Dementia Symptoms among Senior Citizens Living in Geriatric Homes of Kathmandu Valley.

Background With increasing age, the older population becomes more susceptible to mental disorders. It is important to recognize and develop an understanding of psychiatric morbidity particularly among the residents of geriatric homes in resource-poor settings. Objective To assess the prevalence and associated factors of dementia symptoms among Nepalese senior citizens living in geriatric homes of Kathmandu valley. Method A cross-sectional study was conducted among 304 senior citizens living in geriatric homes of Kathmandu valley. Cognitive Impairment Test (CIT), was used to assess dementia symptoms. Bivariate and multivariate logistic regressions were performed. All the variables that were significant at p < 0.05 level in the bivariate analysis were included in the multivariate regression model and statistical significance was declared at p < 0.05 with a 95.00% confidence interval (CI). Result This study showed 75.65%, of the participants, had dementia symptoms. In the multivariate logistic regression analysis, female respondents (AOR=2.94, 95% CI=1.31-6.57), respondents never received geriatric allowances (AOR=2.46, 95% CI=1.22-4.98), respondent's history of alcohol consumption habits (AOR=2.04, 95% CI=1.01-4.11) and non-vegetarian diet habits (AOR= 2.31, 95% CI=1.12-4.76) were found more likely to had higher dementia symptoms whereas, literate participants (AOR=0.19, 95% CI=0.08-0.43) were less likely to had dementia symptoms. Conclusion The high prevalence of dementia symptoms among senior citizens living in geriatric homes in the Kathmandu valley indicates an urgent need for early diagnosis and treatment of mental disorders among senior citizens to improve their quality of life and well-being.

[Involvement of intestinal dysbiosis in the etiopathogenesis and treatment of autism spectrum disorder: a bibliographic review]. / Implicación de la disbiosis intestinal en la etiopatogenia y el tratamiento del trastorno del espectro autista: una revisión bibliográfica.

INTRODUCTION: Autism spectrum disorder is a neurodevelopmental disorder with phenotypic heterogeneity and variable symptomatic course of partly unknown etiology. The prevalence of gastrointestinal disorders in autism leads to investigate the role that intestinal microbiota may have as a causal factor and to propose specific therapeutic interventions. The role of microbiota in brain development and function, demonstrated in animal models, justifies its investigation in this neuropsychiatric disorder. OBJECTIVE: The aim was to investigate the relationship between altered microbiota composition and autism spectrum disorder, and to assess the therapeutic role of prebiotics, probiotics and fecal transplantation in this neurodevelopmental disorder. DEVELOPMENT: A literature review was conducted in PubMed, Cochrane Library and Google Scholar to select relevant articles related to the topic that were published between January 2012 and April 2020. Thirty-five relevant articles were selected. In 23 of them, significant differences were found in the composition and diversity of the microbiota in children with ASD, as well as in the biomolecules involved in certain metabolic pathways. The other 12 investigations reported gastrointestinal and behavioral improvements after therapeutic intervention. CONCLUSIONS: It is reasonable to state that there is enough evidence to support the existence of a relationship between intestinal microbiota and autism spectrum disorders. This fact should be explored in depth to assess the etiopathogenic burden of dysbiosis and the possible therapeutic tools.

