AIM: To investigate hepatitis virus, genetic and environmental factors, and their interactions in predisposing patients to liver diseases in Northeast India.
METHODS: A total of 104 jaundice patients and 124 community controls were included. Serological analysis was performed by routine enzyme-linked immunosorbent assay, and nucleic acid testing for hepatitis viruses was done by polymerase chain reaction (PCR), followed by PCR direct sequencing for viral genotyping. Cytochrome P450 2E1 (CYP2E1) polymorphism was studied by PCR-restriction fragment length polymorphism. Nitrite and volatile nitrosamines in indigenous foods consumed routinely by the Northeast Indian ethnic population were estimated by Griess’s reagent and GC-MS, respectively.
RESULTS: Hepatitis A virus (HAV) infection was predominantly prevalent (36.5%) in our cohort, followed by hepatitis B virus (HBV), hepatitis E virus (HEV) and hepatitis C virus. HBV genotype D and HEV genotype 1 were the most dominant. CYP2E1 c1/c2 genotype frequency was comparatively higher in alcoholic (P < 0.0001, OR = 30.5) and cryptogenic (P = 0.014, OR = 8.714) patients, and was associated with significantly higher hepatitis risk (P = 0.0.007, OR = 6.489). Mutant C allele of Cyp2E1 DraI frequency was comparatively higher in HAV (P = 0.006), alcoholic (P = 0.003) and cryptogenic (P = 0.014) cases, and was associated with overall hepatitis risk (P = 0.026, OR = 5.083). Indigenous foods, Gundruk, Kharoli, betel leaf and nuts were found to have the highest nitrite content.
CONCLUSION: Apart from viral factors, CYP2E1 polymorphism might be associated with increased risk of liver diseases in Northeast India. Indigenous foods that contain nitrite and nitrosamine might be an associated risk factor.
Viral hepatitis; Cytochrome P450 2E1; Gene polymorphism; Nitrites; Nitrosamines
Yersinia enterocolitica is a food-borne pathogen that preferentially infects the Peyer's patches and mesenteric lymph nodes, causing an acute inflammatory reaction. Even though Y. enterocolitica induces a robust inflammatory response during infection, the bacterium has evolved a number of virulence factors to limit the extent of this response. We previously demonstrated that interleukin-1α (IL-1α) was critical for the induction of gut inflammation characteristic of Y. enterocolitica infection. More recently, the known actions of IL-1α are becoming more complex because IL-1α can function both as a proinflammatory cytokine and as a nuclear factor. In this study, we tested the ability of Y. enterocolitica to modulate intracellular IL-1α-dependent IL-8 production in epithelial cells. Nuclear translocation of pre-IL-1α protein and IL-1α-dependent secretion of IL-8 into the culture supernatant were increased during infection with a strain lacking the 70-kDa virulence plasmid compared to the case during infection with the wild type, suggesting that Yersinia outer proteins (Yops) might be involved in modulating intracellular IL-1α signaling. Infection of HeLa cells with a strain lacking the yopP gene resulted in increased nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 similar to what is observed with bacteria lacking the virulence plasmid. YopP is a protein acetylase that inhibits mitogen-activated protein kinase (MAP kinase)- and NF-κB-dependent signal transduction pathways. Nuclear translocation of pre-IL-1α and IL-1α-dependent secretion of IL-8 in response to Yersinia enterocolitica infection were dependent on extracellular signal-regulated kinase (ERK) and p38 MAP kinase signaling but independent of NF-κB. These data suggest that Y. enterocolitica inhibits intracellular pre-IL-1α signaling and subsequent proinflammatory responses through inhibition of MAP kinase pathways.
The antimicrobial activity of alcoholic, butanolic and chloroform extracts of leaves and roots of the plant Acanthus ilicifolius ware studied. Ampicillin and clotrimazole were used as standard antibacterial and antifungal agents respectively. The result of the study revealed that the alcoholic extract and chloroform extract of leaves exhibited strong inhibitory action against Bacillus subtilis, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Aspergillus niger and moderate inhibitory action against Pseudomonas aeruginosa and Proteus vulgaris. The rest of the extracts showed moderate activity.
