine durability. It is critical for the potential vaccines to generate both protective T- and B-cell immune responses in a sustained manner.

Targeting pheochromocytoma/paraganglioma with polyamine inhibitors.

BACKGROUND: Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting. METHODS: Metabolomic analysis was performed on human hPheo1 cells and shRNA SDHB knockdown hPheo1 (hPheo1 SDHB KD) cells. Additional analysis of 115 human fresh frozen samples was conducted. In vitro studies using N1,N11-diethylnorspermine (DENSPM) and N1,N12- diethylspermine (DESPM) treatments were carried out. DENSPM efficacy was assessed in human cell line derived mouse xenografts. RESULTS: Components of the polyamine pathway were elevated in hPheo1 SDHB KD cells compared to wild-type cells. A similar observation was noted in SDHx PCC/PGLs tissues compared to their non-mutated counterparts. Specifically, spermidine, and spermine were significantly elevated in SDHx-mutated PCC/PGLs, with a similar trend in hPheo1 SDHB KD cells. Polyamine pathway inhibitors DENSPM and DESPM effectively inhibited growth of hPheo1 cells in vitro as well in mouse xenografts. CONCLUSIONS: This study demonstrates overactive polyamine pathway in PCC/PGL with SDHB mutations. Treatment with polyamine pathway inhibitors significantly inhibited hPheo1 cell growth and led to growth suppression in xenograft mice treated with DENSPM. These studies strongly implicate the polyamine pathway in PCC/PGL pathophysiology and provide new foundation for exploring the role for polyamine analogue inhibitors in treating metastatic PCC/PGL. PRéCIS: Cell line metabolomics on hPheo1 cells and PCC/PGL tumor tissue indicate that the polyamine pathway is activated. Polyamine inhibitors in vitro and in vivo demonstrate that polyamine inhibitors are promising for malignant PCC/PGL treatment. However, further research is warranted.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase.

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe3O4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 degrees C and pH 7.2 of the reaction mixture before addition of H2O2 (3% w/w), 2% (w/v) PEG(6000) and 0.062:1 molar ratio of PEG to FeCl2 x 4H2O. Further statistical optimization using response surface methodology yielded an R2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

High-Frequency Lithium Acetate Transformation of Schizosaccharomyces pombe.

The introduction of ectopic DNA, such as plasmids, into yeast cells has for decades been a critical protocol for the study of this eukaryotic model system. We describe here an efficient transformation procedure for use in the fission yeast Schizosaccharomyces pombe. This method relies on chemical agents (lithium acetate, and polyethylene glycol) and temperature stresses, which ultimately facilitate transfer of the genetic material through the cell wall and plasma membrane without significant impact on the transferred DNA or the recipient cell. Using this protocol, we consistently see transformation efficiencies between 1.0 × 103 and 1.0 × 104 transformants per microgram of the plasmid with 108 S. pombe cells. The principal benefits and advantages of this method are its simplicity, efficiency, and relative speed of completion.

Duplication and Transformation of the Schizosaccharomyces pombe Collection of Deletion Strains.

We present an efficient and organized method of lithium acetate and polyethylene glycol-based transformation of plasmid DNA into the commercially available collection of Schizosaccharomyces pombe with single-gene deletions. We also describe how to prepare a duplicate collection of the deletion strains in order to preserve the longevity of the master set. These protocols are adapted to the 96-well format of the 3004 strains of the Version 2.0 Bioneer set but can also be used for later releases of the collection. This transformation method typically yields efficiencies in the range between 1.0 × 103 and 1.0 × 104 transformants per microgram of plasmid DNA. However, some deletion strains transformed with significantly lower efficiencies. We provide a list of these difficult-to-transform strains. Applications for this methodology include the transformation of the deletion set with plasmids necessary for genetic screens.

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Loss of the SWI/SNF ATPase subunits BRM and BRG1 drives lung cancer development.

