High-resolution optical microscopy for characterising microstructural deformation in microtensile testing.

Imaging surface deformation of a coupon specimen in microtensile testing with an optical microscope presents challenges due to the narrow depth of field (DoF) of optical microscopes. Materials being heterogeneous at microscopic length scale, the sample surface deforms into a complex 3D surface texture, evolving continuously as the loading increases. Because of the narrow DoF, the region that is in focus within the field of view (FoV) decreases substantially in size with the increasing out-of-plane heterogeneous deformation. To address this challenge, a method based on image blending and stabilisation of the captured image frames is proposed. Image blending combines the partial regions that are in focus from a set of successive image frames captured at different working distances from the object surface plane to construct a single image that has a large part of the FoV in focus. The blended images are then obtained at different levels of macroscopic strains, that is the global homogeneous strain, in order to characterise the evolution of the heterogeneous deformation. The image stabilisation removes any misalignments of the blended images by spatially realigning them choosing a common feature as a reference point. The validation of the proposed method with conventionally and additively manufactured stainless steel 316L (SS 316L) specimens demonstrates excellent improvement in image quality. Almost 100% of the FoV is maintained in focus regardless of the amount of out-of-plane heterogeneous deformation caused during tensile testing, which is quite remarkable for optical microscopy imaging. Consequently, the blended and stabilised images enhanced the accuracy of digital image correlation (DIC). Time-lapse videos of the deformation generated using these images captured the evolution of the slip bands and their transmission through twinning boundaries in the stainless steel microstructure. Overall, this study demonstrates the feasibility of using image-processing techniques to advance optical microscopy to image complex 3D surfaces evolving with time.

The association of postoperative pulmonary complications in 109,360 patients with pressure-controlled or volume-controlled ventilation.

We thought that the rate of postoperative pulmonary complications might be higher after pressure-controlled ventilation than after volume-controlled ventilation. We analysed peri-operative data recorded for 109,360 adults, whose lungs were mechanically ventilated during surgery at three hospitals in Massachusetts, USA. We used multivariable regression and propensity score matching. Postoperative pulmonary complications were more common after pressure-controlled ventilation, odds ratio (95%CI) 1.29 (1.21-1.37), p < 0.001. Tidal volumes and driving pressures were more varied with pressure-controlled ventilation compared with volume-controlled ventilation: mean (SD) variance from the median 1.61 (1.36) ml.kg-1 vs. 1.23 (1.11) ml.kg-1 , p < 0.001; and 3.91 (3.47) cmH2 O vs. 3.40 (2.69) cmH2 O, p < 0.001. The odds ratio (95%CI) of pulmonary complications after pressure-controlled ventilation compared with volume-controlled ventilation at positive end-expiratory pressures < 5 cmH2 O was 1.40 (1.26-1.55) and 1.20 (1.11-1.31) when ≥ 5 cmH2 O, both p < 0.001, a relative risk ratio of 1.17 (1.03-1.33), p = 0.023. The odds ratio (95%CI) of pulmonary complications after pressure-controlled ventilation compared with volume-controlled ventilation at driving pressures of < 19 cmH2 O was 1.37 (1.27-1.48), p < 0.001, and 1.16 (1.04-1.30) when ≥ 19 cmH2 O, p = 0.011, a relative risk ratio of 1.18 (1.07-1.30), p = 0.016. Our data support volume-controlled ventilation during surgery, particularly for patients more likely to suffer postoperative pulmonary complications.

Mice lacking the chromodomain helicase DNA-binding 5 chromatin remodeler display autism-like characteristics.