Acanthus ilicifolius; agar cup-plate method; antimicrobial activity; mangrove
The steroidogenic acute regulatory protein (StAR) is required for adrenal and gonadal steroidogenesis and for male sexual differentiation. StAR acts on the outer mitochondrial membrane (OMM) to facilitate movement of cholesterol from the OMM to the inner mitochondrial membrane to be converted to pregnenolone, the precursor of all steroid hormones. The mechanisms of the action of StAR remain unclear; the peripheral benzodiazepine receptor, an OMM protein, appears to be involved, but the identity of OMM proteins that interact with StAR remain unknown. Here we demonstrate that phosphorylated StAR interacts with voltage-dependent anion channel 1 (VDAC1) on the OMM, which then facilitates processing of the 37-kDa phospho-StAR to the 32-kDa intermediate. In the absence of VDAC1, phospho-StAR is degraded by cysteine proteases prior to mitochondrial import. Phosphorylation of StAR by protein kinase A requires phosphate carrier protein on the OMM, which appears to interact with StAR before it interacts with VDAC1. VDAC1 and phosphate carrier protein are the first OMM proteins shown to contact StAR.
Autism spectrum disorder (ASD) is a heterogeneous disorder with substantial heritability, most of which is unexplained. ASD has a population prevalence of one percent and affects four times as many males as females. Patients with fragile X E (FRAXE) intellectual disability, which is caused by a silencing of the X-linked gene AFF2, display a number of ASD-like phenotypes. Duplications and deletions at the AFF2 locus have also been reported in cases with moderate intellectual disability and ASD. We hypothesized that other rare X-linked sequence variants at the AFF2 locus might contribute to ASD. We sequenced the AFF2 genomic region in 202 male ASD probands and found that 2.5% of males sequenced had missense mutations at highly conserved evolutionary sites. When compared with the frequency of missense mutations in 5545 X chromosomes from unaffected controls, we saw a statistically significant enrichment in patients with ASD (OR: 4.9; P < 0.014). In addition, we identified rare AFF2 3′ UTR variants at conserved sites which alter gene expression in a luciferase assay. These data suggest that rare variation in AFF2 may be a previously unrecognized ASD susceptibility locus and may help explain some of the male excess of ASD.
Bone disorders are of significant concern due to increase in the median age of our population. Traditionally, bone grafts have been used to restore damaged bone. Synthetic biomaterials are now being used as bone graft substitutes. These biomaterials were initially selected for structural restoration based on their biomechanical properties. Later scaffolds were engineered to be bioactive or bioresorbable to enhance tissue growth. Now scaffolds are designed to induce bone formation and vascularization. These scaffolds are often porous, biodegradable materials that harbor different growth factors, drugs, genes or stem cells. In this review, we highlight recent advances in bone scaffolds and discuss aspects that still need to be improved.
bone scaffolds; biomolecule delivery; osteoinduction; vascularization; biomechanical properties; in vitro and in vivo studies
The bursa aurealis transposon has been used to create transposon insertion libraries of Bacillus anthracis and Staphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessential S. aureus genes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study of S. aureus.
β2 (CD18) integrins with α-chains CD11a, -b, -c, and -d are important adhesion molecules necessary for leukocyte migration and cellular interactions. CD18 deficiency leads to recurrent bacterial infections and poor wound healing due to reduced migration of leukocytes to inflammatory sites. CD8 T cells also upregulate CD11a, CD11b, and CD11c upon activation. However, the role these molecules play for CD8 T cells in vivo is not known. To determine the function of individual β2 integrins, we examined CD8 T cell responses to Listeria monocytogenes infection in CD11a-, CD11b-, and CD11c-deficient mice. The absence of CD11b or CD11c had no effect on the generation of antigen-specific CD8 T cells. In contrast, the magnitude of the primary CD8 T cell response in CD11a-deficient mice was significantly reduced. Moreover, the response in CD11a−/− mice exhibited reduced differentiation of short-lived effector cells (KLRG1hi CD127lo), although cytokine and granzyme B production levels were unaffected. Notably, CD11a deficiency resulted in greatly enhanced generation of CD62L+ central memory cells. Surprisingly, CD8 T cells lacking CD11a mounted a robust secondary response to infection. Taken together, these findings demonstrated that CD11a expression contributes to expansion and differentiation of primary CD8 T cells but may be dispensable for secondary responses to infection.
The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.
Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes.
Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.
Tumour hypoxia is associated with impaired apoptosis, resistance to therapy and poor prognosis. We previously reported that high stromal expression of the endogenous marker of hypoxia, carbonic anhydrase IX (CAIX), is associated with significantly reduced survival in oral squamous cell carcinoma (OSCC). In addition to hypoxia, CAIX expression is regulated by proliferation-associated signalling. We hypothesised that incorporating Ki67, a proliferation marker, into our existing CAIX-based stratification of OSCC would identify patients with the least favourable prognosis.
