Clinical and Epidemiological Characteristics of Mpox Cases Identified Through Case-based Surveillance in India, July 2022-January 2023.

BACKGROUND: Of the 43 mpox cases reported by the WHO in South East Asia between January 2022 and March 2023, 24 (56%) were from India. OBJECTIVES: We describe the clinical and epidemiological profile of cases identified through India's hospital case-based surveillance. MATERIALS AND METHODS: We identified mpox cases as a positive result for mpox virus polymerase-chain-reaction assay, reported through surveillance from July 1, 2022 to January 7, 2023. Cases and clinicians were interviewed, and data were abstracted from the medical records. We conducted contact tracing among family, close social networks, and healthcare personnel staff for the first 17 cases. We collected the data on sociodemographics, clinical findings, and behavior, and described data using summary statistics. RESULTS: We identified 24 laboratory-confirmed cases (42% females, median age 30 years, range 22-38), including one death (case fatality rate 4.2%). We collected clinical and behavioural data from 21 of 24 cases. All had rashes with vesicles and genital lesions; 7 (33%) reported genital lesions as the first symptom; and 3 (13%) reported complications. Among the 21 cases, all were sexually active, none self-identified as men having sex with men (MSM), and 6 (29%) reported multiple sex partners. We identified 51 contacts of the first 17 reported cases, none reported symptoms suggestive of mpox. CONCLUSION: The clinical and behavioral characteristics of mpox cases in India are consistent with the global 2022 outbreak, with the exception that no cases in India reported MSM. Most were sexually active young adult economic migrants and developed genital lesions.

Preparation of glycopeptide-modified pH-sensitive liposomes for promoting antigen cross-presentation and induction of antigen-specific cellular immunity.

Cross-presentation, exogenous antigen presentation onto major histocompatibility complex class I molecules on antigen presenting cells, is crucially important for inducing antigen-specific cellular immune responses for cancer immunotherapy and for the treatment of infectious diseases. One strategy to induce cross-presentation is cytosolic delivery of an exogenous antigen using fusogenic or endosomolytic molecule-introduced nanocarriers. Earlier, we reported liposomes modified with pH-responsive polymers to achieve cytosolic delivery of an antigen. Polyglycidol-based or polysaccharide-based pH-responsive polymers can provide liposomes with delivery performance of antigenic proteins into cytosol via membrane fusion with endosomes responding to acidic pH, leading to induction of cross-presentation. Mannose residue was introduced to pH-responsive polysaccharides to increase uptake selectivity to antigen presenting cells and to improve cross-presentation efficiency. However, direct introduction of mannose residue into pH-responsive polysaccharides suppressed cytoplasmic delivery performance of liposomes. To avoid such interference, for this study, mannose-containing glycans were incorporated separately into pH-responsive polysaccharide-modified liposomes. Soybean agglutinin-derived glycopeptide was used as a ligand for lectins on antigen presenting cells. Incorporation of glycopeptide significantly increased the cellular uptake of liposomes by dendritic cell lines and increased cross-presentation efficiency. Liposomes incorporated both glycopeptide and pH-responsive polysaccharides exhibited strong adjuvant effects in vitro and induced the increase of dendritic cells, M1 macrophages, and effector T cells in the spleen. Subcutaneous administration of these liposomes induced antigen-specific cellular immunity, resulting in strong therapeutic effects in tumor-bearing mice. These results suggest that separate incorporation of glycopeptides and pH-responsive polysaccharides into antigen-loaded liposomes is an effective strategy to produce liposome-based nanovaccines to achieve antigen cross-presentation and induction of cellular immunity towards cancer immunotherapy.

Tumor-specific migration routes of xenotransplanted human glioblastoma cells in mouse brain.

