A concept of dual-responsive prodrugs based on oligomerization-controlled reactivity of ester groups: an improvement of cancer cells versus neutrophils selectivity of camptothecin.

Many known chemotherapeutic anticancer agents exhibit neutropenia as a dose-limiting side effect. In this paper we suggest a prodrug concept solving this problem for camptothecin (HO-cpt). The prodrug is programmed according to Boolean "AND" logic. In the absence of H2O2 (trigger T1), e.g. in the majority of normal cells, it exists as an inactive oligomer. In cancer cells and in primed neutrophils (high H2O2), the oligomer is disrupted forming intermediate (inactive) lipophilic cationic species. These are accumulated in mitochondria (Mit) of cancer cells, where they are activated by hydrolysis at mitochondrial pH 8 (trigger T2) with formation of camptothecin. In contrast, the intermediates remain stable in neutrophils lacking Mit and therefore a source of T2. In this paper we demonstrated a proof-of-concept. Our prodrug exhibits antitumor activity both in vitro and in vivo, but is not toxic to normal cell and neutrophils in contrast to known single trigger prodrugs and the parent drug HO-cpt.

Disease spectrum and its molecular characterisation in the lentil production system of lower-Indo Gangetic plains.

Lentil is a food legume grown in the Indo-Gangetic plains including lower Gangetic Bengal (LGB). Lentil productivity in this zone is severely impeded because of the prevalence of several biotic cues. Plausible reports regarding the status of disease scenario and the associated risk factors are missing. Therefore, judicious crop management strategies are lacking. An intensive survey of 267 farmers' fields was conducted over 3 years in major lentil-growing districts of LGB to evaluate the disease incidence and prevalence. Additional insights were generated, apprehending isolation and characterisation of associated pathogens through spore morphology and molecular markers as well as elucidating the role of biophysical factors in influencing disease development. Climate change has shifted the disease dimension of lentil and precipitated new disease complexes of great risk, which was reflected through geospatial mapping results in the present study. The prevalence of three major diseases, namely collar rot (Sclerotium rolfsii), lentil blight complex (LBC) incited by both Alternaria and Stemphylium, and lentil rust (Uromyces viciae-fabae), was ascertained through cultural and molecular studies and contextualised through pathogenicity appraisal. This study is the first to investigate the complex mixed infection of Alternaria alternata and Stemphylium botryosum, successfully isolating S. botyrosum in India, and confirming the pathogens through sequencing by using internal transcribed spacer (ITS) primers and Stemphylium-specific Glycerol-3-phosphate dehydrogenase 1 (gpd1) and gpd2 primers. Unlike late planting, early planting promoted collar rot infestation. LBC and rust incidence were magnified in late planting. Soil texture resulted in the spatial distribution of collar rot disease. The surveyed data also highlighted the potential role of resistant cultivars and cropping pattern intervention to ensure associational resistance towards addressing the disease bottleneck in lentil.

Genetic diversity of Ralstonia solanacearum causing vascular bacterial wilt under different agro-climatic regions of West Bengal, India.

The bacterial wilt disease of solanaceous crops incited by Ralstonia solanacearum is a menace to the production of solanaceous vegetables all over the world. Among the agro climatic zones of West Bengal, India growing solanaceous vegetables, the maximum and minimum incidence of bacterial wilt was observed in Red and Lateritic zone (42.4%) and Coastal and Saline zone (26.9%), respectively. The present investigation reports the occurrence of bacterial wilt of Bottle gourd by R. solanacearum Sequevar 1-48 for the first time in India. Two new biovars (6 and 3b) along with biovar 3 have been found to be prevalent in West Bengal. Under West Bengal condition, the most predominant Sequevar was I-48 (75%) followed by I-47 (25%). Low genetic variation (18.9%) among agro climatic zones (ACZs) compared to high genetic variation (81.1%) within revealed occurrence of gene flow among these ACZs. Standard genetic diversity indices based on the concatenated sequences of the seven genes revealed ACZ-6 as highly diverse among five agro climatic zones. The multi locus sequence analysis illustrated occurrence of synonymous or purifying selection in the selected genes in West Bengal and across world. Under West Bengal conditions maximum nucleotide diversity was observed for the gene gyrB. Occurrence of significant recombination was confirmed by pairwise homoplasy test (θ = 0.47*) among the RSSC isolates of West Bengal, belonging to Phylotype I. Phylotype I isolates of West Bengal are involved in exchange of genetic material with Phylotype II isolates. In case of worldwide RSSC collection, eleven significant recombination events were observed among the five phylotypes. Phylotype IV was genetically most diverse among all the Phylotypes. The most recombinogenic phylotype was Phylotype III. Further, the most diverse gene contributing to the evolution of RSSC worldwide was observed to be endoglucanase (egl).

