Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

Identification of interactions involved in the generation of nucleophilic reactivity and of catalytic competence in the catalytic site Cys/His ion pair of papain

Understanding the roles of noncovalent interactions within the enzyme molecule and between enzyme and substrate or inhibitor is an essential goal of the investigation of active center chemistry and catalytic mechanism. Studies on members of the papain family of cysteine proteinases, particularly papain (EC 3.4.22.2) itself, continue to contribute to this goal. The historic role of the catalytic site Cys/His ion pair now needs to be understood within the context of multiple dynamic phenomena. Movement of Trp177 may be necessary to expose His159 to solvent with consequent decrease in its degree of electrostatic solvation of (Cys25)-S(-). Here we report an investigation of this possibility using computer modeling of quasi-transition states and pH-dependent kinetics using 3,3'-dipyridazinyl disulfide, its n-propyl and phenyl derivatives, and 4,4'-dipyrimidyl disulfide as reactivity probes that differ in the location of potential hydrogen-bonding acceptor atoms. Those interactions that influence ion pair geometry and thereby catalytic competence, including by transmission of the modulatory effect of a remote ionization with pK(a) 4, were identified. A key result is the correlation between the kinetic influence of the modulatory trigger of pK(a) 4 and disruption of the hydrogen bond donated by the indole N-H of Trp177, the hydrophobic shield of the initial "intimate" ion pair. This hydrogen bond is accepted by the amide O of Gln19-a component of the oxyanion hole that binds the tetrahedral species formed from the substrate during the catalytic act. The disruption would be expected to contribute to the mobility of Trp177 and possibly to the effectiveness of the binding of the developing oxyanion.     

Salicylic acid-mediated changes in photosynthesis,nutrients content and antioxidant metabolism in two mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) cultivars differing in salt tolerance

Mustard (Brassica juncea L.) cultivars Alankar (salt-tolerant) and PBM16 (salt-sensitive) plants were grown with 50 mM NaCl and were sprayed with 0.1, 0.5, and 1.0 mM salicylic acid (SA) to study the physiological processes determining salt tolerance and to observe the influence of SA application on the alleviation of NaCl-induced adverse effects. The content of leaf Na⁺, Cl⁻, H₂O₂, TBARS, and electrolyte leakage and the activity of SOD were higher in PBM16 than Alankar. In contrast, nutrients content, activity of APX and GR, glutathione content, photosynthetic and growth characteristics were higher in Alankar. Treatment of 50 mM NaCl resulted in increase of Na⁺ and Cl⁻, oxidative stress, activity of antioxidant enzymes and glutathione content, while nutrients content, photosynthetic, and growth characteristics decreased in both the cultivars. Application of 0.5 mM SA alleviated the negative effects of 50 mM NaCl maximally, but 1.0 mM SA proved inhibitory. The effect of SA was more conspicuous in Alankar than PBM16. It is concluded that the higher tolerance of Alankar was due to its lower leaf Na⁺ and Cl⁻ content, higher nutrients content, and efficient antioxidant metabolism. The application of 0.5 mM SA substantially alleviated salt-induced adverse effects in Alankar.     

Generation of nucleophilic character in the Cys25/His159 ion pair of papain involves Trp177 but not Asp158

Studies on papain (EC 3.4.22.2), the most thoroughly investigated member of the cysteine proteinase superfamily, have contributed substantially to our understanding of the roles of noncovalent interactions in enzyme active center chemistry. Previously, we reported evidence that the long-held view that catalytic competence develops synchronously with formation of the catalytic site (Cys25)-S-/(His159)-Im+H ion pair is incorrect and that conformational rearrangement is necessary for each of the partners to play its role in catalysis. A decrease in the level of mutual solvation of the partners of the noncatalytic "intimate" ion pair should release the nucleophilic character of (Cys25)-S- and allow association of (His159)-Im+H with the leaving group of a substrate to provide its general acid-catalyzed elimination. Hypotheses by which this could be achieved involve electrostatic modulation of the ion pair and perturbation of its hydrophobic shielding from solvent by Trp177. The potential electrostatic modulator closest to the catalytic site is Asp158, the mutation of which to Ala substantially decreases catalytic activity. Here we report an investigation of these hypotheses by a combination of computer modeling and stopped-flow pH-dependent kinetic studies using a new series of cationic aminoalkyl 2-pyridyl disulfide time-dependent inhibitors as reactivity probes. These probes 2-4 (n = 2-4), which exist as equilibrium mixtures of H3N+-[CH2]n-S-S-2-pyridyl+H and H3N+-[CH2]n-S-S-2-pyridyl which predominate in acidic and weakly alkaline media, respectively, were shown by modeling and kinetic analysis to bind with various degrees of effectiveness near Asp158 and in some cases also near Trp177. Kinetic analysis of the reactions of 2-4 and of the reaction of CH3-[CH2]2-S-S-2-pyridyl+H <==>CH3-[CH2]2-S-S-2-pyridyl 1 and normal mode calculations lead to the conclusion that Asp158 is not involved in the generation of nucleophilic character in the ion pair and demonstrates a key role for Trp177.     