TITLE: Implicación de la disbiosis intestinal en la etiopatogenia y el tratamiento del trastorno del espectro autista: una revisión bibliográfica.Introducción. El trastorno del espectro autista es un trastorno del neurodesarrollo con heterogeneidad fenotípica y curso sintomático variable de etiología parcialmente desconocida. La prevalencia de trastornos gastrointestinales en este perfil de pacientes invita a investigar el papel que la microbiota intestinal puede tener como factor causal y a plantear intervenciones terapéuticas específicas. El papel de la microbiota en el desarrollo y la función cerebral, demostrado en modelos animales, justifica su investigación en este trastorno neuropsiquiátrico. Objetivo. Investigar la relación entre la alteración en la composición de la microbiota y el trastorno del espectro autista, y evaluar el papel terapéutico de prebióticos, probióticos y trasplante fecal en este trastorno del neurodesarrollo. Desarrollo. Se realizó una revisión bibliográfica en PubMed, Cochrane Library y Google Scholar con el fin de seleccionar los artículos relevantes relacionados con el tema que se publicaron entre enero de 2012 y abril de 2020. Se seleccionaron 35 artículos relevantes. En 23 de ellos se encontraron diferencias significativas en la composición y la diversidad de la microbiota en niños con TEA, así como en biomoléculas involucradas en determinadas rutas metabólicas. Las otras 12 investigaciones describieron mejorías gastrointestinales y comportamentales tras la intervención terapéutica. Conclusiones. Resulta razonable afirmar que existe evidencia suficiente para apoyar la existencia de una relación entre la microbiota intestinal y los trastornos del espectro autista. Esta vinculación ha de ser explorada en profundidad para perfilar el peso etiopatogénico de la disbiosis y las posibles herramientas terapéuticas.

Implicación de la disbiosis intestinal en la etiopatogenia y el tratamiento del trastorno del espectro autista: una revisión bibliográfica / Involvement of intestinal dysbiosis in the etiopathogenesis and treatment of autism spectrum disorder: a bibliographic review

Introducción: El trastorno del espectro autista es un trastorno del neurodesarrollo con heterogeneidad fenotípica y curso sintomático variable de etiología parcialmente desconocida. La prevalencia de trastornos gastrointestinales en este perfil de pacientes invita a investigar el papel que la microbiota intestinal puede tener como factor causal y a plantear intervenciones terapéuticas específicas. El papel de la microbiota en el desarrollo y la función cerebral, demostrado en modelos animales, justifica su investigación en este trastorno neuropsiquiátrico. Objetivo: Investigar la relación entre la alteración en la composición de la microbiota y el trastorno del espectro autista, y evaluar el papel terapéutico de prebióticos, probióticos y trasplante fecal en este trastorno del neurodesarrollo. Desarrollo: Se realizó una revisión bibliográfica en PubMed, Cochrane Library y Google Scholar con el fin de seleccionar los artículos relevantes relacionados con el tema que se publicaron entre enero de 2012 y abril de 2020. Se seleccionaron 35 artículos relevantes. En 23 de ellos se encontraron diferencias significativas en la composición y la diversidad de la microbiota en niños con TEA, así como en biomoléculas involucradas en determinadas rutas metabólicas. Las otras 12 investigaciones describieron mejorías gastrointestinales y comportamentales tras la intervención terapéutica. Conclusiones:Resulta razonable afirmar que existe evidencia suficiente para apoyar la existencia de una relación entre la microbiota intestinal y los trastornos del espectro autista. Esta vinculación ha de ser explorada en profundidad para perfilar el peso etiopatogénico de la disbiosis y las posibles herramientas terapéuticas.(AU)

Introduction: Autism spectrum disorder is a neurodevelopmental disorder with phenotypic heterogeneity and variable symptomatic course of partly unknown etiology. The prevalence of gastrointestinal disorders in autism leads to investigate the role that intestinal microbiota may have as a causal factor and to propose specific therapeutic interventions. The role of microbiota in brain development and function, demonstrated in animal models, justifies its investigation in this neuropsychiatric disorder. Objective: The aim was to investigate the relationship between altered microbiota composition and autism spectrum disorder, and to assess the therapeutic role of prebiotics, probiotics and fecal transplantation in this neurodevelopmental disorder. Development: A literature review was conducted in PubMed, Cochrane Library and Google Scholar to select relevant articles related to the topic that were published between January 2012 and April 2020. Thirty-five relevant articles were selected. In 23 of them, significant differences were found in the composition and diversity of the microbiota in children with ASD, as well as in the biomolecules involved in certain metabolic pathways. The other 12 investigations reported gastrointestinal and behavioral improvements after therapeutic intervention. Conclusions: It is reasonable to state that there is enough evidence to support the existence of a relationship between intestinal microbiota and autism spectrum disorders. This fact should be explored in depth to assess the etiopathogenic burden of dysbiosis and the possible therapeutic tools.(AU)