A noncovalent hybrid of [Pd(phen)(OAc)2] and st-DNA for the enantioselective hydroamination of ß-nitrostyrene with methoxyamine.

We developed a novel Pd-catalysed enantioselective synthesis of C-N bonds using the chiral scaffold of DNA. The non-covalently linked [Pd(phen)(OAc)2] with st-DNA catalysed the Markonicov hydroamination of ß-nitrostyrene with methoxyamine for the first time with >75% enantiomeric excess (ee) in an aqueous buffer (pH 7.4) at room temperature.

Inheritance pattern of okra enation leaf curl disease among cultivated species and its relationship with biochemical parameters.

Okra production in eastern India at present is severely threatened by whitefly-mediated okra enation leaf curl disease (OELCuD). Identification of resistant genotype and understanding the genetic control and biochemical relationship of OELCuD resistance are prerequisite for developing an effective breeding strategy. This study was conducted employing six populations (P1, P2, F1, F2, BC1 and BC2) of two selected (resistant x susceptible (RxS)) crosses. Associationship between severity of OELCuD and biochemical parameters of parents and hybrids at preflowering and flowering stages was studied. Segregation pattern of the genotypes in F2 generation showing OELCuD reaction of two crosses suggested that two duplicate recessive genes was operative for resistance to OELCuD. Generation mean analysis revealed involvement of both additive and nonadditive effects in the inheritance of disease resistance. Hence, postponement of selection in later generations or intermating among the selected segregates followed by one or two generations of selfing to break the undesirable linkage and allow the accumulation of favourable alleles could be suggested for the development of stable resistant genotype against this disease. Higher peroxidase activity and total phenol content in leaf emerged as reliable biochemical markers for early selection of genotype resistant to OELCuD.

Red light-triggered photoreduction on a nucleic acid template.

Conjugate Sn(iv)(pyropheophorbide a)dichloride-(peptide nucleic acid) catalyzes reduction of azobenzene derivatives in the presence of complementary nucleic acid (NA) upon irridiation with red light (660 nm). This is the first red light-induced NA-templated photoreduction. It is highly sensitive to single mismatches in the NA-template and can detect down to 5 nM NAs.

Current Knowledge on Pathogenicity and Management of Stemphylium botryosum in Lentils (Lens culinaris ssp. culinaris Medik).

Stemphylium blight (SB) caused by Ascomycete, Stemphylium botryosum Wallr. has been a serious threat to lentil cultivation, mainly in Bangladesh, Nepal, India, and Canada since its first outbreak in Bangladesh in 1986. The genus Stemphylium Wallr., a dematiaceous hyphomycete, comprises up to 150 species, and is pathogenic on a wide range of plants infecting leguminous as well as nonleguminous crops. In recent years, studies indicated overlapping in morphological characters among the different species under the genus Stemphylium, making the identification and description of species difficult. This necessitates different molecular phylogenetic analysis in species delimitation. Therefore, a detailed understanding of spatial diversity and population structure of the pathogen is pertinent for producing source material for resistance breeding. The role of different weather variables as predisposing factors for the rapid spread of the pathogen necessitates devising a disease predictive model for the judicial application of fungicides. A dearth of information regarding spore biology, epidemiology, race diversity, host-pathogen interaction, and holistic disease management approach necessitates immediate attention towards more intensive research efforts. This is the first comprehensive review on the current state of knowledge and research efforts being made for a better understanding of the SB resistance through cognizing biology, ecology, and epidemiology of S. botryosum and effective disease management strategies to prevent widespread outbreaks of SB. The information regarding the biology and epidemiology of S. botryosum is also crucial for strengthening the "Integrated Disease Management" (IDM) programme. The need for a regional research network is advocated where the disease is becoming endemic.

Chirality Dependence of Amyloidâ€ ß Cellular Uptake and a New Mechanistic Perspective.

Amyloidâ€…ß is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloidâ€…ß and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms.

Genetic control of yellow vein mosaic virus disease tolerance in Abelmoschus esculentus (L.) Moench.

Okra's (Abelmoschus esculentus (L.) Moench) commercial cultivation is threatened in the tropics due to high incidence of yellow vein mosaic virus (YVMV) disease. Okra geneticists across the world tried to understand the inheritance pattern of YVMV disease tolerance without much success. Therefore, the inheritance pattern of YVMV disease in okra was revisited by employing sixgenerations (P1, P2, F1, F2, BC1 and BC2) of four selected crosses (one tolerant × tolerant, two tolerant × susceptible and one susceptible × susceptible) using two tolerant (BCO-1 and Lal Bhendi) and two susceptible (Japanese Jhar Bhendi and PAN 2127) genotypes. Qualitative genetic analysis was done on the basis of segregation pattern of tolerant and susceptible plants in F2 and backcross generations of all the four crosses. It revealed that a single dominant gene along with some minor factors governed the disease tolerant trait in both the tolerant parents used. However, it was observed that genes governing disease tolerance identified in both the tolerant variety used was different. It could be concluded that the gene governing YVMV disease tolerance in okra wasgenotype specific. Further, duplicate gene action as evident from an approximate ratio of 15:1 (tolerant:susceptible) in the F2 population in the cross of two tolerant varieties gave a scope of increasing the tolerance level of the hybrid plants when both the tolerant genes are brought together. However, generation mean analysis revealed involvement of both additive and nonadditive effects in the inheritance of disease tolerance. Thus, the present study confirms that a complicated genetic inheritance pattern is involved in the disease tolerance against YVMV trait. The major tolerance genes could be transferred to other okra varieties, but the tolerance breaking virus strains might not allow them to achieve tolerance in stable condition. Therefore, accumulation of additional genes may be needed for a sustainable tolerance phenotype in okra.

Host Infection beyond the Traditional Range of Sclerotium (Athelia) rolfsii with Physalis minima.

Physalis minima is an herbaceous plant and inhabitant of the porous and organic matter containing soil of bunds in crop fields, wastelands, around the houses, and on the roadsides. S. rolfsii is soil borne and it can infect over 500 plant species of different families. It is of interest to study the pathogenesis of S. rolfsii on P. minima. The S. rolfsii isolated from P. minima (physr1) was characterized by morphology and sequence of Internal Transcribed Spacer (ITS) region. The population structure determination and phylogenetic analysis showed the isolate physr1 significantly differs from other isolates. The null hypothesis of equal evolutionary rate was rejected throughout the Maximum likelihood (ML) tree topology of different S. rolfsii ITS sequences. The site-specific mean (relative) evolutionary rate analysis showed that most of the sites (80.59 % sites) evolved at a slower rate than average. Finally, the result of Tajima's neutrality test indicated that the population of S. rolfsii has recently begun to expand and that's why the pathogen was infecting the new host P. minima and pose a serious threat of infecting several other cropped and non-cropped hosts.

Suppression of Oligomer Formation and Formation of Non-Toxic Fibrils upon Addition of Mirror-Image Aß42 to the Natural l-Enantiomer.

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aß) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aß42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aß42 should reduce the concentration of toxic oligomers formed from natural l-Aß42. We synthesized l- and D-Aß42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aß42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aß42 at concentrations up to 50 µm. The mixing of Aß42 enantiomers thus accelerates the formation of non-toxic fibrils.

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (Aß), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aß42) and amyloid beta 40 (Aß40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aß42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aß40 peptide with identical success and without the need to alter the method.

Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aß42.

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to ß-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high ß-sheet characteristics. Our approach can be extended to other residues of Aß, as well as further amyloidogenic peptides.

Introduction of d-Glutamate at a Critical Residue of Aß42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity.

The amyloid beta peptide 42 (Aß42) is an aggregation-prone peptide that plays a pivotal role in Alzheimer's disease. We report that a subtle perturbation to the peptide through a single chirality change at glutamate 22 leads to a pronounced delay in the ß-sheet adoption of the peptide. This was accompanied by an attenuated propensity of the peptide to form fibrils, which was correlated with changes at the level of the fibrillary architecture. Strikingly, the incorporation of d-glutamate was found to stabilize a soluble, ordered macromolecular assembly with enhanced cytotoxicity to PC12 cells, highlighting the importance of advanced prefibrillary Aß aggregates in neurotoxicity.

Phylogenetic analysis based evolutionary study of 16S rRNA in known Pseudomonas sp.