Surgically resected tumours from 60 OSCC patients were analysed for CAIX, Ki67 and BAX expression using fluorescence immunohistochemistry and automated quantitative analysis (AQUA).
In patients expressing high stromal CAIX (sCAIX), stratification by tumour Ki67 expression revealed significantly distinct survival outcomes (P=0.005). In our OSCC cohort, below-median Ki67 and top-quartile sCAIX expression (Ki67losCAIXhi) were associated with significantly worse disease-specific survival in univariate (HR 7.2 (2.5–20.4), P=0.001) and multivariate (HR 4.2 (1.4–12.8), P=0.011) analyses. Hypoxia is associated with decreased BAX expression; the Ki67losCAIXhi group was more strongly associated with low BAX expression than high sCAIX alone.
These data suggest that combined analysis of tumour Ki67 and sCAIX expression may provide a more clinically relevant assessment of tumour hypoxia in OSCC.
AQUA; CAIX; Ki67; hypoxia; oral squamous cell carcinoma; head and neck squamous cell carcinoma
Seroepidemiological studies of tetanus in Africans have focused mainly on adults especially pregnant women and data on children are scarcely reported. We investigated the seroprevalence of protective immunity level, determined risk factors for non-protection against tetanus, and evaluated the performance of Tetanos Quick Stick® (TQS) among hospitalized children aged 1–9 years in Nigeria. Blood IgG antibody levels to tetanus was determined using enzyme-linked immunosorbent assay (ELISA) in the laboratory and TQS (an immunochromatographic test) at the bedside for 304 children admitted into emergency unit of a tertiary hospital in Ibadan, Nigeria. Demographic information and vaccination history were also collected. TQS results were compared with anti-tetanus antibody measured by ELISA using seroprotection cut-off of 0.1 IU/ml. Seroprevalence of protective level of immunity against tetanus using ELISA and TQS methods was 44.7 and 45.4% respectively. Protective level of immunity increased as age increases. Of the seven potential factors assessed, male gender and being second or more position among mother’s children were independent predictors of non-protective level of immunity. Absence of history of recent tetanus toxoid injection was significantly associated with non-protective level of immunity in univariate analysis but not logistic regression model. The agreement between the ELISA and the TQS results was good with a k coefficient of 0.931. TQS sensitivity was 95.7%, specificity 97.6%, positive predictive value 98.0%, and negative predictive values 96.0%. This study showed that lack of protective immunity against tetanus is common; few demographic characteristics correctly predict non-protection and IgG antibody levels to tetanus was accurately detected by TQS.
protective immunity; tetanus; tetanos quick stick; hospitalized children; rapid screening test
We report the results of whole genome and transcriptome sequencing of tumor and adjacent normal tissue samples from 17 patients with non-small cell lung carcinoma (NSCLC). We identified 3,726 point mutations and over 90 indels in the coding sequence, with an average mutation frequency more than 10-fold higher in smokers than in never-smokers. Novel alterations in genes involved in chromatic modification and DNA repair pathways were identified along with DACH1, CFTR, RELN, ABCB5, and HGF. Deep digital sequencing revealed diverse clonality patterns in both never smokers and smokers. All validated EFGR and KRAS mutations were present in the founder clones, suggesting possible roles in cancer initiation. Analysis revealed 14 fusions including ROS1 and ALK as well as novel metabolic enzymes. Cell cycle and JAK-STAT pathways are significantly altered in lung cancer along with perturbations in 54 genes that are potentially targetable with currently available drugs.
Enzymes are the large biomolecules that are required for the numerous chemical interconversions that sustain life. They accelerate all the metabolic processes in the body and carry out a specific task. Enzymes are highly efficient, which can increase reaction rates by 100 million to 10 billion times faster than any normal chemical reaction. Due to development in recombinant technology and protein engineering, enzymes have evolved as an important molecule that has been widely used in different industrial and therapeutical purposes. Microbial enzymes are currently acquiring much attention with rapid development of enzyme technology. Microbial enzymes are preferred due to their economic feasibility, high yields, consistency, ease of product modification and optimization, regular supply due to absence of seasonal fluctuations, rapid growth of microbes on inexpensive media, stability, and greater catalytic activity. Microbial enzymes play a major role in the diagnosis, treatment, biochemical investigation, and monitoring of various dreaded diseases. Amylase and lipase are two very important enzymes that have been vastly studied and have great importance in different industries and therapeutic industry. In this review, an approach has been made to highlight the importance of different enzymes with special emphasis on amylase and lipase in the different industrial and medical fields.
Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study we have evaluated the effects of 1.0 wt% strontium (Sr) and 1.0 wt% magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr doped β-TCP samples at day 8 which was absent on Mg doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor αvβ3 integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell mediated degradation, however; significantly restricted for Mg doped β-TCP samples. Our present results indicated substrate chemistry controlled osteoclast differentiation and resorptive activity which can be used in designing TCP based resorbable bone substitutes with controlled degradation properties.
Beta-tricalcium phosphate; Dopants; Osteoclast-like cells; Osteoclastogenesis; Resorption lacunae
An elective or a prophylactic lymph node dissection is the removal of the lymph nodes that are normal on physical examination and on radiographic imaging. This type of dissection is not based on the visible disease in the targeted nodal basins, but on the potential of a radiographically occult tumour which can exist. The pathologic results of an elective lymph node dissection may help in predicting the risk of a future recurrence and, in some solid tumours, guide the delivery of the adjuvant therapy and as in this case, may contribute to a pathological diagnosis. The decision to proceed with an elective node dissection is based on the assessment of the risks and benefits of the procedure. The morbidity of the regional lymph node dissection must be balanced against the potential benefit of the procedure. A thyroid papillary microcarcinoma is defined according to the WHO criteria and Shaha as a thyroid tumour which is smaller than 1–1.5cm. Different terms are currently used to define this thyroid cancer such as small, tiny, minute, minimal or occult papillary carcinomas of the thyroid, impalpable thyroid carcinoma and incidental thyroid papillary cancer. A common clinical scenario is the incidental diagnosis of papillary thyroid microcarcinoma (PTMC) on the histology of the resected thyroid, following the surgery which was done for a presumably benign thyroid disease. PTMC was diagnosed in 7.1% of the patients with a presumably benign thyroid disease. It may be possible that this is an underestimation of the true incidence, because we did not use the serial sectioning technique and maybe because the PTMC which was present was so small that it was grossly not identified and sectioned. Herein, a case which was clinically suspicious and was radiologically and cytologically diagnosed as a case of retrosternal multinodular goitre underwent a near total thyroidectomy and a paratracheal lymphnode dissection. The node was found to have micrometastasis of the follicular variant of a papillary carcinoma and the thyroid, on a retrospective step sectioning, revealed an incidental PTMC. This case has been presented, to highlight the possibility of an incidental PTMC in the thyroid cases which were resected for benign disease and the importance of elective lymphnode dissection in contributing to the diagnosis of PTMC.
Incidental papillary microcarcinoma; Prophylactic lymphadenectomy
The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.
apoptosis; ING1; mitochondria; BAX
Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.
Paramyxoviruses comprise a large family of significant pathogens including Newcastle disease virus (NDV), parainfluenza viruses 1-5 (PIV1-5), respiratory syncytial virus, the highly transmissible measles virus, and the emerging and deadly Nipah and Hendra viruses. Five paramyxoviruses are U.S. Department of Health and Human Services and U.S. Department of Agriculture “select agents,” and prevention and/or treatment of these viruses is a public health priority. Paramyxoviruses infect host cells through the concerted action of a “mushroom-shaped” receptor binding protein (HN, H, or G) and fusion protein (F) on the viral surface. However, despite numerous biochemical and structural insights, many details remain unknown about how these proteins interact and the mechanism by which the interaction triggers membrane fusion. Here we present the X-ray crystal structure of the PIV5 HN ectodomain comprised of a large fragment of the stalk and complete head domains. The structure reveals a unique conformation that is a hybrid of that seen in previous NDV ectodomain and PIV5 attachment protein head domain structures. A high-resolution view of the different orientations that head domains can adopt combined with recent biochemical data suggest a simple mechanism for paramyxovirus fusion. These new insights will help guide vaccine and inhibitor discovery efforts for paramyxoviruses.