The migration of neural progenitor cells (NPCs) to their final destination during development follows well-defined pathways, such as along blood vessels. Cells originating from the highly malignant tumor glioblastoma (GBM) seem to exploit similar routes for infiltrating the brain parenchyma. In this report, we have examined the migration of GBM cells using three-dimensional high-resolution confocal microscopy in brain tumors derived from eight different human GBM cell lines xenografted into immunodeficient mice. The primary invasion routes identified were long-distance migration along white matter tracts and local migration along blood vessels. We found that GBM cells in the majority of tumors (6 out of 8) did not exhibit association with blood vessels. These tumors, derived from low lamin A/C expressing GBM cells, were comparatively highly diffusive and invasive. Conversely, in 2 out of 8 tumors, we noted perivascular invasion and displacement of astrocyte end-feet. These tumors exhibited less diffusive migration, grew as solid tumors, and were distinguished by elevated expression of lamin A/C. We conclude that the migration pattern of glioblastoma is distinctly tumor cell-specific. Furthermore, the ability to invade the confined spaces within white matter tracts may necessitate low expression of lamin A/C, contributing to increased nuclear plasticity. This study highlights the role of GBM heterogeneity in driving the aggressive growth of glioblastoma.

Canvassing Prospects of Glyco-Nanovaccines for Developing Cross-Presentation Mediated Anti-Tumor Immunotherapy.

In view of the severe downsides of conventional cancer therapies, the quest of developing alternative strategies still remains of critical importance. In this regard, antigen cross-presentation, usually employed by dendritic cells (DCs), has been recognized as a potential solution to overcome the present impasse in anti-cancer therapeutic strategies. It has been established that an elevated cytotoxic T lymphocyte (CTL) response against cancer cells can be achieved by targeting receptors expressed on DCs with specific ligands. Glycans are known to serve as ligands for C-type lectin receptors (CLRs) expressed on DCs, and are also known to act as a tumor-associated antigen (TAA), and, thus, can be harnessed as a potential immunotherapeutic target. In this scenario, integrating the knowledge of cross-presentation and glycan-conjugated nanovaccines can help us to develop so called 'glyco-nanovaccines' (GNVs) for targeting DCs. Here, we briefly review and analyze the potential of GNVs as the next-generation anti-tumor immunotherapy. We have compared different antigen-presenting cells (APCs) for their ability to cross-present antigens and described the potential nanocarriers for tumor antigen cross-presentation. Further, we discuss the role of glycans in targeting of DCs, the immune response due to pathogens, and imitative approaches, along with parameters, strategies, and challenges involved in cross-presentation-based GNVs for cancer immunotherapy. It is known that the effectiveness of GNVs in eradicating tumors by inducing strong CTL response in the tumor microenvironment (TME) has been largely hindered by tumor glycosylation and the expression of different lectin receptors (such as galectins) by cancer cells. Tumor glycan signatures can be sensed by a variety of lectins expressed on immune cells and mediate the immune suppression which, in turn, facilitates immune evasion. Therefore, a sound understanding of the glycan language of cancer cells, and glycan-lectin interaction between the cancer cells and immune cells, would help in strategically designing the next-generation GNVs for anti-tumor immunotherapy.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Sensing the interactions between carbohydrate-binding agents and N-linked glycans of SARS-CoV-2 spike glycoprotein using molecular docking and simulation studies.

A recent surge in finding new candidate vaccines and potential antivirals to tackle atypical pneumonia triggered by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) needs new and unexplored approaches in solving this global pandemic. The homotrimeric transmembrane spike (S) glycoprotein of coronaviruses which facilitates virus entry into the host cells is covered with N-linked glycans having oligomannose and complex sugars. These glycans provide a unique opportunity for their targeting via carbohydrate-binding agents (CBAs) which have shown their antiviral potential against coronaviruses and enveloped viruses. However, CBA-ligand interaction is not fully explored in developing novel carbohydrate-binding-based antivirals due to associated unfavorable responses with CBAs. CBAs possess unique carbohydrate-binding specificity, therefore, CBAs like mannose-specific plant lectins/lectin-like mimic Pradimicin-A (PRM-A) can be used for targeting N-linked glycans of S glycoproteins. Here, we report studies on the binding and stability of lectins (NPA, UDA, GRFT, CV-N and wild-type and mutant BanLec) and PRM-A with the S glycoprotein glycans via docking and MD simulation. MM/GBSA calculations were also performed for docked complexes. Interestingly, stable BanLec mutant (H84T) also showed similar docking affinity and interactions as compared to wild-type BanLec, thus, confirming that uncoupling the mitogenic activity did not alter the lectin binding activity of BanLec. The stability of the docked complexes, i.e. PRM-A and lectins with SARS-CoV-2 S glycoprotein showed favorable intermolecular hydrogen-bond formation during the 100 ns MD simulation. Taking these together, our predicted in silico results will be helpful in the design and development of novel CBA-based antivirals for the SARS-CoV-2 neutralization.Communicated by Ramaswamy H. Sarma.