Although autism spectrum disorders (ASDs) share a core set of nosological features, they exhibit substantial genetic heterogeneity. A parsimonious hypothesis posits that dysregulated epigenetic mechanisms represent common pathways in the etiology of ASDs. To investigate this hypothesis, we generated a novel mouse model resulting from brain-specific deletion of chromodomain helicase DNA-binding 5 (Chd5), a chromatin remodeling protein known to regulate neuronal differentiation and a member of a gene family strongly implicated in ASDs. RNA sequencing of Chd5-/- mouse forebrain tissue revealed a preponderance of changes in expression of genes important in cellular development and signaling, sociocommunicative behavior and ASDs. Pyramidal neurons cultured from Chd5-/- cortex displayed alterations in dendritic morphology. Paralleling ASD nosology, Chd5-/- mice exhibited abnormal sociocommunicative behavior and a strong preference for familiarity. Chd5-/- mice further showed deficits in responding to the distress of a conspecific, a mouse homolog of empathy. Thus, dysregulated chromatin remodeling produces a pattern of transcriptional, neuronal and behavioral effects consistent with the presentation of ASDs.

Development of a geometrically accurate and adaptable finite element head model for impact simulation: the Naval Research Laboratory-Simpleware Head Model.

This study demonstrates a novel model generation methodology that addresses several limitations of conventional finite element head models (FEHM). By operating chiefly in image space, new structures can be incorporated or merged, and the mesh either decimated or refined both locally and globally. This methodology is employed in the development of a highly bio-fidelic FEHM from high-resolution scan data. The model is adaptable and presented here in a form optimised for impact and blast simulations. The accuracy and feasibility of the model are successfully demonstrated against a widely used experimental benchmark in impact loading and through the investigation of potential brain injury under blast overpressure loading.

Predictive value of serum actin-free Gc-globulin for complications and outcome in acute liver failure.

This prospective study was designed to evaluate whether early changes in actin-free Gc-globulin levels were associated with complications and outcomes and to identify factors associated with persistent low actin-free Gc-globulin levels in acute liver failure (ALF). Thirty-two consecutive ALF patients admitted from October 2011 to December 2012 were followed up until death or complete recovery. All had serum actin-free Gc-globulin estimation at admission and at day three or expiry. Logistic regression analysis was performed to identify independent predictors of mortality. A receiver operating characteristic curve analysis was also performed. Nonsurvivors had significantly lower median actin-free Gc-globulin levels than survivors (87.32 vs 180 mg/L; P < 0.001). A receiver operating characteristic curve analysis revealed an area under curve (AUC) of 0.771 and showed that serum actin-free Gc-globulin level of ≤124 mg/L would predict mortality with 92% sensitivity and 71.4% specificity. Patients with lower serum actin-free Gc-globulin levels and decreasing trend in serum actin-free Gc-globulin levels were found to have more mortality and developed more complications. Logistic regression analysis showed that serum actin-free Gc-globulin, total leucocyte count and serum creatinine at admission were independent predictors of mortality. Incorporating these variables, a score predicting mortality risk at admission was derived. The scoring system was compared to MELD score and King's College Criteria as individual predictor of mortality. Serum actin-free Gc-globulin level at presentation is predictive of outcome and can be used for risk stratification. Its persistent low-level predicts mortality and is correlated with various complications.

4-N-pyridin-2-yl-benzamide nanotubes compatible with mouse stem cell and oral delivery in Drosophila.

p-aminobenzoic acid (PABA), a structural moiety of many commercial drugs, is self-assembled with linker alkyl side chains to form tubular nanostructures. The tubes exhibited fluorescence either intrinsic or from fluorescent molecules embedded in the wall during self-assembly. Uptake and inter-cellular delivery of the conjugated nanotubes in human cancer cells and in mouse embryonic stem cells were demonstrated by fluorescence imaging and flow cytometry. Biocompatibility, cytotoxicity and clearance were monitored both ex vivo in mouse multipotent embryonic stem cells and in vivo in adult Drosophila. Accumulation of nanotubes had no adverse effects and abnormalities on stem cell morphology and proliferation rate. A distinct distribution of two separate nanotubes in various internal organs of Drosophila interprets that accumulation of nanomaterials might be interdependent on the side chain modifications and physiological settings of cell or tissue types. Unlike carbon nanomaterials, exposure of PABA nanotubes does not produce any hazards including locomotion defects and mortality of adult flies. Despite differential uptake and clearance from multiple live tissues, the use of self-assembled nanotubes can add new dimensions and scope to the development of dual-purpose oral carriers for the fulfilment of many biological promises.

