Side chain inset of neurogenerative amino acids to metalloproteins: a therapeutic signature for huntingtin protein in Huntington's disease.

OBJECTIVE: Huntington's disease is a dominant autosomal inherited neurodegenerative disease that results in progressive impairment, characterized by dementia, chorea, and behavioral and cognitive decline. The objective of this study was to investigate the potential activity of metalloproteins against the huntingtin protein using various insertion-based engineering computational methods. Metalloproteins, metal protein complexes involved in important biochemical and physiological processes, were explored as potential drug candidates for Huntington's disease. MATERIALS AND METHODS: A total of 18 metalloproteins were selected as drug candidates and studied to assess their potential inhibitory effects on the huntingtin protein. The screening process was based on the lowest binding energy. The metalloprotein with the lowest docking score was chosen for side chain insertion of neurogenerative amino acids. The engineered metalloprotein was then evaluated based on physiochemical properties, allergenicity, toxicity, and surface accessibility. Cloning and expression analysis was performed to further investigate its potential as a therapeutic agent. RESULTS: The metalloprotein chosen for side chain insertion, cytochrome C oxidase, showed promising results. It was computed as a probable non-allergen and exhibited no toxic domains, indicating its non-toxic nature. Additionally, it demonstrated a strong binding affinity with the huntingtin protein, with a binding energy of -1,253.3 Kcal/mol. CONCLUSIONS: Metal-based proteins, when engineered with additional neurogenerative amino acids, hold potential as drug candidates for treating neurodegenerative diseases such as Huntington's disease. The successful development of these engineered metalloproteins could offer therapeutic advantages. Further testing, both in vitro and in vivo, is necessary to evaluate their efficacy and validate their potential activity as novel drugs for the treatment of neurodegenerative diseases.

Imidazopyridine, a promising scaffold with potential medicinal applications and structural activity relationship (SAR): recent advances.

Imidazopyridine scaffold has gained tremendous importance over the past few decades. Imidazopyridines have been expeditiously used for the rationale design and development of novel synthetic analogs for various therapeutic disorders. A wide variety of imidazopyridine derivatives have been developed as potential anti-cancer, anti-diabetic, anti-tubercular, anti-microbial, anti-viral, anti-inflammatory, central nervous system (CNS) agents besides other chemotherapeutic agents. Imidazopyridine heterocyclic system acts as a key pharmacophore motif for the identification and optimization of lead structures to increase medicinal chemistry toolbox. The present review highlights the medicinal significances of imidazopyridines for their rationale development as lead molecules with improved therapeutic efficacies. This review further emphasis on the structure-activity relationships (SARs) of the various designed imidazopyridines to establish a relationship between the key structural features versus the biological activities.Communicated by Ramaswamy H. Sarma.

Human Fascioliasis: Report of Two Cases from Kashmir Valley.

Although few case reports of human fascioliasis have been reported from different parts of India, there is no case reported from the Kashmir valley to date. Herein we report two cases of human fascioliasis. Both patients presented with fever, marked eosinophilia, and liver lesions on imaging. Hepatobiliary imaging showed vague features like mild biliary dilatation and liver lesions representing burrows. A liver biopsy in one of the patients revealed eosinophilic granuloma. Both patients were diagnosed definitively with endoscopic retrograde cholangiopancreatography (ERCP) by demonstrating live adult fasciola worms. Any patient presenting with fever, marked eosinophilia, and liver lesions on imaging should be evaluated for fascioliasis.

Treatment of Chronic Hepatitis C Genotype 3 With Ledipasvir and Sofosbuvir: An Observational Study.