Plant lectins and their usage in preparing targeted nanovaccines for cancer immunotherapy.

Plant lectins, a natural source of glycans with a therapeutic potential may lead to the discovery of new targeted therapies. Glycans extracted from plant lectins are known to act as ligands for C-type lectin receptors (CLRs) that are primarily present on immune cells. Plant-derived glycosylated lectins offer diversity in their N-linked oligosaccharide structures that can serve as a unique source of homogenous and heterogenous glycans. Among the plant lectins-derived glycan motifs, Man9GlcNAc2Asn exhibits high-affinity interactions with CLRs that may resemble glycan motifs of pathogens. Thus, such glycan domains when presented along with antigens complexed with a nanocarrier of choice may bewilder the immune cells and direct antigen cross-presentation - a cytotoxic T lymphocyte immune response mediated by CD8+ T cells. Glycan structure analysis has attracted considerable interest as glycans are looked upon as better therapeutic alternatives than monoclonal antibodies due to their cost-effectiveness, reduced toxicity and side effects, and high specificity. Furthermore, this approach will be useful to understand whether the multivalent glycan presentation on the surface of nanocarriers can overcome the low-affinity lectin-ligand interaction and thereby modulation of CLR-dependent immune response. Besides this, understanding how the heterogeneity of glycan structure impacts the antigen cross-presentation is pivotal to develop alternative targeted therapies. In the present review, we discuss the findings on structural analysis of glycans from natural lectins performed using GlycanBuilder2 - a software tool based on a thorough literature review of natural lectins. Additionally, we discuss how multiple parameters like the orientation of glycan ligands, ligand density, simultaneous targeting of multiple CLRs and design of antigen delivery nanocarriers may influence the CLR targeting efficacy. Integrating this information will eventually set the ground for new generation immunotherapeutic vaccine design for the treatment of various human malignancies.

Cockroach exposure and its allergy sensitization in asthma patients.

Asthma is a heterogeneous disease with distinct phenotypes. Serum tIgE, SSIgE and SPT are the methods of evaluating allergen sensitization. The present study evaluates the exposure and sensitization to cockroach (Periplaneta americana) antigens in asthma patients in a metropolitan city of India. The study enrolled 200 consecutive bronchial asthma patients, diagnosed as per GINA guidelines. As per history of exposure to cockroaches, the patients are divided in two groups as exposed and non-exposed asthmatic. All the enrolled subjects underwent SPT against common aeroallergens including cockroach, spirometry and estimation of tIgE level and SSIgE against cockroach. Out of 200 asthma patients, a total of 114 (57%) asthmatic were found SPT positive against one of the common aeroallergens, of which 68 (34%) showed SPT sensitivity against cockroach. A total of 103 (51.5%) patients were found exposed to cockroaches. In the cockroach exposed group, the mean serum tIgE was found significantly higher than the non-exposed group (569.31±224.64 vs 479.29±237 IU/ml; p=0.007). The mean SSIgE against cockroach in exposed groups was found not significant than non-expose group (4.87±11.19 vs 4.11±8.39 KUA/L; p=0.589). The mean tIgE was also not significant in atopic compared to non-atopic asthmatic (553.25±218.12 IU/ml vs 489.1±251.16 IU/ml; p=0.056). The mean SSIgE against cockroach was 5.66±10.45 KUA/L for atopic and 2.96±8.98 KUA/L for non-atopic (p=0.054). The airway obstruction was almost the same in both groups.  Asthmatic patients who were exposed to cockroach and atopic had high tIgE, SSIgE levels and SPT positivity against cockroach antigen compared to non-exposed patients.

Carbohydrate-Binding Agents: Potential of Repurposing for COVID-19 Therapy.