Cross-referral between HIV counselling and testing centres and smear microscopy centres in Tamil Nadu.

BACKGROUND: The combined tuberculosis and human immunodeficiency virus (TB-HIV) epidemic demands effective and urgent action. OBJECTIVE: To assess the effectiveness of the system of referral of TB suspects from the integrated HIV counselling and testing centres (ICTCs) to the designated microscopy centres (DMCs) in Tamil Nadu, and to identify reasons for dropping out. DESIGN: ICTC counsellors identified TB suspects among clients (excluding pregnant women and children) in six districts of Tamil Nadu in 2007 and referred them to DMCs, irrespective of their HIV status. From the records at ICTCs and DMCs, we collected information on the number of referrals to the DMCs, TB suspects attending DMCs and smear-positive TB cases with or without HIV. Clients who did not attend the DMCs were interviewed to elicit reasons for dropping out. RESULTS: Of 18329 clients counselled, 1065 (6%) were identified as TB suspects and referred to DMCs. Of these, 888 (83%) attended and 177 (17%) dropped out; 81% of the drop-outs were interviewed. Reasons for dropping out were multiple: 51% were due to the health system, 62% due to the disease and 62% due to personal reasons. Twelve per cent of DMC attendees were smear-positive. CONCLUSION: The ICTC-to-DMC referral system makes a significant contribution to the detection of TB cases. Reasons for dropping out were multiple, but are correctable. This study also probes into current policies on programme coordination and recommends strategies for strengthening the collaboration between the TB and HIV programmes.

Phosphotidylinositol-3 kinase-mediated signals in mice immunized with the 57 kDa major antigenic outer-membrane protein of Shigella dysenteriae type 1.

Antigen-specific T-cell signalling via T-cell antigen receptor stimulation was carried out in BALB/c mice immunized with the 57 kDa major antigenic component of Shigella dysenteriae 1 outer-membrane proteins. In presence of anti-CD3, the 57 kDa antigen was found to increase the level of IL-2 significantly instead of IL-4. IL-2 production in T cells was consistent with an increase in intracellular free Ca(2+) [(Ca(2+))i] concentration. The antigen-specific modulation was observed during T-cell signalling, with enhanced release of [(Ca(2+))i]. IL-2-receptor stimulation via IL-2 did not significantly induce the release of IL-2 with consistent intracellular Ca(2+) production. Furthermore, the protein tyrosine kinase was activated during anti-CD3 stimulation, which up-regulated the phosphatidylinositol kinase of p85-mediated serine kinase protein kinase-C of p70. Phosphoinositide-specific kinases are regulated by the phosphorylation of tyrosine kinase through the activation of the T-cell antigen receptor. The above findings indicate that phosphotidylinositol-3 kinase-mediated signals are up-regulated through [(Ca(2+))i], which is essential for Th1-type responses.

Evaluation of a dot-immunoblot assay for detecting leishmanial antigen in naturally infected Phlebotomus argentipes (Diptera: Psychodidae).

A simple and highly reproducible dot-immunoblot assay was developed to detect leishmanial antigen in Phlebotomus argentipes that were naturally infected with Leishmania donovani. The test was sensitive to as little as 10 ng of antigenic protein (equivalent to the gut content of one laboratory-infected sandfly) and also appeared to be specific, in that it gave a positive result with some P. argentipes (the primary vector of L. donovani in India) and L. donovani but not with P. papatasi or other pathogens. When used to investigate a large number of sandflies collected from two areas of the Indian state of Bihar where visceral leishmaniasis is endemic, the assay appeared sufficiently sensitive and specific to detect the naturally infected insects. The simplicity, reproducibility, high sensitivity and high specificity of the assay should make it useful for field studies, particularly in determining the prevalence of sandfly infection, the local level of transmission, and the impact of vector-control programmes.