OBJECTIVE: Sofosbuvir/ledipasvir (SOF/LED) is recommended for treatment of genotypes 1, 4, 5 and 6. Despite some preliminary data from the ELECTRON-2 trial regarding use of SOF/LED combination in chronic hepatitis C genotype 3, there are no guidelines recommending this combination in such patients. We conducted this study to evaluate the efficacy of the overall sustained virologic response at 12 weeks (SVR 12) and safety of SOF/LED in chronic hepatitis C genotype 3 infection in our population. METHODS: It was a prospective, hospital-based observational study. All patients with chronic hepatitis C genotype 3 treated with SOF/LED were divided into two groups: patients with cirrhosis and without cirrhosis. Patients without cirrhosis received SOF/LED (90/400 mg) for 12 weeks; however, patients with cirrhosis received treatment for 24 weeks. RESULTS: We enrolled 104 patients with chronic hepatitis C over a period of 24 months. Of the total, 66 were women (63.5%) and 38 were men (36.5%). The average age was 40 years (range: 18-76 years). Of 104 patients, 86 (82.7%) were of genotype 3, 15 (14.9%) were of genotype 1 and 3 (2.9%) were of genotype 4. Ninety-two (88%) were noncirrhotic and 12 (11.5%) were cirrhotic. Ninety-five (95.2%) were treatment naïve. Among genotype 1 and 4, all patients achieved rapid virologic response and SVR 12. Of 86 genotype 3 patients, 78 (90.6%) were noncirrhotic and 8 (9.3%) were cirrhotic. Among genotype 3 patients without cirrhosis, 75 (96%) achieved SVR 12 while 6 (75%) with cirrhosis achieved SVR 12. All patients tolerated the combination well; however, some patients experienced nausea (26%), headache (25%) and fatigue (21%). No patient had to discontinue therapy due to adverse drug reactions. CONCLUSIONS: Single tablet LED and SOF combination is safe and effective in genotype 3 patients without cirrhosis even without ribavirin. Being effective in genotype 3, the combination can be used as a pangenotypic drug in patients without cirrhosis.

Clinical Profile and Efficacy of Antivirals in Hepatitis B Virus Reactivation, in Patients With Cancer Receiving Chemotherapy.

BACKGROUND/PURPOSE: Hepatitis B virus reactivation (HBVR) is common in patients withcancer. The aim of the present study was to find out clinical profile of patients with cancer receiving chemotherapy with HBVR and to study the efficacy of entecavir (ETV) and tenofovir in the treatment of HBVR. METHODS: This is a prospective study in which all consecutive patients with cancer with evidence of HBVR were included. HBVR was defined as: New onset transaminitis with alanine aminotransferase (ALT) >3 times upper limit of normal and >10 fold increase in HBV DNA levels from baseline levels or detection of HBV DNA ≥100,000 IU/ml in patients with no baseline HBV DNA. Patients with HBVR were put on ETV or tenofovir and were closely monitored for efficacy and safety for minimum of 1 year. RESULTS: Of 204 Hepatitis B surface antigen (HBsAg)-positive patients with different cancers, 92 met the inclusion criteria. Of 92, 46 received ETV 0.5 mg/day and 46 received tenofovir disoproxil fumarate (TDF) 300 mg/day. At 6 months, there was 4.7 log reduction in HBV DNA level in the ETV group and 5.2 log reduction in the TDF group (P = 0.029). Proportion of patients with undetectable HBV DNA (75.7% vs 87.5%), ALT normalization (89.2% Vs 87.5%), HBsAg negativity (25% vs 28.1%), and seroconversion (2.8% vs 3.1%) at 1 year were almost similar in both groups with P value > 0.05 for all efficacy end points. There was no HBVR-related mortality in any group. CONCLUSION: Both ETV and tenofovir are very effective in the treatment of HBVR and reduce the liver-related mortality and morbidity in such patients.

Entecavir is Safe and Effective in Long Term for the Treatment of Hepatitis B in Immunocompromised Children.