Polymorphic analysis of MHClinked Heat Shock Protein 70 genes: Their susceptibility and prognostic implication in Kangri cancer cases of Kashmiri population

Kangri cancer is a unique thermally-induced squamous cell carcinoma (SCC) of skin that develops due to persistent use of Kangri (a brazier), used by Kashmiri people, to combat the chilling cold during winter months. We designed a large scale case-control study to characterize the frequency of two polymorphisms within the MHC class III-linked HSP70genes, Hsp70-2 and Hsp70-hom, in order to find any association of these genotypic variants for predisposition to and clinical outcome of Kangri cancer patients from Kashmir valley in North India. Polymerase Chain Reaction and restriction enzymes were utilized to characterize the frequency of two polymorphisms with in Hsp70-2 and Hsp70-hom genes in 118 Kangri carcinoma cases and 95 healthy controls from the same population of Kashmir. Association of high frequency allelic variants of Hsp70genes with various clinicopathological features of prognostic significance was assessed by Chi-square test using SPSS software. In this study, allelic frequency of Hsp70-2 A/G heterozygote (0.87) (P = 0.012) was found to be significantly high in Kangri cancer cases compared to control (0.736) with a Relative Risk of 2.45 fold. Conversely, the allelic frequency of Hsp70-2 A/A allele in homozygous condition was significantly low in Kangri cancer cases and worked out to be 0.084 (Vs 0.252 in control) with P is equal to 0.001, implicating it as a protective allele against Kangri cancer in subjects with this genotype. Similarly, significantly high frequency of 0.50 (Vs 0.29 in control) of Hsp70-homC/C allele was found in homozygous condition in Kangri cancer cases suggestive of a positive relative risk associated with this genotype (RR is equal to 2.47) (P is equal to 0.002). The overall allele frequency data analysis of Hsp70-2 and Hsp70-hom genes was significant (χ² is equal to 12.38, P is equal to 0.002; and χ² is equal to 12.21, P is equal to 0.002). The study also reveals considerable association of high frequency alleles of HSP70 genes, especially of Hsp70-2 A/G or G/G in Kangri tumors with clinico-pathological features of poor prognosis. These results indicate that the relative risk of Kangri cancer associated with Hsp70-2 and Hsp70- hom gene polymorphisms is confined to Hsp70-2 A/G or G/G and Hsp70homC/C haplotype in our population. The study, therefore, suggests Hsp70-2 A/G or G/G and Hsp70homC/C genotypes as potential susceptibility markers and independent prognostic indicators in Kangri carcinoma patients in Kashmiri population.     

Salicylic acid alleviates decreases in photosynthesis under salt stress by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean cultivars

Salicylic acid (SA) is known to affect photosynthesis under normal conditions and induces tolerance in plants to biotic and abiotic stresses through influencing physiological processes. In this study, physiological processes were compared in salt-tolerant (Pusa Vishal) and salt-sensitive (T44) cultivars of mungbean and examined how much these processes were induced by SA treatment to alleviate decrease in photosynthesis under salt stress. Cultivar T44 accumulated higher leaf Na⁺ and Cl⁻ content and exhibited greater oxidative stress than Pusa Vishal. Activity of antioxidant enzymes, ascorbate peroxidase (APX) and glutathione reductase (GR) was greater in Pusa Vishal than T44. Contrarily, activity of superoxide dismutase (SOD) was greater in T44. The greater accumulation of leaf nitrogen and sulfur through higher activity of their assimilating enzymes, nitrate reductase (NR) and ATP-sulfurylase (ATPS) increased reduced glutathione (GSH) content more conspicuously in Pusa Vishal than T44. Application of 0.5 mM SA increased nitrogen and sulfur assimilation, GSH content and activity of APX and GR. This resulted in the increase in photosynthesis under non-saline condition and alleviated the decrease in photosynthesis under salt stress. It also helped in restricting Na⁺ and Cl⁻ content in leaf, and maintaining higher efficiency of PSII, photosynthetic N-use efficiency (NUE) and water relations in Pusa Vishal. However, application of 1.0 mM SA resulted in inhibitory effects. The effect of SA was more pronounced in Pusa Vishal than T44. These results indicate that SA application alleviates the salt-induced decrease in photosynthesis mainly through inducing the activity of NR and ATPS, and increasing antioxidant metabolism to a greater extent in Pusa Vishal than T44.     