Estimating Risk of Breast Cancer Occurrences at Different Ages: Application of Survival Techniques

Background: Awareness is the primary means to control breast cancer occurrence. The purpose of the present work is to study the risk of breast cancer occurrence in different age group, for the study area, Assam, India, by means of survival analysis techniques. Methods: Survival and hazard functions are key concepts in survival analysis for describing the distribution of event times. In the present research a new individialized model has been proposed for cumulative hazard function, taking gamma probability distribution as probability distribution of breast cancer occurrences. Kaplan Meier Survival method has been applied to find out the probability of diseases occurrence in the early menarche and late menarche group. The data used for implementation were collected from the Record Department of a prime local cancer institute, for the period 2010-2012. The information for the risk factor age at menarche were collected from the patients registered during August 2011 to February 2012. Results: The study reveals that in the study area, cumulative hazard of the women belonging to 35 to 50 years is higher than the early and late aged women. The cumulative hazard plot with shape parameter 0.5, 1 and 10 shows that cumulative risk for early aged women are greater than the late age women but when this values is increased from 10, the opposite trend is observed. Further, the median age of disease occurrence among early menarche group is 52 years and for late menarche it is 54 years. Conclusion: The model developed could successfully point out the age group for women lying at higher risk of breast cancer occurrence. Additionally the important risk factor, age at menarche, was effectively applied to supplement to this calculation. It is hoped that practical use of this method would enhance not only awareness but also early detection of the said disease.

Optimal Timing of Pulmonary Banding for Newborns with Single Ventricle Physiology and Unrestricted Pulmonary Blood Flow.

The aim of this study was to determine the optimal timing of pulmonary artery band (PAB) placement in neonates with single ventricle physiology, unrestricted pulmonary blood flow, and no systemic outflow tract obstruction. Retrospective chart review of all patients who underwent isolated PAB for single ventricle physiology between January 2005 and December 2014 was carried out. The influence of age at the time of PAB on operative mortality, the need for reoperation to adjust the PAB, the preparedness of the pulmonary vascular bed prior to the second-stage bidirectional cavopulmonary shunt (BCPS), and the outcomes following BCPS were studied. The study cohort included 54 subjects (34 males). The median age at the time of PAB was 18 days. The overall mortality following PAB was 4 % (2/54). Reoperation for PAB adjustment was 7 % (4/54). Younger age at the time of PAB was not associated with mortality or increased risk of reoperation. There was a mild positive correlation between the age at PAB and the mean pulmonary artery pressure prior to BCPS. There was also a weak positive correlation between the age at PAB and the duration of ventilation following BCPS. Age at the time of PAB did not influence pulmonary vascular resistance (PVR) prior to BCPS or the mortality and hospital stay following BCPS. PAB can be done safely and effectively soon after birth in neonates with single ventricle physiology, increased pulmonary blood flow, and no potential or actual systemic outflow tract obstruction. It may not be necessary to wait for a few weeks after birth for the neonatal PVR to fall before placing a PAB.

A matched case control study of risk indicators of breast cancer in assam, India.

Breast cancer is the leading cause of cancer death among women worldwide especially in a developing country like India. It also occupies the highest place with relative proportion 17.5% in the Hospital Based Cancer Registry in progress in the Dr. B. Borooah Cancer Research Institute. Dr. B. Borooah Cancer Research Institute is the Regional Cancer Care Center for entire North East region of India. With this background a matched case control study of 100 cases of breast cancer and 100 controls was carried out to investigate the role of different Socio economic, Female Reproductive and Life style related factors and to understand the etiology of breast cancer in Assam. Controls are matched to the cases by age at diagnosis (±5 years), family income and place of residence with matching ratio 1:1. Data were collected using questionnaire and then conditional logistic regression analysis is used to estimate the odd ratios for several factors. Study revealed that breast cancer occurrence has statistical association with the factors chewing habits (p=0.003), number of children (p=0.080), age at marriage (p=0.014), age at first child birth (p=0.007), age at menarche (p=0.010).

Negative pressure wound therapy for sternal wound infections following congenital heart surgery.

OBJECTIVE: This study examines the efficacy of a comprehensive, multidisciplinary wound management team and negative pressure wound therapy (NPWT) for the treatment of sternal wound infections in congenital heart surgery patients. METHOD: A single-institution retrospective review of all congenital heart surgery patients with post-operative sternal wound infections who were treated with NPWT was performed. Patients were evaluated based on (a) whether NPWT occurred before or after the establishment of a multidisciplinary wound management team, and (b) whether NPWT was initiated early (within 2 days) or late (greater than 2 days) after diagnosis of a sternal wound infection. RESULTS: The median duration of NPWT was 12 days (range 2-50 days). NPWT was successfully initiated in patients as young as 15 days of age. There was a trend toward shorter duration of both NPWT and antibiotic use following (a) the implementation of the multidisciplinary wound management team, and (b) in patients with early use of NPWT; however, these results did not achieve statistical significance. CONCLUSION: NPWT can be successfully utilised in congenital heart surgery patients, including young neonates, for the treatment of sternal wound infections. The trends observed in the reduction of wound therapy duration and antibiotic duration with early implementation of negative pressure therapy and multidisciplinary wound management require further investigation to verify their clinical efficacy in patient care.

Effect of Methanolic Leaf Extract of Ocimum basilicum L. on Benzene-Induced Hematotoxicity in Mice.

The aim of the present study was to investigate the protective role of methanolic leaf extract of Ocimum basilicum L. against benzene-induced hematotoxicity in Swiss albino mice. GC analysis and subacute toxicity level of the extract were tested. Mice were randomly divided into three groups among which II and III were exposed to benzene vapour at a dose 300 ppm × 6 hr/day × 5 days/week for 2 weeks and group I was control. Group III of this experiment was treated with the leaf methanolic extract at a dose of 100 mg/kg body weight, a dose in nontoxic range. Hematological parameters (Hb%, RBC and WBC counts), cell cycle regulatory proteins expression and DNA fragmentation analysis of bone marrow cells was performed. There was an upregulation of p53 and p21 and downregulation of levels of CDK2, CDK4, CDK6, and cyclins D1 and E in leaf extract-treated group. DNA was less fragmented in group III compared to group II (P < 0.05). The present study indicates that the secondary metabolites of O. basilicum L. methanolic leaf extract, comprising essential oil monoterpene geraniol and its oxidized form citral as major constituents, have modulatory effect in cell cycle deregulation and hematological abnormalities induced by benzene in mice.

Characterization of HCV-specific CD4+Th17 immunity in recurrent hepatitis C-induced liver allograft fibrosis.

Hepatitis C virus (HCV) recurrence with accelerated fibrosis following orthotopic liver transplantation (OLT) is a universal phenomenon. To evaluate mechanisms contributing to HCV induced allograft fibrosis/cirrhosis, we investigated HCV-specific CD4+Th17 cells and their induction in OLT recipients with recurrence utilizing 51 HCV+ OLT recipients, 15 healthy controls and 9 HCV- OLT recipients. Frequency of HCV specific CD4+ Tcells secreting IFN-γ, IL-17 and IL-10 was analyzed by ELISpot. Serum cytokines and chemokines were analyzed by LUMINEX. Recipients with recurrent HCV induced allograft inflammation and fibrosis/cirrhosis demonstrated a significant increase in frequency of HCV specific CD4+Th17 cells. Increased pro-inflammatory mediators (IL-17, IL-1ß, IL-6, IL-8 and MCP-1), decreased IFN-γ, and increased IL-4, IL-5 and IL-10 levels were identified. OLT recipients with allograft inflammation and fibrosis/cirrhosis demonstrated increased frequency of Foxp3+ regulatory T cells (Tregs) that inhibited HCV specific CD4+Th1 but not Th17 cells. This suggests that recurrent HCV infection in OLT recipients induces an inflammatory milieu characterized by increased IL-6, IL-1ß and decreased IFN-γ which facilitates induction of HCV specific CD4+Th17 cells. These cells are resistant to suppression by Tregs and may mediate an inflammatory cascade leading to cirrhosis in OLT recipients following HCV recurrence.