OBJECTIVE: Hepatitis B infection is common in patients with cancer, and prompt treatment is necessary; otherwise, it can result in life-threatening complications. The objective of this study was to assess the long-term safety and efficacy of entecavir in immunocompromised children with hepatitis B. METHODS: This single-center prospective study was conducted on children with different malignancies referred to our department with evidence of hepatitis B infection. Only those children were included in the study who had HBsAg positive and alanine aminotransferase (ALT) more than 2 times the upper limit of normal and whose hepatitis B virus (HBV) DNA was more than 20,000IU/ml. These children were put on entecavir and prospectively observed upto 192 weeks. Primary efficacy end point was the proportion of patients who achieved undetectable HBV DNA at 48 weeks of treatment. Other efficacy end points were the proportion of patients with HBeAg seroconversion, undetectable HBV DNA, and ALT normalization at weeks 48 and 96 weeks. RESULTS: A total of 41 children met the inclusion criteria, of which 5 children died because of malignancy and 5 were lost to follow-up. Mean log DNA was 7.67 at the start which after starting entecavir reduced to 4.1, 2.8, 1.19, 1.09, and 0.84 at 12, 24, 48, 72, and 96 weeks, respectively (P value < 0.0001). Mean ALT decreased from 332.5 which reduced to 190, 115, 63, and 46 at 4, 12, 24, and 48 weeks, respectively (P < 0.0001). 67.7% achieved the primary outcome and had undetectable DNA at 48 weeks which increased to 26 (83.9%) at 96 weeks. At 48 weeks, 80.6% patients achieved ALT normalization. Thirty percent developed HBeAg seroconversion. Two patients developed virological breakthrough, one at 96 weeks and another at 192 weeks. No significant adverse effects were observed. CONCLUSION: Entecavir is safe and effective in long term for the treatment of hepatitis B in immunocompromised children.

Genotype 1 and 3 Response to Velpatasvir and Sofosbuvir in Chronic Hepatitis C in the Kashmiri Population: An Observational Study.

BACKGROUND: Our data is one of the earliest study from the Indian subcontinent on Velpatasvir/Sofosbuvir (VEL/SOF) combination in chronic hepatitis C (CHC). The primary end point was to evaluate sustained virologic response (SVR) 12 in CHC-infected patients and to determine its effect in patients with hepatitis C virus-related cirrhosis. The secondary end point was to observe any adverse events related to treatment. METHODS: All patients with CHC were randomized into two groups: noncirrhotic and cirrhotic. The combination of VEL/SOF was given as recommended. RESULTS: One hundred patients with CHC infection treated with the VEL/SOF regimen were evaluated. A total of 79 (79%) of 100 patients were noncirrhotic, and 21 (21%) were cirrhotic. We achieved SVR12 in 99 (99%) of 100 patients. Among cirrhotics, the mean serum bilirubin (mg/dl), albumin (g/dl), and platelet count (×10³/µL) improved from baseline 1.82 ± 0.87, 3.22 ± 0.69, and 80.19 ± 46.03 to 1.74 ± 0.87, 3.48 ± 0.72, and 85.05 ± 42.50, respectively, at SVR12 (P-value > 0.05). Mean serum alanine aminotransferase (ALT) (U/L) improved from baseline 71.28 ± 59.17 to 35.38 ± 17.39 at SVR12 (P-value < 0.024). Baseline mean liver stiffness measurement (LSM) in cirrhotic patients was 28.24 ± 10.87 kPa, which decreased to 24.04 ± 9.33 kPa at SVR12 (P-value, 0.02). The baseline Model for End-Stage Liver Disease (MELD) score was 13.47 ± 3.66, which decreased to 12.33 ± 5.46 at SVR12 (P-value, 0.28). The Child-Turcotte-Pugh score improved by 1 point in 33.33% (7/21) patients and 2 points in 9.52% (2/21) patients, and in the majority, that is, 38.09% (8/21), the score remained as it is. CONCLUSION: A single daily dose of the tablet SOF/VEL combination is safe and effective in all types of CHC. There was a significant improvement in the mean transaminase level and LSM at SVR12. And the MELD score improved by 1 point at SVR12 among cirrhotics.

