In vitro and in vivo anticancer activity of Indigofera cassioides Rottl. Ex. DC

ObjectiveTo evaluate the antitumor, cytotoxic and antioxidant activities of methanolic leaf extract of Indigofera cassioides (MEIC) against transplantable tumors and human cancer cell lines.MethodsMEIC was investigated for its short term cytotoxicity on EAC and DLA cells by trypan blue dye exclusion method and in vitro cytotoxicity on HeLa, HEp-2, HEpG-2, MCF-7, HT-29, Vero and NIH 3T3 cells by MTT assay. In vivo antitumor activity was studied on EAC and DLA tumor bearing mice. Activity was assessed by monitoring the mean survival time, effect on hematological parameters, antioxidant enzyme levels and solid tumor volume.ResultsMEIC exhibit potent in vitro cytotoxicity against all the tested cancer cell lines, but it was found to be safe on normal cells. The extract significantly (P < 0.001) increase the mean survival time and also have a protective effect on the hemopoietic system at the tested dose levels (200 and 400 mg/kg). The extract prevented lipid peroxidation and restored the antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase in the liver of tumor control animals. It also significantly (P < 0.01) reduce the solid tumor volume.ConclusionsThe results strongly support that MEIC shows potent antitumor and cytotoxic effects against EAC, DLA and human cancer cell lines. The extract prevents lipid peroxidation and promotes the enzymatic antioxidant defense system in tumor bearing animals which might be due to activities like scavenging of free radicals by the phytochemicals in MEIC.     

Multi-dimensional MR spectroscopy: towards a better understanding of hepatic encephalopathy

Hepatic encephalopathy (HE) is normally diagnosed by neuropsychological (NP) tests. The goals of this study were to quantify cerebral metabolites, separate glutamate (Glu) from glutamine (Gln) in patients with minimal hepatic encephalopathy (MHE) as well as healthy subjects using the prior-knowledge fitting (ProFit) algorithm on data acquired by two-dimensional (2D) localized correlated spectroscopy (L-COSY) on two different MR scanners, and to correlate the metabolite changes with neuropsychological (NP) tests. We studied 14 MHE patients and 18 healthy controls using a GE 1.5?T Signa MR scanner. Another group of 16 MHE patients and 18 healthy controls were studied using a Siemens 1.5?T Avanto MR scanner. The following parameters were used for L-COSY: TR/TE?=?2?s/30?ms, 3?×?3?×?3?cm(3) voxel size, 96 Δt(1) increments with 8 averages per Δt(1). Using the ProFit algorithm, we were able to differentiate Gln from Glu on the GE 1.5?T data in the medial frontal white/gray matter. The ratios of myo-inositol (mI), Glu, total choline, scyllo-inositol (sI), phosphoethanolamine (PE), and total N-acetyl aspartate (NAA) showed statistically significant decline in HE patients compared to healthy controls, while the ratio of Gln was significantly increased. Similar trend was seen in the ProFit quantified Siemens 1.5?T data in the frontal and occipito-parietal white/gray regions. Among the NP domain scores, motor function, cognitive speed, executive function and the global scores showed significant differences. Excellent correlations between various NP domains and metabolite ratios were also observed. ProFit based cerebral metabolite quantitation enhances the understanding and basis of the current hypothesis of MHE.     

Phage lysin to supplement phagebiotics to decontaminate processed sputum specimens

The overgrowth of normal flora escaping the action of sputum processing chemicals is the major problem in broth-based tuberculosis (TB) detection systems. The use of phages to control the overgrowth of normal flora in processed sputum samples has already been established. Phage lysin and its supplementation to phagebiotics for the effective control of normal flora in sputum specimens were evaluated. Crude lysin was prepared from phage host mixture using standard procedures. About 120 sputum samples processed with 4% NaOH were collected and used to evaluate the effect of lysin, phagebiotics and phagebiotics supplemented with lysin on the overgrowth of normal flora. The effect of phagebiotics and lysin on the growth and retrieval of Mycobacterium tuberculosis was studied by conventional methods and the luciferase reporter phage (LRP) assay. Lysin alone and phagebiotics supplemented with lysin arrested the growth of normal flora in a significantly greater number of samples than phagebiotics alone. Lysin and phagebiotics did not show any inhibitory activity on M. tuberculosis. The use of antibiotics can be replaced by lysin or phagebiotics supplemented with lysin to control the overgrowth of normal flora in processed sputum samples without hampering the viability of M. tuberculosis.     