With the emergence of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the whole world is suffering from atypical pneumonia, which resulted in more than 559,047 deaths worldwide. In this time of crisis and urgency, the only hope comes from new candidate vaccines and potential antivirals. However, formulating new vaccines and synthesizing new antivirals are a laborious task. Therefore, considering the high infection rate and mortality due to COVID-19, utilization of previous information, and repurposing of existing drugs against valid viral targets have emerged as a novel drug discovery approach in this challenging time. The transmembrane spike (S) glycoprotein of coronaviruses (CoVs), which facilitates the virus's entry into the host cells, exists in a homotrimeric form and is covered with N-linked glycans. S glycoprotein is known as the main target of antibodies having neutralizing potency and is also considered as an attractive target for therapeutic or vaccine development. Similarly, targeting of N-linked glycans of S glycoprotein envelope of CoV via carbohydrate-binding agents (CBAs) could serve as an attractive therapeutic approach for developing novel antivirals. CBAs from natural sources like lectins from plants, marine algae and prokaryotes and lectin mimics like Pradimicin-A (PRM-A) have shown antiviral activities against CoV and other enveloped viruses. However, the potential use of CBAs specifically lectins was limited due to unfavorable responses like immunogenicity, mitogenicity, hemagglutination, inflammatory activity, cellular toxicity, etc. Here, we reviewed the current scenario of CBAs as antivirals against CoVs, presented strategies to improve the efficacy of CBAs against CoVs; and studied the molecular interactions between CBAs (lectins and PRM-A) with Man9 by molecular docking for potential repurposing against CoVs in general, and SARSCoV- 2, in particular.

Engineering anti-cancer nanovaccine based on antigen cross-presentation.

Dendritic cells (DCs) present exogenous antigens on major histocompatibility complex (MHC) class I molecules, thereby activating CD8+ T cells, contributing to tumor elimination through a mechanism known as antigen cross-presentation. A variety of factors such as maturation state of DCs, co-stimulatory signals, T-cell microenvironment, antigen internalization routes and adjuvants regulate the process of DC-mediated antigen cross-presentation. Recently, the development of successful cancer immunotherapies may be attributed to the ability of DCs to cross-present tumor antigens. In this review article, we focus on the underlying mechanism of antigen cross-presentation and ways to improve antigen cross-presentation in different DC subsets. We have critically summarized the recent developments in the generation of novel nanovaccines for robust CD8+ T-cell response in cancer. In this context, we have reviewed nanocarriers that have been used for cancer immunotherapeutics based on antigen cross-presentation mechanism. Additionally, we have also expressed our views on the future applications of this mechanism in curing cancer.

Effect of laser application in the healing of intrabony defects treated with bioactive glass.

AIMS: Presence of bacteria within the environment of infrabony pockets affects healing during their treatment. Present investigation utilized a diode laser for pocket sanitization before the placement of bone biomaterial with the aim of enhancing the healing. MATERIALS AND METHODS: Twelve patients with bilateral intrabony defects participated in a split-mouth study design. Control group received biomaterial application only after surgical debridement. Infrabony pockets in the test group were irradiated with 810-nm diode laser at 0.8 W, continuous wave for 20 s before surgical debridement and biomaterial application. Healing was assessed using clinical and radiologic parameters. RESULTS: Control group showed mean probing depth (PD) reduction of 3.25 ± 0.62 at 3, 4.08 ± 0.90 mm at 6 months. 3.00 ± 0.73 at 3, 3.91 ± 0.66 mm at 6 months reduction in mean PD was seen in the test group (P < 0.001). No statistically significant differences between the groups were observed. A gain of 2.50 ± 0.67 at 3, 3.25 ± 0.62 mm at 6 months in relative clinical attachment level was seen in the control and of 2.33 ± 0.77 at 3, 3.16 ± 0.57 mm at 6 months in the test group (P < 0.001) without significant differences between groups. 1.33 ± 0.57 and 0.95 ± 0.68 mm hard-tissue fill (difference in the radiographic distance between cementoenamel junction and base of the intrabony defect pre- and post-operative) at 6 months was observed in the control and test groups, respectively (P < 0.001). Between groups differences (0.22 ± 0.24 mm) were not significant. CONCLUSIONS: Similar reduction in soft- and hard-tissue parameters in both groups indicates that adjunctive pocket sanitization with diode laser did not improve the healing of intrabony defects treated with bioactive glass.

Transcriptome data of cultivated tetraploid and hexaploid wheat variety during grain development.