Effectiveness and safety of ferric carboxymaltose compared to iron sucrose in women with iron deficiency anemia: phase IV clinical trials.

BACKGROUND: Iron deficiency anemia (IDA) is a significant problem worldwide particularly in women. The aim of the study was to evaluate the effectiveness and safety of intravenous ferric carboxymaltose (FCM) in comparison to iron sucrose (IS) in women with IDA. METHOD: Two hundred patients at Department of Obstetrics and Gynaecology, Sher-i-Kashmir Institute of Medical Sciences Medical College and Hospital, Jammu & Kashmir, India identified with IDA were enrolled for the study. Intravenous FCM and IS were both given as per the protocol. Change in the laboratory parameters such as hemoglobin (Hb), mean corpuscular value, and serum ferritin levels at two weeks and four weeks interval after the treatment was recorded. RESULT: A significant increase in the mean Hb was observed from 7.76 ± 0.709 to 13.25 ± 0.606 in patients treated with FCM and 7.64 ± 0.710 to 11.59 ± 0.733 g/dL (P < 0.001) in patients treated with IS after four weeks of therapy. The rise in mean corpuscular volume was from 66.82 ± 5.24 to 86.76 ± 3.765 and 68.05 ± 5.56 to 93.80 ± 3.80 and rise in serum ferritin levels were from 8.32 ± 1.787 to 38.94 ± 6.095 µg/L and 8.16 ± 1.540 to 27 ± 8.175 µg/L in patients treated with FCM and IS respectively after four weeks of therapy. No serious adverse effects were reported. CONCLUSION: Parenteral therapy is effective in IDA, but FCM elevates hemoglobin level and restored iron stores faster than IS with minimum adverse drug reactions. TRIAL REGISTRATION NUMBER: ISRCTN14484575 Dated: 15-12-2017 retrospectively registered. https://doi.org/10.1186/ISRCTN14484575.

¹H, ¹5N, and ¹³C backbone chemical shift assignment of titin domains A59-A60 and A60 alone.

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 µm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or 'super-repeats'. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619-628, 2012). Here we report backbone (15)N, (13)C and (1)H chemical shift and (13)Cß assignments for the A59-A60 domain tandem from the titin A59-A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59-A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59-A60.

Evidence of Bacterial Biofilms among Infected and Hypertrophied Tonsils in Correlation with the Microbiology, Histopathology, and Clinical Symptoms of Tonsillar Diseases.

Diseases of the tonsils are becoming more resistant to antibiotics due to the persistence of bacteria through the formation of biofilms. Therefore, understanding the microbiology and pathophysiology of such diseases represent an important step in the management of biofilm-related infections. We have isolated the microorganisms, evaluated their antimicrobial susceptibility, and detected the presence of bacterial biofilms in tonsillar specimens in correlation with the clinical manifestations of tonsillar diseases. Therefore, a total of 140 palatine tonsils were collected from 70 patients undergoing tonsillectomy at University Malaya Medical Centre. The most recovered isolate was Staphylococcus aureus (39.65%) followed by Haemophilus influenzae (18.53%). There was high susceptibility against all selected antibiotics except for cotrimoxazole. Bacterial biofilms were detected in 60% of patients and a significant percentage of patients demonstrated infection manifestation rather than obstruction. In addition, an association between clinical symptoms like snore, apnea, nasal obstruction, and tonsillar hypertrophy was found to be related to the microbiology of tonsils particularly to the presence of biofilms. In conclusion, evidence of biofilms in tonsils in correlation with the demonstrated clinical symptoms explains the recalcitrant nature of tonsillar diseases and highlights the importance of biofilm's early detection and prevention towards better therapeutic management of biofilm-related infections.

Left-sided hepatic resection for hepatolithiasis: a longitudinal study of 110 patients.

BACKGROUND: Hepatolithiasis affecting the left hepatobiliary system is common in the Asia Pacific region. This aim of this study was to describe an experience with the diagnosis and treatment of patients with isolated left-sided hepatolithiasis. METHODS: One hundred and ten patients with isolated left-sided hepatolithiasis who underwent a left-sided hepatic resection between January 1999 and February 2010 were included for further analysis. The clinical profile, cholangiograms, operative procedures and early and late results were examined. RESULTS: Analysis of magnetic resonance cholangiopancreatography (MRCP) and endoscopic retrograde cholangiopancreatography (ERCP) findings revealed left hepatic ductal anatomical details in 101 patients. Three types of left hepatic ductal variations were observed (type 1 in 90.1%, type 2 in 5.9% and type 3 in 4.0% patients). Eighty-four (76.4%) patients had the presence of strictures in the left hepatic ductal system and in 26 (23.6%) patients no strictures could be seen. Out of 84 patients with strictures, 78 could be classified (type I in 89.7% and type II in 10.3%). Of the 110 patients, 75 (68.2%) underwent a left lateral sectionectomy, 33 (30%) a left hepatectomy and 2 (1.8%) patients were treated with a left hepatectomy combined with a caudate lobe resection. Of the patients who underwent a left hepatectomy 11.4% developed a bile leak. Eight per cent of patients who underwent a left lateral sectionectomy had infective complications. During a median follow-up period of 63 (range 3-134) months, 2 (2.1%) patients were discovered to have residual stones and five (5.2%) others possessed recurrent stones. CONCLUSIONS: This study highlights the impact of the presence or absence of bile duct stricture on the clinical and histological profile of patients as well as their operative and the post-operative behaviour. It is concluded that hepatic resection is an appropriate treatment modality in localized left-sided hepatolithiasis.

In vitro activity of tigecycline against Acinetobacter baumannii isolates from a teaching hospital in Malaysia.

The In vitro susceptibility of clinical and environmental isolates of Acinetobacter baumannii to tigecycline and other antibiotics was determined by disk diffusion method. The E-test was used to determine the minimum inhibitory concentration (MIC). The growth curves of tigecycline treated environmental and clinical strains were established. Fifty-seven percent and 75% of the clinical and environmental isolates were MDR strains, respectively. Ninety-five percent of the clinical isolates were susceptible to tigecycline and 5% showed intermediate resistance with MIC ranging between 0.032 and 3 mg/l. Tigecycline susceptible and intermediate resistance among the environmental isolates were 40% and 60%, respectively, with a significantly lower MIC range of 0.5-4 mg/l. The bacterial growth curves demonstrated the higher ability of the environmental strains to tolerate the antibiotic effects than the clinical strains. The relatively high resistance profile among the environmental isolate suggests an insidious emergence of tigecycline resistance amongst A. baumannii. Strict infection control procedures are imperative to prevent the dissemination of tigecycline-resistant A. baumannii strains in the hospital environment.

¹H, ¹5N and ¹³C backbone chemical shift assignment of the titin A67-A68 domain tandem.

Single molecules of the giant protein titin extend across half of the muscle sarcomere, from the Z-line to the M-line, and have roles in muscle assembly and elasticity. In the A-band titin is attached to thick filaments and here the domain arrangement occurs in regular patterns of eleven called the large super-repeat. The large super-repeat itself occurs eleven times and forms nearly half the titin molecule. Interactions of the large super-repeats with myosin are consistent with a role in thick filament assembly. Here we report backbone assignments of the titin A67-A68 domain tandem (Fn-Ig) from the third super-repeat (A65-A75) completed using triple resonance NMR experiments.

Phylogenetic characterization of archaea in saltpan sediments.

A study was undertaken to investigate the presence of archaeal diversity in saltpan sediments of Goa, India by 16S rDNA-dependent molecular phylogeny. Small subunit rRNA (16S rDNA) from saltpan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. 10 unique phylotypes were obtained by PCR based RFLP of 16S rRNA genes using endonuclease Msp 1, which was most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain archaea including both crenarchaeota and euryarcheaota. None of the retrieved crenarchaeota sequences could be grouped with previously cultured crenarchaeota however; two sequences were related with haloarchaea. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of low archaeal population but showed yet unclassified species, may specially adapted to the salt pan sediment of Goa.

Hydatid cyst of the pancreas. An experience with six cases.

CONTEXT: The pancreas is an infrequent site of hydatid disease. OBJECTIVE: This study aims at giving better insight into the diagnostic and managerial approach to the disease. PATIENTS: Six patients with hydatid cysts of the pancreas. DESIGN: Retrospective review of the clinical records. RESULTS: The six patients (four men, two women) ranged in age from 18 to 68 years. Five of the cysts were primary while one had an associated cyst in the liver. Abdominal pain, vomiting, abdominal mass and dyspeptic symptoms were seen in cysts involving the body and tail. Two patients having cysts in the head of the pancreas presented with obstructive jaundice. An indirect hemagglutination test and an enzyme-linked immunoabsorbent assay were positive for the presence of specific hydatid antibodies in four patients. Abdominal ultrasonography, computed tomography and magnetic resonance cholangiopancreatography (MRCP) successfully imaged the cysts and also defined the relationship of the lesion with the pancreatic duct. All patients underwent surgical exploration. Three patients had intraoperative fine needle aspiration cytology of the cystic lesion for microscopic and electrolyte analysis. A preoperative diagnosis was possible in two patients and, in the other four, the diagnosis was made intraoperatively and confirmed on histopathological examination. PATIENTS: with cysts located in the tail underwent a distal pancreatectomy with a splenectomy while those with cysts in the body had a pericystectomy or central pancreatectomy. Cysts of the head were treated with evacuation, partial cystectomy and tube drainage. There were no postoperative complications, and no evidence of cyst recurrence was observed during the follow-up period. All the patients were followed up at three-month intervals with a mean follow-up time of 58.7 months (rang: 4-120 months); no patient had cyst recurrence or dissemination. CONCLUSION: A hydatid cyst is an uncommon cause of cystic lesions in the pancreas and should be included in the differential diagnosis of cystic lesions of the pancreas, especially in endemic areas. Intraoperative fine needle aspirate for microscopic and electrolyte estimation seems to be an effective method for establishing a proper diagnosis. MRCP, which can depict the communication of the cystic lesion with the pancreatic duct, helps in defining the type of surgical treatment. Cysts in body and tail are best treated by resectional methods whereas, for those in the head region, a cystectomy with simple drainage is a simple, quick and effective solution.

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L.

BACKGROUND: Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide. RESULTS: Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 microg/ml for yeasts, 125 to 500 microg/ml for Aspergillus species, and 7.81 to 62.5 microg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 x MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 x MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 x to 8 x MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol. CONCLUSIONS: The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.

Functional cloning and predictive structural modeling of a novel esterase from Bacillus subtilis strain, RRL 1789.

We have recently reported the purification and characterization of a novel esterase from the Bacillus subtilis strain. In the present study we report the genomic DNA cloning and predictive structural modeling of this novel esterase. Tributyrin- and Rhodamine B-based functional screen of a Bacillus subtilis genomic library led to the identification of a potential lipolytic gene. DNA sequence analysis of the cloned gene showed that it encodes a protein of 489 amino acid residues. Sequence homology search and multiple sequence alignment showed that the protein was highly homologous to known esterases. Secondary structure-driven multiple sequence alignment with the homologous esterase of known three-dimensional structures was performed and a 3D structure model of this enzyme was constructed. Based on the topological organization of the secondary structures, this protein belongs to the alpha/beta hydrolase superfamily. Moreover, the presence of serine in the context of amino acid sequence G/A-X-S-X-G (with X an arbitrary amino acid residue) in the protein indicates that it belong to the class of serine hydrolases of this superfamily.

