The structural insights of 16.3 kDa heat shock protein (HSP16.3) from Mycobacterium tuberculosis via in silico study

Mycobacterium tuberculosis (Mtb) is capable of surviving in dormancy before developing to tuberculosis (TB). One of the major challenges of TB management is the identification of patients, making TB diagnosis critical for disease management. This study focuses on the 16 kDa heat shock protein (HSP16.3; a potential biomarker for latent TB infection) that is expressed during the latent phase of Mtb growth. In order to explore the dynamics and interactions of HSP16.3, the 3-D structure of HSP16.3 was built via comparative modelling. The predicted structure shows a predominantly beta-sheet dodecamer with alpha-helical folds at its N-terminal. A known protein-hydrophobic probe (1,1′-Bi(4-anilino)naphthalene-5,5′-disulfonic acid; bisANS) was docked to the HSP16.3 model. Interacting residues predicted from docking and MD simulations are in good accordance with experimental data reported in the literature. MMPBSA calculation from MD simulation also showed favourable binding free energy of ?29.90 kcal/mol, driven mainly by van der waals and non-polar solvation energies. The statistical evaluation and results from the computational study on HSP16.3 indicate the reliability of the built model, which is potentially useful for further structural studies of HSP16.3 for latent TB diagnostics.     

Multiplexed labeling system for high-throughput cell sorting

Flow cytometry and fluorescence activated cell sorting techniques were designed to realize configurable classification and separation of target cells. A number of cell phenotypes with different functionalities have recently been revealed. Before simultaneous selective capture of cells, it is desirable to label different samples with the corresponding dyes in a multiplexing manner to allow for a single analysis. However, few methods to obtain multiple fluorescent colors for various cell types have been developed. Even when restricted laser sources are employed, a small number of color codes can be expressed simultaneously. In this study, we demonstrate the ability to manifest DNA nanostructure-based multifluorescent colors formed by a complex of dyes. Highly precise self-assembly of fluorescent dye-conjugated oligonucleotides gives anisotropic DNA nanostructures, Y- and tree-shaped DNA (Y-DNA and T-DNA, respectively), which may be used as platforms for fluorescent codes. As a proof of concept, we have demonstrated seven different fluorescent codes with only two different fluorescent dyes using T-DNA. This method provides maximum efficiency for current flow cytometry. We are confident that this system will provide highly efficient multiplexed fluorescent detection for bioanalysis compared with one-to-one fluorescent correspondence for specific marker detection.     

Phenotyping of UGT1A1 Activity Using Raltegravir Predicts Pharmacokinetics and Toxicity of Irinotecan in FOLFIRI

Background

Irinotecan toxicity correlates with UGT1A1 activity. We explored whether phenotyping UGT1A1 using a probe approach works better than current genotyping methods.

Methods

Twenty-four Asian cancer patients received irinotecan as part of the FOLFIRI regimen. Subjects took raltegravir 400 mg orally and intravenous midazolam 1 mg. Pharmacokinetic analyses were performed using WinNonLin and NONMEM. Genomic DNA was isolated and screened for the known genetic variants in UGT1A1 and CYP3A4/5.

Results

SN-38G/SN-38 AUC ratio correlated well with Raltegravir glucuronide/ Raltegravir AUC ratio (r = 0.784 p<0.01). Midazolam clearance correlated well with irinotecan clearance (r = 0.563 p<0.01). SN-38 AUC correlated well with Log10Nadir Absolute Neutrophil Count (ANC) (r = -0.397 p<0.05). Significant correlation was found between nadir ANC and formation rate constant of raltegravir glucuronide (r = 0.598, P<0.005), but not UGT1A1 genotype.

Conclusion

Raltegravir glucuronide formation is a good predictor of nadir ANC, and can predict neutropenia in East Asian patients. Prospective studies with dose adjustments should be done to develop raltegravir as a probe to optimize irinotecan therapy.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00808184","term_id":"NCT00808184"}}NCT00808184     

Analysis of the recurrence risk factors for the patients with hepatocellular carcinoma meeting University of California San Francisco criteria after curative hepatectomy

We report a rare male case of an undifferentiated carcinoma with osteoclast-like giant cells originating in an indeterminate mucin-producing cystic neoplasm of the pancreas. A 59-year-old Japanese man with diabetes visited our hospital, complaining of fullness in the upper abdomen. A laboratory analysis revealed anemia (Hemoglobin; 9.7 g/dl) and elevated C-reactive protein (3.01 mg/dl). Carbohydrate antigen 19-9 was 274 U/ml and Carcinoembryonic antigen was 29.6 ng/ml. A computed tomography scan of the abdomen revealed a 14-cm cystic mass in the upper left quadrant of the abdomen that appeared to originate from the pancreatic tail. The patient underwent distal pancreatectomy/splenectomy/total gastrectomy/cholecystectomy. The mass consisted of a multilocular cystic lesion. Microscopically, the cyst was lined by cuboidal or columnar epithelium, including mucinous epithelium. Sarcomatous mononuclear cells and multinucleated osteoclast-like giant cells were found in the stroma. Ovarian-type stroma was not seen. We made a diagnosis of osteoclast-like giant cell tumor originating in an indeterminate mucin-producing cystic neoplasm of the pancreas. All surgical margins were negative, however, two peripancreatic lymph nodes were positive. The patient recovered uneventfully. Two months after the operation, multiple metastases occurred in the liver. He died 4 months after the operation.     

Expression of perforin by natural killer cells within first trimester endometrium in humans.

The lymphocyte pore-forming protein (PFP)--perforin, also named cytolysin--is a potent mediator of cytotoxicity found in the granules of cytotoxic T lymphocytes and natural killer (NK) cells. Granulated metrial gland (GMG) cells found in the pregnant mouse uterus express perforin and are thought to be highly activated cytolytic lymphocytes related to NK cells. Their role in pregnancy is unknown. Human endometrial granulocytes (EGs) are phenotypically similar to murine GMG cells and, like them, express NK cell markers. However, up to now it was not known whether EGs also express perforin. By means of immunohistochemical analysis, using antisera specific for perforin and monoclonal antibodies to CD56 (NKH-1), CD2 (T11), CD3 (Leu-4), CD4 (Leu-3a), and CD8 (OKT-8), we demonstrated that perforin is present in EGs in the decidualized endometrial stroma and decidual tissue of first-trimester gestational endometrium. In fact, double immunohistochemical labeling demonstrated the co-expression of perforin and NKH-1. This population also expressed some T-cell surface antigens (Leu-4 and T11), but not Leu-3a or OKT-8. Chorionic villi, in contrast, lack perforin+ cells. The presence of a potent cytolytic mediator in NK-like cells in both murine and human pregnant uterus raises the issue of the function of such cells in pregnancy.     

Predictive evaluation for the preparation of a synthetic Y-shaped DNA nanostructure

With the advent of deoxyribonucleic acid (DNA) nanotechnology, the Y-shaped DNA nanostructure (Y-DNA) as a basic block was first created. Due to their characteristic selectivity and specificity, Y-DNA-based materials have been utilized in a variety of scientific fields including multiplexed nanobarcoding. Basically, the tripod DNA nanostructure was prepared by simple hybridization of three different single stranded DNA (ssDNA). Before the synthetic process, the optical densities (OD) of the three ssDNAs were measured to accurately estimate the concentration. Through repeated temperature fluctuations, three ssDNAs were hybridized into a Y-shaped block with both a central junction and three blunt ended arms. After the reaction, the ODs of the synthesized DNA products were measured and compared with the theoretical OD values calculated by a MATLAB program (‘matrix laboratory’) with different molar concentrations and volumes to predict the presence of Y-DNA. Simultaneously, the product was analyzed by agarose gel electrophoresis to confirm the YDNA structure. The measured ODs of the solutions with confirmed Y-DNA structures were close to the theoretical maximum OD values. This article provides means to help understand and prepare Y-DNA by performing OD measurements. It is highly expected that this guide will be an excellent starting point for structural DNA nanotechnology.     

Induction of Type I Interferon Signaling Determines the Relative Pathogenicity of Staphylococcus aureus Strains

The tremendous success of S. aureus as a human pathogen has been explained primarily by its array of virulence factors that enable the organism to evade host immunity. Perhaps equally important, but less well understood, is the importance of the intensity of the host response in determining the extent of pathology induced by S. aureus infection, particularly in the pathogenesis of pneumonia. We compared the pathogenesis of infection caused by two phylogenetically and epidemiologically distinct strains of S. aureus whose behavior in humans has been well characterized. Induction of the type I IFN cascade by strain 502A, due to a NOD2-IRF5 pathway, was the major factor in causing severe pneumonia and death in a murine model of pneumonia and was associated with autolysis and release of peptidogylcan. In contrast to USA300, 502A was readily eliminated from epithelial surfaces in vitro. Nonetheless, 502A caused significantly increased tissue damage due to the organisms that were able to invade systemically and trigger type I IFN responses, and this was ameliorated in Ifnar ^-/- mice. The success of USA300 to cause invasive infection appears to depend upon its resistance to eradication from epithelial surfaces, but not production of specific toxins. Our studies illustrate the important and highly variable role of type I IFN signaling within a species and suggest that targeted immunomodulation of specific innate immune signaling cascades may be useful to prevent the excessive morbidity associated with S. aureus pneumonia.     

Adaptive evolution of tetrodotoxin resistance in animals

Tetrodotoxin (TTX), first isolated from pufferfish (tetraodontids), is a highly potent neurotoxin that selectively binds to voltage-gated sodium channels (Na(v)) in muscle and nerve tissues causing paralysis and death. Saxitoxin (STX) is a TTX-related neurotoxin produced by dinoflagellates. Recent investigations have implicated diverse substitutions in the P-loop regions of skeletal muscle and neuronal Na(v) channels in the convergent evolution of neurotoxin resistance in pufferfish, garter snakes and softshell clams, which has enabled them to feed on TTX- and STX-bearing organisms.