Wheat is a major food crop and an important component of human diet throughout the world. There are two major types of cultivated wheat; one is tetraploid durum (pasta) wheat and another one is hexaploid bread wheat. Wheat grain is the reservoir of two major dietary components - carbohydrate and protein, which get accumulated during seed maturation and directly affects yield and quality. Hexaploid, having 6 copies of each chromosome differs to a great extent from tetraploid having 4 copies of each chromosome. Studying the gene expression pattern in developing grain would help in understanding the difference in metabolic process as well as involvement of the genes in these two types of wheat. A transcriptional comparison of developing grains was carried out between the two wheat genotypes; tetraploid (AABB:PDW233) and hexaploid (AABBDD:PBW343) using RNA-seq. Approximately 194 million raw reads were obtained from both libraries. After removal of contaminations, a huge proportion (>99%), of high quality reads were obtained, were aligned to reference genome. A total of 2324 up-regulated and 522 down-regulated genes were identified as differentially expressed between PDW233 vs PBW343. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes between durum and bread wheat. This information will help in understanding process grain reserve in tetraploid and hexaploid wheat in relation to their nutritional quality.

Proliferative verrucous leukoplakia misdiagnosed as oral leukoplakia.

Correct diagnosis of white lesions of the oral cavity is sometimes difficult, because some oral white lesions behave differently and tend to change their appearance with time. Clinicians often wrongly diagnose such lesions as oral leukoplakias and treat simply. Lesions recur and turn malignant. Proliferative verrucous leukoplakia (PVL) is a distinct form of oral leukoplakia characterized by a high recurrence rate and high rate of transformation into oral squamous cell carcinoma. We present herein a case of PVL which was misdiagnosed as oral leukoplakia and progressed to oral carcinoma.

Self-medication practice among preclinical university students in a medical school from the city of Pokhara, Nepal.

BACKGROUND: In developing countries like Nepal medicines can be acquired from the chemist's without of a prescription which sometime may have many drawbacks due to intake of excessive drugs without a proper diagnosis. The primary objective of the study was to find out the pattern of self-medication practice among the preclinical medical students at Manipal College of Medical Sciences. MATERIALS AND METHODS: This was a cross sectional study carried out using structured questionnaire at Manipal College of Medical Sciences, Pokhara, Nepal between November 2012- July 2014. RESULTS: The overall response rate of this study was 95.31%. 81.35% of the students were practicing self-medication in this institution. Most common group of drugs that were consumed were antipyretics 31%, antibiotics 26.2%, analgesics 18.89%, antihistaminics 10.1% respectively. Paracetamol was the most common drug used for self-medication 31%, followed by Azithromycin 17.6% and combination of Paracetamol and Ibuprofen 15.6%, Cetirizine 8.6%, Amoxicillin 6.5%, Omeprazole 6.3%, Albendazole 3.3%, Mefenemic acid 2.8%, Cefpodoxime2% respectively. CONCLUSION: Medical student should be educated through awareness programme regarding pros and cons of self-medication practice and they should be motivated regarding the rationale use of antibiotics. .

Integrin signaling via FAK-Src controls cytokinetic abscission by decelerating PLK1 degradation and subsequent recruitment of CEP55 at the midbody.

Adhesion to extracellular matrix is required for cell cycle progression through the G1 phase and for the completion of cytokinesis in normal adherent cells. Cancer cells acquire the ability to proliferate anchorage-independently, a characteristic feature of malignantly transformed cells. However, the molecular mechanisms underlying this escape of the normal control mechanisms remain unclear. The current study aimed to identify adhesion-induced reactions regulating the cytokinesis of non-transformed human fibroblasts.The adhesion-dependent control of cytokinesis was found to occur at a late stage close to the abscission, during which the endosomal sorting complex required for transport (ESCRT) severs the thin intercellular bridge connecting two nascent daughter cells. CEP55, a key protein involved in the abscission process, was localized at the midbody in both adherent and non-adherent fibroblasts, but it was unable to efficiently recruit ALIX, TSG101, and consequently the ESCRT-III subunit CHMP4B was missing in the non-adherent cells. PLK1, a kinase that prevents premature recruitment of CEP55 to the midbody, disappeared from this site more rapidly in the non-adherent cells. A FAK-Src signaling pathway downstream of integrin-mediated cell adhesion was found to decelerate both PLK1 degradation and CEP55 accumulation at the midbody. These data identify the regulation of PLK1 and CEP55 as steps where integrins exert control over the cytokinetic abscission.