Immune modulation and apoptosis induction: Two sides of antitumoural activity of a standardised herbal formulation of Withania somnifera.

Deregulated apoptosis and suppressed tumour reactive immunity render tumour cells to grow amok in the host body. Traditionally used botanicals may offer potential anticancer chemo-immunotherapeutic leads. We report in this study a chemically standardised herbal formulation (WSF) of Withania somnifera possessing anticancer and Th1 immune up-regulatory activities. WSF produced cytotoxicity in a panel of human cancer cell lines in vitro. The molecular mechanism of cell cytotoxicity, IC(50) 48h approximately 20mug/ml, was investigated in HL-60, where it induced apoptosis by activating both intrinsic and extrinsic signalling pathways. It induced early generation of reactive nitrogen and oxygen species (RNOS), thus producing oxidative stress mediated mitochondrial membrane potential (MMP) loss leading to the release of cytochrome c, the translocation of Bax to mitochondria and apoptosis-inducing factor to the nuclei. These events paralleled the activation of caspase-9, -3 and PARP cleavage. WSF also activated caspase-8 through enhanced expression of TNF-R1 and DR-4, suggesting also the involvement of extrinsic pathway of apoptosis. WSF at 150mg/kg, i.p., inhibited >50% tumour growth in the mouse tumour models. In tumour-bearing mice, WSF inhibited the expression of pStat-3, with a selective stimulation of Th1 immunity as evidenced by enhanced secretion of IFN-gamma and IL-2. In parallel, it enhanced the proliferation of CD4(+)/CD8(+) and NK cells along with an increased expression of CD40/CD40L/CD80. In addition, WSF also enhanced T cell activation in camptothecin treated tumour-bearing mice. WSF being safe when given orally up to 1500mg/kg to rats for 6 months may be found useful in the management of malignancy by targeting at multiple pathways.

Anticancer activity of an essential oil from Cymbopogon flexuosus.

The essential oil from a lemon grass variety of Cymbopogon flexuosus was studied for its in vitro cytotoxicity against twelve human cancer cell lines. The in vivo anticancer activity of the oil was also studied using both solid and ascitic Ehrlich and Sarcoma-180 tumor models in mice. In addition, the morphological changes in tumor cells were studied to ascertain the mechanism of cell death. The in vitro cytotoxicity studies showed dose-dependent effects against various human cancer cell lines. The IC(50) values of oil ranged from 4.2 to 79 microg/ml depending upon the cell line. In 502713 (colon) and IMR-32 (neuroblastoma) cell lines, the oil showed highest cytotoxicity with IC(50) value of 4.2 and 4.7 microg/ml, respectively. Intra-peritoneal administration of the oil significantly inhibited both ascitic and solid forms of Ehrlich and Sarcoma-180 tumors in a dose-dependent manner. The tumor growth inhibition at 200 mg/kg (i.p.) of the oil observed with both ascitic and solid tumor forms of Ehrlich Ascites carcinoma was 97.34 and 57.83 respectively. In case of Sarcoma-180, the growth inhibition at similar dose of oil was 94.07 and 36.97% in ascitic and solid forms respectively. Morphological studies of the oil treated HL-60 cells revealed loss of surface projections, chromatin condensation and apoptosis. The mitochondria showed apparent loss of cristae in the cells undergoing apoptosis. The morphological studies of Sarcoma-180 solid tumor cells from animals treated with the oil revealed condensation and fragmentation of nuclei typical of apoptosis. Morphological studies of ascites cells from animals treated with the oil too revealed the changes typical of apoptosis. Our results indicate that the oil has a promising anticancer activity and causes loss in tumor cell viability by activating the apoptotic process as identified by electron microscopy.