Cytokine release induced by killed bacteria associated with anti-IFN-gamma antibody in Shigella infection.

An effort was made to analyze the effect of in vitro stimulation on macrophages using killed Shigella dysenteriae type-1 (KSD1) coupled with anti-Interferon Gamma (anti-IFN-gamma) antibody. The stimulated macrophages were co-cultured with primed or non-primed T-cells from Shigella infected patients. T-cell cultures were also established by co-culturing KSD1 coupled with or without PHA stimulated macrophages. Emulsified KSD1 coupled with anti-IFN-gamma antibody was found to act as a potent immunogen, inducing the release of Th1 cytokine from primed T-cells cultured in acute stage of the disease. It was observed that the levels of IFN-gamma and IL-2 production rather than IL-4 and IL-6 were increased as the disease became more severe. On comparison, the subsequent values of IL-6, IFN-gamma, IL-4 and IL-2 were found to be less significant in healthy primed T-cell cultures. This was also associated with a substantial production of superoxide ions (O2-), which probably inhibits the colonization of intracellular Shigella due to the presence of anti-IFN-gamma antibodies. On the other hand KSD1 with or without PHA failed to induce such responses. The above findings reflect that in the presence of anti-IFN-gamma antibody, KSD1 acts as a potential immunogen for eliciting cellular immunity against shigellosis.

Role of anti-CD3 in modulation of Th1-type immune response in Shigella dysenteriae infection.

A murine model was used to evaluate the role of anti-CD3 in modulating a Th1-type response by restimulation of T-cells after immunization with the 57 kDa immunodominant antigen of Shigella dysenteriae 1 outer-membrane proteins (OMPs), followed by Shigella infection after immunization. To observe the effect of anti-CD3, other T-cell cultures were also established following anti-CD1, anti-IL2 and phytohaemagglutinin stimulation. Anti-CD3 stimulation of reconstituted T-cells showed 'mean' levels of CD4 and CD25 were enhanced by 34.5 and 31.1 % in immunized mice, which was comparable to 53.2 and 50.7 %, respectively, in challenged-immunized mice, and were dominant over CD8+ T-cells. Levels of IL2 generated by anti-CD3-stimulated T-cells of immunized mice were greater than those of unstimulated T-cells and were significantly elevated in challenged-immunized mice. The reactivity of T-cells indicated their complete responsiveness, as anti-CD3 antibody might not inhibit the migration of the macrophages but rather inhibit IL4. These macrophage factors synergistically act with anions towards an activated response, which in turn provokes IL2 secretion with a low degree of internalization of its receptor. Thus, sharing of IL2 to form a high-affinity receptor complex with CD4+ T-cells through motive signals suggested a generalized T-cell activation with increased humoral responses. Macrophage migration inhibition factor (MIF) and IL4 responses during anti-CD3 stimulation of immunized mice indicated that the role of anti-CD3 in generation of O2- is due to a synergistic effect by Th1 subsets of Th0 cells. The above findings should have implications for understanding the immunoregulatory role of anti-CD3 associated with 57 kDa antigen in immunoprophylactic measures.

The Bispectral Index and induced hypothermia--electrocerebral silence at an unusually high temperature.

The optimal temperature for deep hypothermic circulatory arrest remains undefined. We present a case in which Bispectral Index monitoring during hypothermic cardiopulmonary bypass showed electrocerebral silence at a higher temperature than previously reported. Bispectral Index monitoring may be a potentially useful tool in surgery employing deep hypothermic circulatory arrest.

Characterization of a retrotransposon-like element from Entamoeba histolytica.