Pathogenicity Islands PAPI-1 and PAPI-2 Contribute Individually and Synergistically to the Virulence of Pseudomonas aeruginosa Strain PA14

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and severe chronic lung infections in cystic fibrosis patients. The reference strains PA14 and PAO1 have been studied extensively, revealing that PA14 is more virulent than PAO1 in diverse infection models. Among other factors, this may be due to two pathogenicity islands, PAPI-1 and PAPI-2, both present in PA14 but not in PAO1. We compared the global contributions to virulence of PAPI-1 and PAPI-2, rather than that of individual island-borne genes, using murine models of acute pneumonia and bacteremia. Three isogenic island-minus mutants (PAPI-1-minus, PAPI-2-minus, and PAPI-1-minus, PAPI-2-minus mutants) were compared with the wild-type parent strain PA14 and with PAO1. Our results showed that both islands contributed significantly to the virulence of PA14 in acute pneumonia and bacteremia models. However, in contrast to the results for the bacteremia model, where each island was found to contribute individually, loss of the 108-kb PAPI-1 island alone was insufficient to measurably attenuate the mutant in the acute pneumonia model. Nevertheless, the double mutant was substantially more attenuated, and exhibited a lesser degree of virulence, than even PAO1 in the acute pneumonia model. In particular, its ability to disseminate from the lungs to the bloodstream was markedly inhibited. We conclude that both PAPI-1 and PAPI-2 contribute directly and synergistically in a major way to the virulence of PA14, and we suggest that analysis of island-minus strains may be a more appropriate way than individual gene knockouts to assess the contributions to virulence of large, horizontally acquired segments of DNA.Pseudomonas aeruginosa is an environmentally ubiquitous opportunistic pathogen that causes a wide range of acute life-threatening infections, especially in immunocompromised patients. Hospitalized patients with damaged airways due to mechanical ventilation, injury, or viral infections are at significant risk of acute pneumonia caused by P. aeruginosa (51). Resulting cases of ventilator-associated pneumonia are associated with very high rates of mortality (12). In addition, this bacterium is a common cause of chronic respiratory infection in patients with cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and non-CF bronchiectasis (43, 50). A large variety of extracellular virulence factors, including proteases, hemolysins, pyocyanin, pili, lipopolysaccharide, alginate, and the type III secretion system (T3SS) effector proteins, ExoS, ExoT, ExoU, and ExoY, are associated with P. aeruginosa (52, 62).P. aeruginosa PA14 is a fully sequenced, highly virulent wild-type (WT) reference strain that has been used extensively to study the contribution of putative virulence factors to disease (31). Furthermore, as defined through study of a large panel of strains representative of this species, PA14 was found to belong to the most common P. aeruginosa global lineage (61). Another fully sequenced strain, PAO1, is widely regarded as a laboratory strain of P. aeruginosa. PA14 has been shown to be much more virulent than PAO1 in a number of diverse models of infection, leading to the hypothesis that PA14 is a multihost pathogen capable of infecting invertebrate and vertebrate animal species and plant species (29). The genomes of individual P. aeruginosa strains have a highly mosaic structure made up of conserved cores and variable accessory genomes (36, 61), resulting in composite genomes of 5.2 to 7 Mb (36). The majority of large-scale genomic differences arise from the presence or absence of an expanding list of genomic islands within conserved “hot spots” throughout the genome (36). PA14 carries two well-characterized pathogenicity islands: PAPI-1, a 108-kb island integrated within a lysine tRNA gene (PA4541.1), and PAPI-2, a much smaller, 11-kb island inserted into a sequence-identical but distinct tRNA^Lys gene (PA0976.1) (19). In addition to targeting integration sites bearing identical sequences, these two islands share a limited but recognizable degree of synteny and code for factors that have been implicated in pathogenesis in murine, plant, and nematode models of infection (19, 31, 37, 52). Interestingly, islands with significant homology to PAPI-1 and/or PAPI-2 have been identified in many P. aeruginosa strains and several other bacterial species (5, 6, 19, 23, 27, 30, 61, 63) (see Tables S2 and S3 and Fig. S2 in the supplemental material).PAPI-1 is a member of the increasingly populated pKLC102/PAGI-2 island family (26). PAPI-1 encodes a number of likely virulence factors, including type IVB pili, the cupD1 to cupD5 fimbrial biogenesis gene cluster (39), and PvrR, a two-component response regulator involved in biofilm synthesis and antibiotic resistance (11, 38). However, the large majority of PAPI-1 predicted proteins have no assigned function, and many of these are encoded by homologues on related islands in other P. aeruginosa strains (27, 63). PAGI-5, a hybrid island that shares 79 of 121 predicted open reading frames (ORFs) with PAPI-1, has recently been implicated in acute murine pneumonia (5). Isogenic mutants harboring deletions within NR-I or NR-II, PAGI-5-specific novel regions, were shown to be attenuated in a model of acute murine pneumonia, demonstrating that an island in the pKLC102/PAGI-2 family played a role in this infection.PAPI-2 is a stably integrated island belonging to the ExoU island family (30). This island encodes the cytotoxin ExoU, a potent phospholipase, and its cognate chaperone SpcU. ExoU has been studied extensively and has been shown to be the most potent of the known T3SS effector proteins in P. aeruginosa (54). The introduction of exoU into strains that lacked this gene resulted in marked increases in virulence in a murine acute pneumonia model (1). Recent work has suggested that ExoU impairs host response in the lung through targeted killing of phagocytes, which play a crucial role during the early phase of infection (10). Additionally, the secretion of ExoU by an infecting strain has been shown to be a marker of high virulence and poor clinical outcome in hospital-acquired pneumonia (52).Since genomic islands are widely believed to be acquired and/or lost as whole units, and since PAPI-1-like islands are known to be widespread and possibly highly mobile (46), we investigated the contributions of PAPI-1 and PAPI-2 to virulence as single unitary modules rather than defining the roles of individual island-borne genes (5, 19). We postulated that this en bloc deletion approach would reveal combinatorial and/or synergistic effects that would be missed by single-gene knockout approaches. To pursue this goal, we created three isogenic whole-island deletion mutants that lacked either PAPI-1 (PA14ΔPAPI-1), PAPI-2 (PA14ΔPAPI-2), or both (PA14Δ1Δ2) in a PA14 background and examined these relative to their wild-type parent and the laboratory strain PAO1, which naturally lacks both islands. Our results revealed that both PAPI-1 and PAPI-2 contributed individually and synergistically to the virulence of P. aeruginosa strain PA14 in murine models of acute pneumonia and bacteremia.     

Impact of Sugar Factory Effluent on the Growth and Biochemical Characteristics of Terrestrial and Aquatic Plants

The physico-chemical characteristics of sugar industry effluent were measured and some were found to be above those limits permissible in the Indian irrigation water standard. A pot study was initially conducted to study the effects of different concentrations (20%, 40%, 60%, 80% and 100%) of sugar factory effluent on seed germination, seedling growth and biochemical characteristics of green gram and maize. A similar study was also carried out using the aquatic plants, water hyacinth and water lettuce. The higher effluent concentrations (above 60%) were found to affect plant growth, but diluted effluent (up to 60%) favored seedling growth.     

Identification and characterization of nematode specific protective epitopes of Brugia malayi TRX towards development of synthetic vaccine construct for lymphatic filariasis

Although multi-epitope vaccines have been evaluated for various diseases, they have not yet been investigated for lymphatic filariasis. Here, we report for the first time identification of two immunodominant B epitopes (TRX_P1 and TRX_P2) from the antioxidant Brugia malayi thioredoxin by studying their immune responses in mice model and human subjects. TRX_P1 was also found to harbor a T epitope recognized by human PBMCs and mice splenocytes. Further, the epitopic peptides were synthesized as a single peptide conjugate (PC1) and their prophylactic efficacy was tested in a murine model of filariasis with L3 larvae. PC1 conferred a significantly high protection (75.14%) (P < 0.0001) compared to control (3.7%) and recombinant TRX (63.03%) (P < 0.018) in experimental filariasis. Our results suggest that multi-epitope vaccines could be a promising strategy in the control of lymphatic filariasis.     

Hydrogen sulfide producing enzymes in pregnancy and preeclampsia

Preeclampsia, a human pregnancy specific disorder is characterized by an anti-angiogenic state. As hydrogen sulfide (H(2)S) has pro-angiogenic and anti-oxidative characteristics, we hypothesized that H(2)S levels could play a role in the pathogenesis of preeclampsia and studied the placental expression of the H(2)S-producing enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS). CBS and CSE protein are expressed in the fetal-placental endothelium and CBS only in Hofbauer cells. CBS mRNA expression is decreased (p = 0.002) in early-onset preeclampsia, while CSE mRNA is unchanged. Thus, down regulation of CBS during early-onset preeclampsia may result in less H(2)S-production and may aid in the anti-angiogenic state.