Infection in primary total joint prostheses is estimated to occur in up to 3% of all surgeries. As a measure to improve the antimicrobial properties of implant materials, silver (Ag) was incorporated into plasma sprayed hydroxyapatite (HA) coatings. To offset potential cytotoxic effects of Ag in the coatings, strontium (Sr) was also added as a binary dopant. HA powder were doped with 2.0 wt% Ag2O, 1.0 wt% SrO and the powder was then heat treated at 800° C. Titanium substrates were coated using a 30 kW plasma spray system equipped with a supersonic nozzle. X-ray diffraction (XRD) confirmed the phase purity and high crystallinity of the coatings. Samples were evaluated for mechanical stability by adhesive bond strength testing. Results show that the addition of dopants did not affect the overall bond strength of the coatings. The antibacterial efficacies of the coatings were tested against Pseudomonas aeruginosa. Samples that contained the Ag2O dopant were found to be highly effective against the bacterial colonization. In vitro cell-material interactions using human fetal osteoblast (hFOB) cells were characterized by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability, field emission scanning electron microscopy (FESEM) for cell morphology and confocal imaging for the important differentiation marker alkaline phosphatase (ALP). Our results showed evidence of cytotoxic effects in the Ag-HA coatings, characterized by poor cellular morphology and cell death and nearly complete impediment of functional ALP activity. The addition of SrO to Ag-HA coatings was able to effectively offset these negative effects and improve the performance when compared to pure HA coated samples.
hydroxyapatite; coating; antibacterial; silver; strontium
Flow fractionation of cells using physical fields to achieve lateral displacement finds wide applications, but its extension to surface molecule-specific separation requires labeling. Here we demonstrate affinity flow fractionation (AFF) where weak, short-range interactions with asymmetric molecular patterns laterally displace cells in a continuous, label-free process. We show that AFF can directly draw neutrophils out of a continuously flowing stream of blood with an unprecedented 400,000-fold depletion of red blood cells, with the sorted cells being highly viable, unactivated, and functionally intact. The lack of background erythrocytes enabled the use of AFF for direct enumeration of neutrophils by a downstream detector, which could distinguish the activation state of neutrophils in blood. The compatibility of AFF with capillary microfluidics and its ability to directly separate cells with high purity and minimal sample preparation will facilitate the design of simple and portable devices for point-of-care diagnostics and quick, cost-effective laboratory analysis.
Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration
(4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.
mitochondrial; quick-prep; restriction enzyme
Influenza A virus (flu) is a respiratory tract pathogen causing high morbidity and mortality among the human population. Nitric oxide (NO) is a cellular mediator involved in tissue damage due to apoptosis of target cells and resulting enhancement of local inflammation. Inducible nitric oxide (iNOS) is involved in the production of NO following infection. Although NO is a key player in the development of exaggerated lung disease during flu infection, the underlying mechanism including the role of NO in apoptosis during infection has not been reported. Similarly, the mechanism of iNOS gene induction during flu infection is not well defined in terms of host trans-activator(s) required for iNOS gene expression. In the current study we have identified kruppel-like factor 6 (KLF6) as a critical transcription factor essential for iNOS gene expression during flu infection. We have also underscored the requirement of iNOS in inducing apoptosis during infection. KLF6 gene silencing in human lung epithelial cells resulted in drastic loss of NO production, iNOS-promoter specific luciferase activity and expression of iNOS mRNA following flu infection. Chromatin immuno-precipitation assay revealed a direct interaction of KLF6 with iNOS promoter during both in vitro and in vivo flu infection of human lung cells and mouse respiratory tract, respectively. Significant reduction in flu mediated apoptosis was noted in KLF6 silenced cells, cells treated with iNOS inhibitor and in primary murine macrophages derived from iNOS knock-out (KO) mice. A similar reduction in apoptosis was noted in the lungs following intra-tracheal flu infection of iNOS KO mice.
Development of Nourseothricin N-acetyl transferase (NAT) as a selection marker for mammalian cells is described. Mammalian cells are acutely susceptible to Nourseothricin, similar to the widely used drug Puromycin, and NAT allows for quick and robust selection of transfected/transduced cells in the presence of Nourseothricin. NAT is compatible with other selection markers puromycin, hygromycin, neomycin, blasticidin, and is a valuable addition to the repertoire of mammalian selection markers.
We have used particulate silver coating on stainless steel to prevent in vivo bacterial infection. Stainless steel is commonly used as an implant material for fracture management. The antimicrobial use of silver has been well documented and studied, therefore the novelty of this research is the use of a particulate coating as well as facing the real world challenges of a fracture repair implant. The variable parameters for applying the coating were time of deposition, silver solution concentration, voltage applied, heat treatment temperature between 400 to 500 °C and time. The resultant coating is shown to be non-toxic to human osteoblasts using an MTT assay for proliferation and SEM images for morphology. In vitro silver release studies of various treatments were done using simulated body fluid. The bactericidal effects were tested by challenging the coatings with P. aeruginosa in a bioreactor and compared against uncoated stainless steel. A 13-fold reduction in bacteria was observed at 24 hours and proved to be statistically significant.
Silver; Antimicrobial; Stainless Steel Implants; Fracture Management