Inactivation of Brg1 and Brm accelerated lung tumor development, shortened tumor latency, and caused a loss of differentiation. Tumors with Brg1 and/or Brm loss recapitulated the evolution of human lung cancer as observed by the development of local tumor invasion as well as distal tumor metastasis, thereby making this model useful in lung cancer studies. Brg1 loss contributed to metastasis in part by driving E-cadherin loss and Vimentin up-regulation. By changing more than 6% of the murine genome with the down-regulation of tumor suppressors, DNA repair, differentiation and cell adhesion genes, and the concomitant up-regulation of oncogenes, angiogenesis, metastasis and antiapoptosis genes, caused by the dual loss of Brg1/Brm further accelerated tumor development. Additionally, this Brg1/Brm-driven change in gene expression resulted in a nearly two-fold increase in tumorigenicity in Brg1/Brm knockout mice compared with wild type mice. Most importantly, Brg1/Brm-driven lung cancer development histologically and clinically reflects human lung cancer development thereby making this GEMM model potentially useful.

BRG1 and BRM loss selectively impacts RB and P53, respectively: BRG1 and BRM have differential functions in vivo.

The SWI/SNF complex is an important regulator of gene expression that functions by interacting with a diverse array of cellular proteins. The catalytic subunits of SWI/SNF, BRG1 and BRM, are frequently lost alone or concomitantly in a range of different cancer types. This loss abrogates SWI/SNF complex function as well as the functions of proteins that are required for SWI/SNF function, such as RB1 and TP53. Yet while both proteins are known to be dependent on SWI/SNF, we found that BRG1, but not BRM, is functionally linked to RB1, such that loss of BRG1 can directly or indirectly inactivate the RB1 pathway. This newly discovered dependence of RB1 on BRG1 is important because it explains why BRG1 loss can blunt the growth-inhibitory effect of tyrosine kinase inhibitors (TKIs). We also observed that selection for Trp53 mutations occurred in Brm-positive tumors but did not occur in Brm-negative tumors. Hence, these data indicate that, during cancer development, Trp53 is functionally dependent on Brm but not Brg1. Our findings show for the first time the key differences in Brm- and Brg1-specific SWI/SNF complexes and help explain why concomitant loss of Brg1 and Brm frequently occurs in cancer, as well as how their loss impacts cancer development.

Characterization and application of a detergent-stable alkaline α-amylase from Bacillus subtilis strain AS-S01a.

A strain AS-S01a, capable of producing high-titer alkaline α-amylase, was isolated from a soil sample of Assam, India and was taxonomically identified as Bacillus subtilis strain AS-S01a. Optimized α-amylase yield by response surface method (RSM) was obtained as 799.0 U with a specific activity of 201.0 U/mg in a process control bioreactor. A 21.0 kDa alkaline α-amylase purified from this strain showed optimum activity at 55°C and pH 9.0, and it produced high molecular weight oligosaccharides including small amount of glucose from starch as the end product. The K(m) and V(max) values for this enzyme towards starch were determined as 1.9 mg/ml and 198.21 µmol/min/mg, respectively. The purified α-amylase retained its activity in presence of oxidant, surfactants, EDTA and various commercial laundry detergents, thus advocating its suitability for various industrial applications.

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency.

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent K(m) and V(max) values of Bafibrinase towards fibrin were determined as 0.24 µM and 2.8 µmol/min, respectively. It showed a K(m) value of 0.139 mM towards a chromogenic substrate for plasmin (D-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,ß-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

Biodegradable and biocompatible epoxidized vegetable oil modified thermostable poly(vinyl chloride): thermal and performance characteristics post biodegradation with Pseudomonas aeruginosa and Achromobacter sp.

The increased production of municipal solid waste by the disposal of plastic materials heightens the urgency to develop biodegradable materials for daily use. In vitro-biodegradation study on poly(vinyl chloride) (PVC) plasticized by epoxidized Mesua ferrea L. seed oil at three different weight percentages (PVC/ENO ratio of 75/25, 50/50 and 25/75) was conducted by using Pseudomonas aeruginosa and Achromobacter sp. bacteria. The test bacterial species were able to grow on the polymer matrix by using it as a source of energy; however the pristine PVC did not support the microbial growth. The PVC/ENO material of 25/75 ratio showed the highest percent (%) of biodegradation compared to other tested systems. The bacterial count and the dry biomass post 180 days of inoculation in 25/75 plasticized PVC suggested bacterial growth at the expense of degradation of the system. The tensile strength of 25/75 PVC/ENO system, post 180 days of inoculation by Pseudomonas aeruginosa and Achromobacter sp. decreased by about 53% and 43% respectively. Further, surface erosion phenomenon and structural change of the matrix after bacterial growth, as studied by FTIR and SEM analysis of PVC/ENO of 25/75 ratio exhibited noticeable deterioration post 180 days of inoculation.