Molecular evolution analysis of 16S rRNA sequences of native Pseudomonas strains and different fluorescent pseudomonads were conducted on the basis of Molecular Evolutionary Genetics Analysis version 5.2 (MEGA5.2). Topological evaluations show common origin for native strains with other known strains with available sequences at GenBank database. Phylogenetic affiliation of different Pseudomonas sp based on 16S rRNA gene shows that molecular divergence contributes to the genetic diversity of Pseudomonas sp. Result indicate direct dynamic interactions with the rhizospheric pathogenic microbial community. The selection pressure acting on 16S rRNA gene was related to the nucleotide diversity of Pseudomonas sp in soil rhizosphere community among different agricultural crops. Besides, nucleotide diversity among the whole population was very low and tajima test statistic value (D) was also slightly positive (Tajima׳s test statistics D value 0.351). This data indicated increasing trends of infection of soil-borne pathogens under gangetic-alluvial regions of West Bengal due to high degree of nucleotide diversity with decreased population of plant growth promoting rhizobacteria like fluorescent Pseudomonads in soil.

Nucleic acid-specific photoactivation of oligodeoxyribonucleotides labeled with deuterated dihydro-N,N,N',N'-tetramethylrhodamine using green light.

We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N',N'-tetramethylrhodamine (F*) with oligodeoxyribonucleotides and a 2'-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD4 and conjugates of oligonucleotides with N,N,N',N'-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the F* is oxidized forming fluorescent F. This reaction is >30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells.

Effect of anti-oxidant on tear film in patients suffering from diabetes mellitus.

To study the effects of anti-oxidant on tear film parameters in patients with diabetes mellitus, a total of 100 patients of diabetes mellitus with defective Schirmer test and tear film break-up time were enrolled in this study and they were given vitamin C (1000 mg/day) and vitamin E (400 IU/day) for 30 days. Schirmer test and tear break-up, time were re-examined after 1 month. There was significant improvement in values for Schirmer test (p < 0.001) and tear break-up time. Oxidative stress has an influence on how diabetes mellitus affects various systems of body. This study clearly depicted im- portant roles of vitamins C and E in improving the well-being of the ocular surface.

Fluorogenic, catalytic, photochemical reaction for amplified detection of nucleic acids.

Photochemical, nucleic acid-induced reactions, which are controlled by nontoxic red light, are well-suited for detection of nucleic acids in live cells, since they do not require any additives and can be spatially and temporally regulated. We have recently described the first reaction of this type, in which a phenylselenyl derivative of thymidine (5'-PhSeT-ODNa) is cleaved in the presence of singlet oxygen (Fülöp, A., Peng, X., Greenberg, M. M., Mokhir, A. (2010) A nucleic acid directed, red light-induced chemical reaction. Chem. Commun. 46, 5659-5661). The latter reagent is produced upon exposure of a photosensitizer 3'-PS-ODNb (PS = Indium(III)-pyropheophorbide-a-chloride: InPPa) to >630 nm light. In 2012 we reported on a fluorogenic version of this reaction (Dutta, S., Flottmann, B., Heilemann, M., Mokhir, A. (2012) Hybridization and reaction-based, fluorogenic nucleic acid probes. Chem. Commun. 47, 9664-9666), which is potentially applicable for the detection of nucleic acids in cells. Unfortunately, its yield does not exceed 25% and no catalytic turnover could be observed in the presence of substrate excess. This problem occurs due to the efficient, competing oxidation of the substrate containing an electron rich carbon-carbon double bonds (SCH═CHS) in the presence of singlet oxygen with formation of a noncleavable product (SCH═CHSO). Herein we describe a related, but substantially improved photochemical, catalytic transformation of a fluorogenic, organic substrate, which consists of 9,10-dialkoxyanthracene linked to fluorescein, with formation of a bright fluorescent dye. In highly dilute solution this reaction occurs only in the presence of a nucleic acid template. We developed three types of such a reaction and demonstrated that they are high yielding and generate over 7.7 catalytic turnovers, are sensitive to single mismatches in nucleic acid targets, and can be applied for determination of both the amount of nucleic acids and potentially their localization.

Hybridization and reaction-based fluorogenic nucleic acid probes.

We developed fluorogenic probes, which are photoactivated in the presence of specific nucleic acid templates with the release of fluorescent dyes. This templated reaction can be used to target specific nucleic acids in complex mixtures. We further demonstrate that this reaction can be monitored by single-molecule fluorescence imaging.

An autocatalytic chromogenic and fluorogenic photochemical reaction controlled by nucleic acids.

The autocatalytic photochemical reaction, which is potentially controlled by any selected nucleic acid, is highly sequence specific and not inhibited by its products, was developed. This reaction generates colored and fluorescent products, which can be monitored by the naked eye.