Effect of starter culture and turmeric on physico-chemical quality of carabeef pastirma.

Carabeef samples were sliced, pressed, cured and divided into 6 groups. Starter cultures (Micrococcus varians M483 (MV), Staphylococcus carnosus (SC), Lactobacillus sakei (LS), M. varians M483+ Lb. sakei and Staph. carnosus + Lb. sakei) were inoculated at the dose of 10(6)-0(7)cfu/g and stored at 10 ± 1°C for 7 days. Uninoculated samples were maintained as control. Samples were then divided into 2 treatment groups. Samples of treatment 1 (T1) were smeared with a paste of turmeric followed by application of a thick layer of the paste of garlic, cumin, black pepper and red pepper whereas, samples of treatment 2 (T2) were applied with a thick layer of spices as above without turmeric. With the gradual fall in pH there was a reduction in water-holding capacity (WHC) of samples. The WHC of samples treated with SC+LS of T1 reduced to 6.3 ± 0.03 cm(2) and those inoculated with MV+LS of T2 to 6.2 ± 0.03 cm(2). The extract release volume (ERV) increased in all samples during storage. The least ERV of 11.7 and 11.6 ml were recorded in samples inoculated with MV of T1 and T2, respectively. The tyrosine (TV) and thiobarbituric acid (TBA) number of turmeric treated samples were significantly lower than non turmeric treated samples. The samples inoculated with LS had the least TV of 30.9 mg tyrosine/100 g of meat and TBA number of 0.06 mg manoladehyde/kg of meat. Samples inoculated with MV and LS of both T1 and T2 were better in physico-chemical qualities.

Impact of donor and recipient factors on allograft survival in lung transplantation: A single-center analysis.

BACKGROUND: It remains unclear which donor and recipient factors influence long-term allograft function in lung transplantation (LTx). METHODS: From October 1988 to February 2005, a total of 280 recipients underwent LTx at our center. Donor data and cause of death (CoD) were analyzed. The CoD was categorized according to rate of increase in intracranial pressure at the time of death. Each donor and recipient factor was correlated with long-term graft function. Recipient details, type of transplant, indication for transplant, and time on waiting list were analyzed. Recipients were stratified based on allograft ischemia time (AIT): 0 to 6, 6 to 8, 8 to 10, and >10 hours. RESULTS: Mean donor age was 30.9 years (36.7% male); 49.8% were cytomegalovirus (CMV) positive. Donor CoD was characterized by a slow rise in intracranial pressure (ICP) in 34.4%, rapid ICP in 18.7%, an intermediate ICP in 44.3%, and with no rise in 2.6%. A graft survival benefit was seen with female donors (P = .048); 34.4% of recipients ultimately developed graft failure at long term follow-up. Mean recipient age was 48 years; 63% were male and mean body-mass index (BMI) was 23.6; 60.2% had single lung transplantation, and mean wait list time was 323 days. Mean AIT totaled 421 minutes. Graft survival was longer with AIT of 8 to 10 hours compared to 6 to 8 hours (P = .03). CONCLUSIONS: Donor factor analysis implied only female donor status conferred a long-term graft survival advantage. Intracranial pressure rise differences appear clinically unimportant. Prolonged cold ischemic time (>10 hours) or low recipient BMI did not adversely affect allograft function in our review.

Photoinduced intramolecular charge transfer reaction in (E)-3-(4-Methylamino-phenyl)-acrylic acid methyl ester: A fluorescence study in combination with TDDFT calculation.

A donor-acceptor substituted aromatic system (E)-3-(4-Methylamino-phenyl)-acrylic acid methyl ester (MAPAME) has been synthesized, and its photophysical behavior obtained spectroscopically has been compared with the theoretical results. The observed dual fluorescence from MAPAME has been assigned to emission from locally excited and twisted intramolecular charge transfer states. The donor and acceptor angular dependency on the ground and excited states potential energy surfaces have been calculated both in vacuo and in acetonitrile solvent using time dependent density functional theory (TDDFT) and TDDFT polarized continuum model (TDDFT-PCM), respectively. Calculation predicts that a stabilized twisted excited state is responsible for red shifted charge transfer emission.

Validation of the proposed International Society for Heart and Lung Transplantation grading system for primary graft dysfunction after lung transplantation.

BACKGROUND: A scoring system was recently proposed to grade the severity of primary graft dysfunction (PGD), a frequent early complication of lung transplantation. The purposes of this study are to: (1) validate the PGD grading system with respect to patient outcomes; and (2) compare the performance of criteria employing the arterial oxygenation to fraction of inspired oxygen (P/F) ratio to an alternative grading system employing the oxygenation index (OI). METHODS: We retrospectively reviewed the medical records of 402 patients having undergone lung transplantation at our institution from 1992 through 2004. The ISHLT PGD grading system was modified and grades were assigned up to 48 hours post-transplantation as follows: Grade 1 PGD, P/F > 300; Grade 2, P/F 200 to 300; and Grade 3, P/F < 200. A worst score T(0-48) was also assigned, which reflects the highest grade recorded between T0 and T48. RESULTS: The prevalence of severe PGD (P/F Grade 3) declined after transplant, from 25% at T0 to 15% at T48. Grouping patients by P/F grade at T48 demonstrated the clearest differentiation of 90-day death rates (Grade 1, 7%; Grade 2, 12%; Grade 3, 33%) (p = 0.0001). T48 OI grade also differentiates 90-day death rates. There was no difference in longer-term survival between patients with PGD Grades 1 and 2. OI grade at T0 qualitatively improved differential mortality between Grades 1 and 2; however, the differences did not reach statistical significance. Patients with a worst score T(0-48) of Grade 3 PGD did have significantly decreased long-term survival, as well as longer ICU and hospital stay, when compared with Grades 1 and 2 PGD. Significant risk factors for short- and long-term mortality in our multivariate model were P/F Grade 3 [worst score T(0-48) as well as T0 grade], single-lung transplant, use of cardiopulmonary bypass and high pre-operative mean pulmonary artery pressure. CONCLUSIONS: There is an increased risk of short- and long-term mortality and length of hospital stay associated with severe (Grade 3) PGD. The proposed ISHLT grading system can rapidly identify patients with poor outcomes who may benefit from early, aggressive treatment. Refinement of the scoring system may further improve patient risk stratification.

Fungal infections in transplant recipients receiving alemtuzumab.

Recently, we have used an anti-T-cell agent, alemtuzumab, as induction or conversion therapy to achieve a calcineurin (CNI) and steroid-free immunosuppressive regimen. We identified recipients who developed systemic fungal infections after the initiation of alemtuzumab and looked at their outcomes. The study population consisted of all pancreas transplant recipients who received alemtuzumab. Only invasive fungal infections were included in the analysis (eg, fungemia, meningitis, or pneumonia; fungal urinary tract infections were excluded). The organism was confirmed by culture, histopathology, or latex antigen test. Between February 2003 and February 2004, a total of 121 pancreas transplant recipients received alemtuzumab-56 as part of induction, and 65 as part of conversion. Of these, 8 (6.6%) developed an invasive fungal infection; 2 (3.6%) recipients as part of induction therapy and 6 (9.2%) as part of conversion therapy. Mean recipient age was 42.1 years. The mean length of time from alemtuzumab administration (first dose) to the diagnosis of the fungal infection was 115.9 days (range 5 to 318). The organisms identified initially were: Cryptococcus, Histoplasma, Aspergillus, and Candida. Overall, 3 (38%) of the eight patients died during ongoing treatment of their fungal infection: two from sepsis, one due to myocardial infarction. The other five recipients were treated successfully and have functioning grafts. The initial therapeutic agents used included: amphotericin B/liposomal AMB (n = 6), voriconazole (n = 3), capsofungin (n = 2), and fluconazole (n = 1). The use of alemtuzumab as induction or conversion therapy in pancreas transplant recipients may predispose patients to the development of systemic fungal infections. It would be important to determine what the most appropriate prophylaxis regimen would be for these patients.