Exogenously-sourced ethylene increases stomatal conductance, photosynthesis, and growth under optimal and deficient nitrogen fertilization in mustard

In order to ascertain the stomatal and photosynthetic responses of mustard to ethylene under varying N availability, photosynthetic characteristics of mustard grown with optimal (80 mg N kg(-1) soil) or low (40 mg N kg(-1) soil) N were studied after the application of an ethylene-releasing compound, ethephon (2-chloroethyl phosphonic acid) at 40 days after sowing (DAS). The availability of N influenced ethylene evolution and affected stomatal conductance and photosynthesis. The effect of ethylene was smaller under deficient N where plants contained higher glucose (Glc) sensitivity, despite high ethylene evolution even in the absence of ethephon, potentially because the plants were less sensitive to ethylene per se. Ethephon application at each level of N increased ethylene and decreased Glc sensitivity, which increased photosynthesis via its effect on the photosynthetic machinery and effects on stomatal conductance. Plants grown with sufficient-N and treated with 200 μl l(-1) ethephon exhibited optimal ethylene, the greatest stomatal conductance and photosynthesis, and growth. These plants made maximum use of available N and exhibited the highest nitrogen-use efficiency (NUE).     

Validation of CSN1S1 transcriptional expression,promoter methylation,and prognostic power in breast cancer using independent datasets

Breast cancer ranked second among most frequent cancer in the world playing a significant role in mortality rate. Having prior knowledge on differentially expressed genes in breast cell carcinoma elucidated important indications to understand the molecular mechanism underneath breast carcinogenesis. In this study we have investigated the distinguished CSN1S1 expression in human breast cancer. We have analyzed CSN1S1 mRNA expression between cancer and normal tissues using TCGA datasets. Moreover, analysis including promoter methylation, mutations, prognosis, co-expression, gene ontology, and pathways of CSN1S1 were performed by the TCGA Wanderer, UCSC Xena, cBioPortal, PrognoScan, UALCAN, and Enricher server. We have observed low mRNA expression and high promoter methylation of CSN1S1 in cancer tissues compared to normal tissues. Furthermore, we have also identified low mRNA expression in clinicopathological patients, as well as 9 deleterious mutations with highly co-expressed protein MRC1, and significantly related signaling pathways. We have found a positive correlation between the lower expression of CSN1S1 and patients surviving with breast cancer. Here we have concluded that CSN1S1 acts as a biomarker for the surveillance and prognosis of breast cancer, and also works as a novel therapeutic target at the molecular and pathway levels.     

Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant

An efficacious and reproducible in vitro regeneration technique for safflower was established using varying concentrations and composition of plant growth regulators (PGRs) supplemented Murashige and Skoog (MS) medium. Successful in vitro seed germination in half strength MS (H-MS) with 1.4 µM GA₃ resulted in procurement of sterile explants (cotyledons, apical meristems) for in vitro study. Callogenesis (2.2 µM BAP?+?2.7 µM NAA), indirect organogenesis of shoot buds (0.54 µM NAA?+?9.08 µM TDZ), somatic embryogenesis (2.2 µM BAP?+?5.4 µM NAA) and somatic embryo germinated plantlets (H-MS?+?1.4 µM GA₃?+?2.2 µM BAP?+?5.4 µM NAA) were successfully obtained. Histological study and scanning electron micrographs of embryogenic callus revealed pre-globular, heart-shaped and torpedo stages of dicot embryogeny. H-MS?+?8 µM NAA showed maximum rhizogenic response with a mean root and shoot length of 17.5 mm and 48.50 mm respectively in 2.2 µM BAP?+?0.54 µM NAA bearing an average of 9 capitula per plantlet with 70% post transplantation survival rate. True to type nature of the regenerates was confirmed using Start Codon Targeted (SCoT) marker, exhibiting 100% and 97.3% monomorphic bands for direct and somatic embryo regenerated plants respectively. Flow cytometry method (FCM) was employed for 2C DNA content analysis. The histogram peaks of 2C nuclear DNA content of in vitro regenerated safflower (direct and embryo derived) were similar to the peak of field grown donor plant. 2C nuclear DNA content of field grown, direct and somatic embryo regenerated C. tinctorius was 2.65?±?0.04 pg, 2.62?±?0.06 pg and 2.68?±?0.04 pg respectively, further verifying genetic homogeneity. All things considered, the above protocol is insusceptible to genetic alteration and can be used for large scale production and sustainable utilization of desired genotype.

     

Discovery of novel Jak2–Stat pathway inhibitors with extended residence time on target

The discovery of the activating mutation V617F in the JH2 domain of Jak2 and the modulation of oncogenic Stat3 by Jak2 inhibitors have spurred a great interest in the inhibition of the Jak2/Stat pathway in oncology. In this Letter, we communicate the discovery of novel inhibitors of the Jak2/Stat5 axis, the N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives. The rationale, synthesis and biological evaluation of these derivatives are reported. Two lead analogs from this series, 6 and 9, displayed prolonged residence time on Jak2, at enzymatic level. Although 6 and 9 exhibited moderate selectivity in a selected kinase panel, we chose to test these inhibitors in vivo as a consequence to their long residence time. However, extended inhibition of Jak2 due to the long residence time, in the form of inhibiting phosphorylation of downstream Stat5, was not recapitulated in an in vivo setting.