Transcription factor ZBED6 affects gene expression, proliferation, and cell death in pancreatic beta cells.

We have investigated whether the recently discovered transcription factor, zinc finger BED domain-containing protein 6 (ZBED6), is expressed in insulin-producing cells and, if so, to what extent it affects beta cell function. ZBED6 was translated from a ZC3H11A transcript in which the ZBED6-containing intron was retained. ZBED6 was present in mouse ßTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. We propose that ZBED6 supports proliferation and survival of beta cells, possibly at the expense of specialized beta cell function-i.e., insulin production-because (i) the nuclear ZBED6 were the predominant forms in rapidly proliferating ßTC-6 cells, but not in human islet cells; (ii) down-regulation of ZBED6 in ßTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell-cycle arrest, increased expression of beta cell-specific genes, and higher rates of apoptosis; (iii) silencing of ZBED6 in the human PANC-1 duct cell line reduced proliferation rates; and (iv) ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis, and apoptosis. Furthermore, it is possible that beta cells, by switching from full length to a truncated form of ZBED6, can decide the subcellular localization of ZBED6, thereby achieving differential ZBED6-mediated transcriptional regulation.

A Scientific Validation of Antihyperglycemic and Antihyperlipidemic Attributes of Trichosanthes dioica.

The present study was undertaken to scientifically validate the antidiabetic activity of aqueous fruit extract of Trichosanthes dioica Roxb. (Family: Cucurbitaceae) which has been traditionally used for managing diabetes mellitus. This plant commonly known as "Sespadula" in English has not been explored scientifically so far for its glycemic potential except by our research group. The study was conducted with variable doses on normal, mild, and severe diabetics models, and several biochemical parameters including blood glucose level (BGL) were assessed. Maximum fall in BGL of 23.8% in normal rats and of 31.3% in mild diabetic rats was observed during their fasting blood glucose (FBG) and glucose tolerance test (GTT) with the dose of 1000 mg kg(-1). In severely diabetic animals after 4 weeks treatment with FBG, postprandial glucose, total cholesterol, and triglyceride levels were reduced by 28.7, 30.7, 57.2, and 18.5%, whereas high density lipoprotein, total protein, hemoglobin, and body weight were increased by 33.0, 36.7, 15.7 and 16.7%, respectively. Moreover, urine sugar was reduced from +4 to +1. Thus, the study scientifically validates the traditional use of T. diocia in diabetes management and could be developed as an effective oral agent for treating diabetes mellitus and complications associated with it.

Fibronectin assembly in the crypts of cytokinesis-blocked multilobular cells promotes anchorage-independent growth.

Anchorage-independent growth is a characteristic feature of cancer cells. However, it is unclear whether it represents a cause or a consequence of tumorigenesis. For normal cells, integrin-mediated adhesion is required for completion of the G1 and cytokinesis stages of the cell cycle. This study identified a mechanism that can drive anchorage-independent growth if the G1 checkpoint is suppressed. Cells with defective G1 checkpoint progressed through several rounds of the cell cycle in suspension in spite of uncompleted cytokinesis, thereby forming bi- and multilobular cells. Aurora B and CEP55 were localized to midbodies between the lobes, suggesting that the cytokinesis process reached close to abscission. Integrin-mediated re-attachment of such cells induced cytokinesis completion uncoupled from karyokinesis in most cells. However, a portion of the cells instead lost the constriction and became binucleated. Also, long-term suspension culture in soft agar produced colonies where the cytokinesis block was overcome. This process was fibronectin-dependent since fibronectin-deficient cells did not form colonies unless fibronectin was expressed or exogenously added. While fibronectin normally is not deposited on non-adherent single cells, bi/multilobular cells accumulated fibronectin in the intussusceptions. Based on our data we conclude: 1) Suppression of the G1 checkpoint allows multiple rounds of the cell cycle in detached cells and thereby enables matrix formation on their surface. 2) Uncompleted cytokinesis due to cell detachment resumes if integrin interactions are re-formed, allowing colony formation in soft agar 3) Such delayed cell division can generate binucleated cells, a feature known to cause chromosomal instability.

The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

Mannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro.