Mechanistic insights into the inhibition of endo-ß 1,4 xyloglucan hydrolase by a classical aspartic protease inhibitor.

This is the first report of inactivation of xyloglucanase from Thermomonospora sp by pepstatin A, a specific inhibitor towards aspartic proteases. The steady state kinetics revealed a reversible, competitive, two-step inhibition mechanism with IC 50 and K i values of 3.5 ± 0.5 µM and 1.25 ± 0.5 µM respectively. The rate constants determined for the isomerization of EI to EI(*) and the dissociation of EI* were 14.5 ± 1.5 × 10(-5) s(-1) and 2.85 ± 1.2 × 10(-8) s(-1) respectively, whereas the overall inhibition constant K i(*) was 27 ± 1 nM. The conformational changes induced upon inhibitor binding to xyloglucanase were monitored by fluorescence analysis and the rate constants derived were in agreement with the kinetic data. The abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled xyloglucanase and far UV analysis suggested that pepstatin binds to the active site of the enzyme. Our results revealed that the inactivation of xyloglucanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis.

Menstrual practices, fertility intentions and decision-making regarding family planning by women belonging to various indigenous groups of Wayanad- A qualitative study.

Background: Among the different social groups in India, the tribes are the most vulnerable and socioeconomically deprived. The tribal population is distinct from the general population in terms of their unique cultural, traditional, and reproductive health practices. Therefore, the aim of the study was to conduct an exploratory analysis of the menstrual practices, fertility intentions and decision-making regarding family planning among various indigenous tribal women of Kerala. Methods: A qualitative study was conducted among the tribal women of Wayanad district using the grounded theory approach. Using purposive sampling 16 in-depth interviews, 6 key informant interviews and 2 focus group discussion (FGD) s were conducted. Interview guides were developed for in-depth interviews, key-informant interviews, and focus group discussion through extensive formative research with literature reviews and taking expert opinions. The interviews were conducted among women of reproductive age and their spouses hailing from Paniya and Kurichiya tribal groups in Kalpetta and Mananthavady areas of Wayanad district. Key informant interviews were also conducted among doctors, pharmacists, and community health workers. Results: The key findings of this study were the identification of a web of cultural practices pertaining to menstruation among persons of tribal origin. Unique traditional practices such as "Valayamapura" and "Thirandukalyanam" were reported and most of the women were keen to carry it forward. Fertility desires among couples were found to be not significantly influenced by any gender bias. Decision-making regarding family size were found to be on a mutual agreement between the spouses. The tribal women were aware of modern spacing methods, but preferred natural methods of contraception for temporary use and tubectomy as the permanent method. Non-contraceptive use of oral contraceptive pills (OCP) was prevalent, and the majority took it occasionally for postponement of menstruation to attend various social events. Conclusion: Menstruation-related myths and practices are prevalent and require educational interventions. More focus needs to be given to male sterilization as the permanent method of contraception. The study underscores the need to address gender inequalities and attitudes among tribal populations and to increase efforts to promote higher education among the tribes for busting cultural myths and practices.

A low-molecular-mass aspartic protease inhibitor from a novel Penicillium sp.: implications in combating fungal infections.

A low-molecular-mass aspartic protease inhibitor was isolated from a novel Penicillium sp. The inhibitor was purified to homogeneity, as shown by reversed-phase HPLC and SDS-PAGE. The M(r) of the inhibitor was 1585 and the amino acid composition showed the presence of D, D, D, E, A, K, L, Y, H, I and W residues. The steady-state kinetic interactions of Aspergillus saitoi aspartic protease with the inhibitor revealed the reversible, competitive, time-dependent tight-binding nature of the inhibitor, with IC(50) and K(i) values of 1.8 and 0.85 µM, respectively. Fluorescence spectroscopy and circular dichroism analysis showed that inactivation of the enzyme was due to binding of the inhibitor to the active site. The inhibitor was found to inhibit mycelial growth and spore germination of Aspergillus fumigatus and Aspergillus niger in vitro with MIC values of 1.65 and 0.30 µg ml(-1), respectively. This study will potentially open the way towards the development of a tight-binding peptidic inhibitor against fungal aspartic proteases to combat human fungal infections.

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144 kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K (m) of 1.67 mg/ml. The enzyme was active at a broad range of pH (5-8) and temperatures (40-80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60 h with half-lives of 14 h, 6 h and 7 min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn(2+), K(+), cysteine, ß-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12 h at 60°C and 6 h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.

Improving the Quality of Transurethral Resection of Bladder Tumour (TURBT) Operative Notes Following the European Association of Urology Guidelines: A Completed Audit Loop Study.

Background The European Association of Urology (EAU) recommends that the operative steps and documentation necessary for successful and appropriate management of bladder cancer include identifying factors necessary to assign disease risk stratification, clinical stage, adequacy of resection and the presence of complications and immediate intravesical chemotherapy administration. Aim To assess and improve the adequacy of current transurethral resection of bladder tumour (TURBT) documentation at a district general hospital in the UK against the EAU 2022 guidelines. Methods Operative notes over a one-year period were assessed for the inclusion of key steps to achieve a comprehensive TURBT as outlined by EAU guidelines. Outcomes included documentation on the details of the operative findings and intervention as well as the perioperative assessment. A standardised template for TURBT procedures was created and surgical staff received training on its usage. The audit was subsequently repeated after six months to assess for improvements. Results TURBT documentation of 78 cases in the first cycle was compared to 37 cases from the second cycle. Significant improvements in the documentation of tumour size (46% to 89%; p<0.05), tumour description (59% to 89%; p <0.05), depth of resection (36% to 89%; p<0.05), administration of chemotherapy (21% to 46%; p<0.05) and assessment for perforation (22% to 68%; p=0.001) were demonstrated. Improvements in pre-operative and post-operative examination rates under anaesthesia also achieved statistical significance (47% & 14% respectively to 89%; p<0.05). There was an increase in the documentation of completeness of resection but this did not achieve statistical significance (59% to 68%; p=0.42). Conclusion The operative note template led to the improvement in the documentation, improving the risk stratification of bladder cancer in patients undergoing TURBT. The use of procedure-specific operative note templates should be adopted for all commonly performed procedures to improve the completeness of documentation.

Pattern of contraceptive use, determinants and fertility intentions among tribal women in Kerala, India: a cross-sectional study.

OBJECTIVES: To assess the pattern of contraceptive use and its determinants, knowledge regarding contraceptives including oral contraceptive pills and fertility intentions among tribal women in the reproductive age group. DESIGN: Community-based cross-sectional study. SETTING: Community development blocks in a predominantly tribal district of Wayanad in Kerala, India. PARTICIPANTS: Women in the reproductive age group (15-49 years) from the tribal groups in the district numbering 2495. OUTCOME MEASURES: PRIMARY: Prevalence of contraceptive use and its determinants. SECONDARY: Knowledge regarding contraceptives in general, oral contraceptives and fertility intentions. RESULTS: The mean age of the study participants was 30.8 years (SD=9.8) and belonged to various tribal groups such as Paniya (59.2%), Kurichiyar (13.6%) and Adiya (10.9%). Current use of contraceptive was reported by about a fourth, 658 (26.4%) (95% CI 27.9 to 24.9) of women. Following logistic regression, belonging to Paniya tribe (adjusted OR (aOR) 2.67, 95% CI 1.49 to 4.77; p<0.001) and age at menarche >13 years (aOR 1.69, 95% CI 1.14 to 2.52; p<0.009) had significantly higher use of contraceptives whereas social vulnerability as indicated by staying in a kutcha house had a lesser likelihood of use of contraceptive (aOR 0.55, 95% CI 0.31 to 0.95; p<0.03). Oral contraceptive use was low (4.8%) among this population and no abuse was observed.Less than half (47%) of the respondents had an above average knowledge on contraception. Multivariable logistic regression indicated that above average knowledge was 2.2 times more likely with higher education (95% CI 1.2 to 3.9), lesser among those who desired more than two children (aOR 0.59; 95% CI 0.38 to 0.94; p<0.02).Two children per family was the preferred choice for 1060 (42.5%) women. No gender bias in favour of the male child was observed. CONCLUSION: Awareness and use of contraceptives are poor though the fertility is not commensurately high. Along with developing targeted responses to contraceptive use among Indigenous people with indigenous data, awareness also requires attention. Ethnographic studies are also necessary to determine the differences in contraceptive use including traditional methods among the various Indigenous groups.

Response of the health system in Nipah outbreak in Ernakulam district: A qualitative analysis.

BACKGROUND: Nipah is an emerging zoonotic disease that is transmitted through contaminated food or directly between people. Recently, Nipah virus infection was confirmed in Kochi, Kerala, making it the fourth outbreak reported in India. However, due to its good epidemic response, the health system of Kerala was able to control it in a timely manner. OBJECTIVE: To qualitatively analyse the response of the health system of Kerala in controlling the Nipah outbreak (2019) by identifying the enabling factors and the challenges faced by it. METHODS: A qualitative study was conducted using grounded theory approach. Key informant interviews were conducted till data saturation was reached. The audio recorded data was translated, transcribed and was manually coded and thematically analysed. RESULTS: The major enablers for its apt response were identified to be effective communication, good line of control, effective division of work, intersectorial coordination, strong leadership, political commitment, resilient public private partnership and support groups, past experiences in disaster management, quick procurement of medicines and availability of lab facilities. On the other hand, the challenges identified were initial confusions, lack of standard operating procedures/guidelines for epidemic management, complacency, lack of effective zoonotic surveillance, media management and community participation. CONCLUSION: This study attributes the success of the outbreak response to the strong leadership and political commitment. In order to prevent recurrences in the future, there is a need to build a resilient health system through capacity building and further strengthening of the surveillance system.

Uncontrolled Blood Pressure and Associated Factors Among Persons With Diabetes: A Community Based Study From Kerala, India.

The coexistence of raised blood pressure (BP) in people with type 2 diabetes mellitus (T2DM) is a major contributor to the development and progression of both macrovascular and microvascular complications. The aim of our study was to determine the prevalence of uncontrolled BP and its associated factors in persons with T2DM in a district in Kerala. METHODS: The study was conducted in Ernakulam district in Kerala, and a total of 3,092 individuals with T2DM were enrolled after obtaining consent. Those with a BP "above or equal to 140 mmHg" and/or "above or equal to 90 mmHg" were thus considered to have uncontrolled BP. If the BP was equal or >140 and/or 90 mmHg, a repeat reading was taken after 30 min and the average of the two was considered. Basic demographic details were enquired along with electronic measurement of BP, HbA1c estimation and screening for diabetic retinopathy, peripheral arterial disease (PAD), and peripheral neuropathy. Quantitative and qualitative variables were expressed as mean (SD) and proportions, respectively. The model for determinants of uncontrolled BP was developed adjusting for age, gender, education, duration of diabetes, occupation, body mass index (BMI) and clustering effect. RESULTS: The mean age of the study population was 59.51 ± 9.84 years. The mean duration of T2DM was found to be 11.3 ± 6.64 years. The proportion of uncontrolled HTN adjusted for clustering was 60% (95% CI 58 and 62%). Among them, only one in two persons (53.3%) had a history of hypertension. Age >60 years [adjusted odds ratio (aOR) 1.48, 95% CI 1.24, 1.76; p < 0.001], unemployment (aOR 1.33, 95% CI 1.01, 1.75; p < 0.01), duration of diabetes > 11 years (aOR 1.42, 95% CI 1.19, 1.68; p < 0.001), and BMI ≥23 (aOR 1.33, 95% CI 1.10, 1.59; p < 0.002) were found to be independent determinants of high BP levels when adjusted for the aforementioned variables, gender, education, and cluster effect. The association between complications, such as peripheral neuropathy, PAD, and retinopathy showed a higher risk among those with uncontrolled BP. Retinopathy was 1.35 times more (95% CI 1.02, 1.7, p < 0.03), PAD was 1.6 times more (95% CI 1.2, 2.07, p < 0.001), and peripheral neuropathy was 1.5 (95% CI 1.14, 1.9, p < 0.003) times more compared to their counterparts. CONCLUSION: Target BP levels were far from being achieved in a good majority of the persons with T2DM. To reduce further macrovascular and microvascular events among people with T2DM, effective awareness and more stringent screening measures need to be employed in this population.

Glutaminase Inhibitors Induce Thiol-Mediated Oxidative Stress and Radiosensitization in Treatment-Resistant Cervical Cancers.

The purpose of this study was to determine if radiation (RT)-resistant cervical cancers are dependent upon glutamine metabolism driven by activation of the PI3K pathway and test whether PI3K pathway mutation predicts radiosensitization by inhibition of glutamine metabolism. Cervical cancer cell lines with and without PI3K pathway mutations, including SiHa and SiHa PTEN-/- cells engineered by CRISPR/Cas9, were used for mechanistic studies performed in vitro in the presence and absence of glutamine starvation and the glutaminase inhibitor, telaglenastat (CB-839). These studies included cell survival, proliferation, quantification of oxidative stress parameters, metabolic tracing with stable isotope-labeled substrates, metabolic rescue, and combination studies with L-buthionine sulfoximine (BSO), auranofin (AUR), and RT. In vivo studies of telaglenastat ± RT were performed using CaSki and SiHa xenografts grown in immune-compromised mice. PI3K-activated cervical cancer cells were selectively sensitive to glutamine deprivation through a mechanism that included thiol-mediated oxidative stress. Telaglenastat treatment decreased total glutathione pools, increased the percent glutathione disulfide, and caused clonogenic cell killing that was reversed by treatment with the thiol antioxidant, N-acetylcysteine. Telaglenastat also sensitized cells to killing by glutathione depletion with BSO, thioredoxin reductase inhibition with AUR, and RT. Glutamine-dependent PI3K-activated cervical cancer xenografts were sensitive to telaglenastat monotherapy, and telaglenastat selectively radiosensitized cervical cancer cells in vitro and in vivo These novel preclinical data support the utility of telaglenastat for glutamine-dependent radioresistant cervical cancers and demonstrate that PI3K pathway mutations may be used as a predictive biomarker for telaglenastat sensitivity.

Interactions of a low molecular weight inhibitor from Streptomyces sp. MBR04 with human cathepsin D: implications in mechanism of inactivation.

Cathepsin D, a lysosomal aspartic protease, is of potential interest as a target for drug design due to its implication in breast and ovarian cancer. The article reports a low molecular weight cathepsin D inhibitor from Streptomyces sp. MBR04. The Mr of the inhibitor was 1,078 Da as determined by MALDI-TOF, and the amino acid analysis showed the presence of Asp, Asp, Gly, Ala, Lys, Leu, Tyr, Trp residues. The steady-state kinetic interactions revealed reversible, competitive, slow-tight-binding nature of the inhibitor with an IC50 and K i values of 3.2 and 2.5 nM, respectively. The binding of the inhibitor with the enzyme and the subsequent conformational changes were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-54 residue. Based on the fluorescence and circular dichroism studies, we demonstrate that the inhibitor binds to the active site of cathepsin D and causes inactivation. All these kinetic, thermodynamic, and quenching studies suggest that the newly isolated peptidic inhibitor could be a potential scaffold to study and can be used to develop new potent therapeutic lead molecule for the development of drugs. The inhibitor will be significant as a potential lead molecule to target cathepsin D.

Inhibition of xyloglucanase from an alkalothermophilic Thermomonospora sp. by a peptidic aspartic protease inhibitor from Penicillium sp. VM24.