An autosomal recessive syndrome of choanal atresia, hypothelia/athelia and thyroid gland anomalies overlapping bamforth syndrome, ANOTHER syndrome and methimazole embryopathy.

Two sibs from an inbred Arab family are described with an autosomal syndrome of choanal atresia, hypothelia/athelia and thyroid gland anomalies overlapping Bamforth syndrome, ANOTHER syndrome and methimazole embryopathy. In one case the syndrome described was lethal. Cases with similar features are reviewed and genetic mutations discussed.

Acid-induced partly folded conformation resembling a molten globule state of xylanase from an alkalothermophilic Bacillus sp.

Nonnative protein structures having a compact secondary, but not rigid tertiary structure, have been increasingly observed as intermediate states in protein folding. We have shown for the first time during acid-induced unfolding of xylanase (Xyl II) the presence of a partially structured intermediate form resembling a molten globule state. The conformation and stability of Xyl II at acidic pH was investigated by equilibrium unfolding methods. Using intrinsic fluorescence and CD spectroscopic studies, we have established that Xyl II at pH 1.8 (A-state) retains the helical secondary structure of the native protein at pH 7.0, while the tertiary interactions are much weaker. At variance, from the native species (N-state), Xyl II in the A-state binds 1-anilino-8-sulfonic acid (ANS) indicating a considerable exposure of aromatic side chains. Lower concentration of Gdn HCl are required to unfold the A-state. For denaturation by Gdn HCl, the midpoint of the cooperative unfolding transition measured by fluorescence for the N-state is 3.5 +/- 0.1 M, which is higher than the value (2.2 +/- 0.1 M) observed for the A-state at pH 1.8. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its structural stability that is characteristic for native proteins.

Artificial chaperone mediated refolding of xylanase from an alkalophilic thermophilic Bacillus sp. Implications for in vitro protein renaturation via a folding intermediate.

To gain insight into the molecular aspects of unfolding/refolding of enzymes from extremophilic organisms, we have used xylanase from an alkalophilic thermophilic Bacillus as the model system. Kinetics of denaturation/renaturation were monitored using intrinsic fluorescence studies. The protein fluorescence measurements suggested a putative intermediate state present in 0.08 M guanidine hydrochloride with an emission maximum of 345 nm; the far-UV circular dichroism spectra revealed content of secondary structure similar to the native enzyme. Studies with the fluorescent apolar probe 1-anilinonapthalene-8-sulfonate (1,8-ANS) were consistent with the presence of increased hydrophobic surfaces as compared with the native or fully unfolded protein. The refolding of Xyl II, was attempted by a relatively new strategy using an artificial chaperone assisted two-step method. The unfolded xylanase was found to bind to the detergent transiently and the subsequent addition of methyl-beta-cyclodextrin helped to strip the detergent and assist in the folding. Our findings suggested that the detergent stabilized a putative intermediate in the folding pathway seemingly equivalent to the folding state described as molten globule. The reactivation of Xyl II was affected by ionic as well as nonionic detergents. However, the cationic detergent cetyltrimethylammonium bromide (CTAB) provided a maximum reactivation (threefold) of the enzyme. The 'delayed detergent addition' experiments revealed that the detergent acts by suppressing the initial aggregate formation and not by dissolving aggregates. The relevance of our findings to the role of artificial chaperones in vivo is discussed.