Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l^?1), Kn (0.5 mg l^?1) and PG (0.5 mg l^?1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l^?1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l^?1) and PG (0.5 mg l^?1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l^?1) + PAA (0.5 mg l^?1) + CC (2 mg l^?1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each.     

Virus-induced gene silencing: A versatile tool for discovery of gene functions in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an antiviral defense mechanism in plants as a tool for plant reverse genetics. VIGS circumvents the need for plant transformation, is methodologically simple and yields rapid results. Various VIGS vectors have been developed and have helped to unravel the functions of genes involved in processes such as disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis.     

Exploring tryptophan dynamics in acid-induced molten globule state of bovine α-lactalbumin: a wavelength-selective fluorescence approach

The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning to be understood. Bovine α-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity (acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general.     

Inflammatory mediators and the failing heart: a translational approach

Recent studies have identified the importance of proinflammatory mediators in regulating cardiac structure in health and disease. Recent studies suggest that cytokines that are expressed within the myocardium in response to a environmental injury, namely tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) and the interleukin-6 (IL-6) family of cytokines play an important role in initiating and integrating homeostatic responses within the heart. However, these "stress-activated" cytokines all have the potential to produce cardiac decompensation when expressed at sufficiently high concentrations. Indeed, there is now a growing appreciation that these molecules may play an important role in mediating disease progression in the failing heart. The growing appreciation of the pathophysiological consequences of sustained expression of proinflammatory mediators in pre-clinical and clinical heart failure models culminated in a series of multicenter clinical trials that utilized "targeted" approaches to neutralize tumor necrosis factor (TNF) in patients with moderate to advanced heart failure. However, these targeted approaches have resulted in worsening heart failure, thereby raising a number of important questions about what role, if any, proinflammatory cytokines play in the pathogenesis of heart failure. This review will summarize the tremendous growth of knowledge that has taken place in this field, with a focus on what we have learned from the negative clinical trials, as well as the potential direction of future research in this area.     

Sortase-catalyzed assembly of distinct heteromeric fimbriae in Actinomyces naeslundii

Two types of adhesive fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.     

In-Silico and In-Vitro Analysis of Human SOS1 Protein Causing Noonan Syndrome - A Novel Approach to Explore the Molecular Pathways

AimsPerform in-silico analysis of human SOS1 mutations to elucidate their pathogenic role in Noonan syndrome (NS).BackgroundNS is an autosomal dominant genetic disorder caused by single nucleotide mutation in PTPN11, SOS1, RAF1, and KRAS genes. NS is thought to affect approximately 1 in 1000. NS patients suffer different pathogenic effects depending on the mutations they carry. Analysis of the mutations would be a promising predictor in identifying the pathogenic effect of NS.MethodsWe performed computational analysis of the SOS1 gene to identify the pathogenic nonsynonymous single nucleotide polymorphisms (nsSNPs) th a t cause NS. SOS1 variants were retrieved from the SNP database (dbSNP) and analyzed by in-silico tools I-Mutant, iPTREESTAB, and MutPred to elucidate their structural and functional characteristics.ResultsWe found that 11 nsSNPs of SOS1 that were linked to NS. 3D modeling of the wild-type and the 11 nsSNPs of SOS1 showed that SOS1 interacts with cardiac proteins GATA4, TNNT2, and ACTN2. We also found that GRB2 and HRAS act as intermediate molecules between SOS1 and cardiac proteins. Our in-silico analysis findings were further validated using induced cardiomyocytes (iCMCs) derived from NS patients carrying SOS1 gene variant c.1654A>G (NSiCMCs) and compared to control human skin fibroblast-derived iCMCs (C-iCMCs). Our in vitro data confirmed that the SOS1, GRB2 and HRAS gene expressions as well as the activated ERK protein, were significantly decreased in NS-iCMCs when compared to C-iCMCs.ConclusionThis is the first in-silico and in vitro study demonstrating that 11 nsSNPs of SOS1 play deleterious pathogenic roles in causing NS.     

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.     

Biosorption and Reuse Potential of a Blue Green Alga for the Removal of Hazardous Reactive Dyes from Aqueous Solutions

The adsorption potential of a nonliving alga Nostoc comminutum for the removal of hazardous dyes from aqueous solutions was assessed. The algal biomass in its native form exhibited the highest dye removal efficiency at a pH of 1 and at a contact time of 50 min. The effect of various chemical pretreatments was studied in order to assess the role of surface chemistry and the underlying mechanism. Combined with Fourier transform infrared (FTIR) studies, it was concluded that the carboxylic and amine groups played a dominant role in the dye binding process. pH studies further revealed that besides the electrostatic mechanism, other physical interactions might be operative, which is also revealed from the Dubinin-Radushkevich (D-R) isotherm model. Kinetic studies indicated that the adsorption process followed the second-order kinetics and particle diffusion mechanisms were operative. Thermodynamic studies revealed that the adsorption of two dyes onto the algal biomass was feasible, spontaneous, and exothermic under the studied conditions. Microwave irradiation was proposed as a green method for the regeneration and reuse of the biomass. A 16% loss in the regeneration efficiency of the biosorbent was observed, which reveals its stability and reuse potential. Column experiments with real textile wastewater established the practicality of the developed system. Finally, a comparative study revealed the efficacy of the biosorbent with other previously reported biosorbents for dye removal.