A bifunctional inhibitor from Penicillium sp VM24 causing inactivation of xyloglucanase from Thermomonospora sp and an aspartic protease from Aspergillus saitoi was identified. Steady state kinetics studies of xyloglucanase and the inhibitor revealed an irreversible, non-competitive, two-step inhibition mechanism with IC(50) and K(i) values of 780 and 500nM respectively. The interaction of o-phthalaldehyde (OPTA)-labeled xyloglucanase with the inhibitor revealed that the inhibitor binds to the active site of the enzyme. Far- and near-UV spectrophotometric analysis suggests that the conformational changes induced in xyloglucanase by the inhibitor may be due to irreversible denaturation of enzyme. The bifunctional inhibitor may have potential as a biocontrol agent for the protection of plants against phytopathogenic fungi.

Slow-tight binding inhibition of pepsin by an aspartic protease inhibitor from Streptomyces sp. MBR04.

The present article reports a low molecular weight aspartic protease inhibitor from a Streptomyces sp. MBR04 exhibiting a two-step inhibition mechanism against pepsin. The kinetic interactions revealed a reversible, competitive, slow-tight binding inhibition with an IC(50) and K(i) values of 4.5 nM and 4 nM respectively. The conformational changes induced upon inhibitor binding to pepsin was monitored by far and near UV analysis, demonstrated that the inhibitor binds to the active site and causes inactivation. Chemical modification of the inhibitor with WRK and TNBS abolished the antiproteolytic activity of the inhibitor.

Enzymatic hydrolysis and ethanol production using xyloglucanase and Debaromyces hansenii from tamarind kernel powder: galactoxyloglucan predominant hemicellulose.

The hydrolysis and ethanol production from tamarind kernel powder (TKP), a rich source of galactoxyloglucan (GXG) was investigated for the first time using xyloglucanase and thermotolerant Debaromyces hansenii. The acid hydrolysis of TKP with 2N H(2)SO(4) at 120 degrees C for 30 min yielded an overall saccharification of 94% based on the total available carbohydrate content and further fermentation at 40 degrees C with thermotolerant D hansenii produced an ethanol yield of 0.35 g/g. A maximum hydrolysis of 55 and 78% for GXG was obtained in 48 h at 50 degrees C using Thermomonospora xyloglucanase (TXy) and accellerase1000, respectively. The synergistic effect of beta-galactosidase and xyloglucanase was demonstrated by the exogenous addition of beta-galactosidase to TXy which improved the overall hydrolysis of GXG by 30%. The rate of hydrolysis of GXG with TXy and accellerase was increased by 15-20% in the presence of chemical surfactants (tween 80 and toluene) or protein additive (BSA). The fermentation of enzymatic hydrolysates of GXG by TXy and accellerase with free cells at 40 degrees C produced an ethanol yield of 0.39 and 0.41 g/g whereas with immobilized cells produced 0.45 and 0.43 g/g, respectively, with a theoretical conversion efficiencies of 78-88%. The immobilized yeast cells were reused six times at 40 degrees C with 100% fermentation efficiency.

Biocatalytic approach for the utilization of hemicellulose for ethanol production from agricultural residue using thermostable xylanase and thermotolerant yeast.

A hydrolysis of 62% and 50% for OSX (Oat spelt xylan) and WBH (Wheat bran hemicellulose) were obtained in 36h and 48h using Accellerase 1000 at 50 degrees C wherein thermostable xylanase from alkalothermophilic Thermomonospora sp. yielded 67% (OSX) in 3h and 58% (WBH) in 24h at 60 degrees C, favouring a reduction in process time and enzyme dosage. The rate of hydrolysis with thermostable xylanase was increased by 20% with the addition of nonionic surfactant tween 80 or biosurfactant sophorolipid. The simultaneous saccharification and fermentation (SSF) of OSX and WBH using thermostable xylanase and D. hansenii in batch cultures produced 9.1g/L and 9.5 g/L of ethanol, respectively and had a shorter overall process time than the separate hydrolysis and fermentation (SHF). The immobilized yeast cells in Ca-alginate matrix produced ethanol with a yield of 0.46 g/g from hemicellulosic hydrolysates and were reused six times with 100% fermentation efficiency.