Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide

Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

A novel role for Bruton's tyrosine kinase in hepatocyte growth factor-mediated immunoregulation of dendritic cells

The limited success of dendritic cell (DC)-based immunotherapy in multiple myeloma is partly due to hepatocyte growth factor (HGF)-induced DC dysfunction. From a therapeutic standpoint, it is important to understand the molecular events involved in inhibition of DC activation/maturation by HGF. Because Bruton's tyrosine kinase (Btk) negatively regulates maturation and immunostimulatory function of DCs, a role for Btk in HGF-induced inhibition of both murine and human DCs was investigated. We demonstrate that Btk is a novel proximal component of HGF-induced c-MET (HGF receptor) signaling. Following HGF treatment, Btk binds to c-MET and becomes activated. Btk activation in turn blocks the NF-κB pathway and subsequent DC activation via the c-Src-PI3K-AKT-mammalian target of rapamycin (mTOR) pathway. Notably, Btk activation is necessary for HGF-induced association of c-Src and PI3K with c-MET. Furthermore, we provide the first evidence that HGF inhibits DC activation by inducing autocrine interleukin (IL)-10 secretion, which requires activation of Btk. Blocking activation of Btk and its downstream the c-Src-PI3K-AKT-mTOR pathway prevents HGF-induced IL-10 secretion by DCs. In addition, neutralization of IL-10 secretion from DCs impaired the inhibitory effect of HGF on DCs. Thus, our study identifies a novel role for Btk in HGF-induced DC inhibition.     

Serotype 1a O-Antigen Modification: Molecular Characterization of the Genes Involved and Their Novel Organization in the Shigella flexneri Chromosome

The factors responsible for serotype 1a O-antigen modification in Shigella flexneri were localized to a 5.8-kb chromosomal HindIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the dsdC and the thrW proA genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.     

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes

Background

Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.

Methodology/Principal findings

Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.

Conclusions/Significance

Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.     

Predictors and Outcome of Hypoxemia in Severely Malnourished Children under Five with Pneumonia: A Case Control Design

Background

There is lack of information in the medical literature on predictors of hypoxemia in severely malnourished children with pneumonia, although hypoxemia is common and is often associated with fatal outcome in this population. We explored the predictors of hypoxemia in under-five children who were hospitalized for the management of pneumonia and severe acute malnutrition (SAM).

Methods

In this unmatched case-control design, SAM children of both sexes, aged 0–59 months, admitted to the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) with radiological pneumonia and hypoxemia during April 2011 to April 2012 were studied. SAM children with pneumonia and hypoxemia (SpO₂<90%) constituted the cases (n = 37), and randomly selected SAM children with pneumonia but without hypoxemia constituted controls (n = 111).

Results

The case-fatality was significantly higher among the cases than the controls (30% vs. 4%; p<0.001). In logistic regression analysis, after adjusting for potential confounders such as nasal flaring, head nodding, inability to drink, and crackles in lungs, fast breathing (95% CI = 1.09–13.55), lower chest wall in-drawing (95% CI = 2.48–43.41), and convulsion at admission (95% CI = 3.14–234.01) were identified as independent predictors of hypoxemia in this population. The sensitivity of fast breathing, lower chest wall in-drawing and convulsion at admission and their 95% confidence intervals (CI) to predict hypoxemia were 84 (67–93)%, 89 (74–96)%, and 19 (9–36)% respectively, and their specificity were 53 (43–63)%, 60 (51–69)% and 98 (93–100)% respectively.

Conclusion and Significance

Fast breathing and lower chest wall in-drawing were the best predictors of hypoxemia in SAM children with pneumonia. There thus, in resources poor settings where pulse oximetry is not available, identification of these simple clinical predictors of hypoxemia in such children could be reliably used for early O₂ supplementation in addition to other appropriate management to reduce morbidity and deaths.     

Catechin and Catechin Fractions as Biochemical Markers to Study the Diversity of Indian Tea (Camellia sinensis (L.) O. Kuntze) Germplasm

The heterogeneous Indian tea germplasm includes ‘China’, ‘Assam’, ‘Cambod’, and their hybrids which were evaluated using biochemical markers viz., total catechin and their fractions, for varietal identification and characterization. Principal component analysis (PCA) of biochemical characters showed that the total catechin and trihydroxylated catechin has higher eigenvalues. The first two principal components (PCs) could differentiate more than 90% of the clones studied. This grouping based on first two principal component matrices differentiated ‘China’, and their hybrids with ‘Assam’ and ‘Cambod’ variety. Morphologically indistinct large‐leaved ‘Cambod’ variety and ‘Assam’ varieties could not be differentiated using biochemical markers, since both varietal types taxonomically belong to a single species. Clones of ‘China’ type showed low total catechin content and catechin ratio which are distinctly grouped. The ‘China–Assam’ and ‘China–Cambod’ hybrids formed intermediate groups between ‘China’ PC group and ‘Cambod’/‘Assam’ PC groups, providing evidence for genetic control of catechin ratio variation. Tea clones which are differentially positioned in the PC group could be explained based on the genetic contribution by other varietal type as parents. This biochemical characterization will be a useful tool in the development of quality‐tea clones with different proportion of total catechin and their fractions.     

Neue chalkon-derivate aus südafrikanischen Helichrysum-arten

The investigation of two South African Helichrysum species afforded in addition to known compounds two new chalcone derivatives, four related dihydrochalcones,5,7,8-trimethoxyflavone and 5-methoxy-7,8-methylenedioxyflavone. The structures are elucidated by spectroscopic methods and some chemical transformations. The chemotaxonomic situation in the large genus Helichrysum is discussed, in the light of these results.