The protozoan parasite Entamoeba histolytica is the causative agent of amoebiasis. The genome organization of this organism is not well understood. We had earlier reported the presence of a multicopy sequence, HMc, in E. histolytica. Subsequent analysis showed that HMc is a member of a retrotransposon family that we have named the E. histolytica retrotransposon-like element (EhRLE). Four other members of this family have been characterized. The EhRLE family is distributed across all chromosomes of the parasite. There are 140 copies, which show minor sequence variation with respect to one another (2--4% from the consensus sequence). From a sequence analysis of five members of the EhRLE family, the complete EhRLE unit is estimated to be 4086 bp in length. It has a 27-mer inverted repeat at its ends. A pairwise comparison with sequences in the database showed a highly significant match of a part of EhRLE with reverse transcriptases (RT), especially those encoded by non-long terminal repeat retrotransposons. There are stop codons in all the five EhRLEs, but a continuous open reading frame of 464 amino acids could be reconstructed by comparing the sequences of several EhRLEs. The reconstructed sequence showed a much better identity with RT as compared with any of the original EhRLE sequences. The non-pathogenic species, Entamoeba dispar, also contains this element, with 85% sequence identity with EhRLE. The data suggest that EhRLE may be a retrotransposon, but many of its members are probably nonfunctional due to the accumulation of mutations.

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani.

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.

Alternative medicine--old wine in a new bottle.

Alternative medicine encompasses ancient medical practices of Ayurveda, Unani, Tibbi, chiropractice, acupuncture and others alike. This part of medicine ignores mechanisms of drug action and relies on antique methods and natural remedies. It has no study of pathology, rather depends on philosophy. One must take note of ethno-botany in the evolution of ancient medicines. Ethnobotany reflects the use of drugs available in natural places. Use of natural plants as medicine by the animals is very interesting to observe. Some religious practices like namaz and yoga constitute body exercises and are encouraged in alternative medicine. Chiropractice is a manual mode of treating in painful conditions of back, ankle and knee. Acupuncture is an age old process and practised mainly in China for various ailments.

The genome of Entamoeba histolytica.

Estimation of genome size of Entamoeba histolytica by different methods has failed to give comparable values due to the inherent complexities of the organism, such as the uncertain level of ploidy, presence of multinucleated cells and a poorly demarcated cell division cycle. The genome of E. histolytica has a low G+C content (22.4%), and is composed of both linear chromosomes and a number of circular plasmid-like molecules. The rRNA genes are located exclusively on some of the circular DNAs. Karyotype analysis by pulsed field gel electrophoresis suggests the presence of 14 conserved linkage groups and an extensive size variation between homologous chromosomes from different isolates. Several repeat families have been identified, some of which have been shown to be present in all the electrophoretically separated chromosomes. The typical nucleosomal structure has not been demonstrated, though most of the histone genes have been identified. Most Entamoeba genes lack introns, have short 3' and 5' untranslated regions, and are tightly packed. Promoter analysis revealed the presence of three conserved motifs and several upstream regulatory elements. Unlike typical eukaryotes, the transcription of protein coding genes is alpha-amanitin resistant. Expressed Sequence Tag analysis has identified a group of highly abundant polyadenylated RNAs which are unlikely to be translated. The Expressed Sequence Tag approach has also helped identify several important genes which encode proteins that may be involved in different biochemical pathways, signal transduction mechanisms and organellar functions.

Lack of a chromosomal copy of the circular rDNA plasmid of Entamoeba histolytica.

A number of small circular DNAs constitute a part of the genome of Entamoeba histolytica. Among them, the 24.5 kb circular DNA encoding rRNA (EhR1) is the most abundant. Pulsed field gel electrophoresis was used to determine if a chromosomal copy of EhR1 exists and what fraction of the total genome is circular. The results show that the chromosomes of E. histolytica are linear, and that no copy of EhR1 could be detected in any of the linear chromosomes.