Crystal structure of the N-terminal domain of MtClpC1 in complex with the anti-mycobacterial natural peptide Lassomycin.

Antibiotics form our frontline therapy against disease-causing bacteria. Unfortunately, antibiotic resistance is becoming more common, threatening a future where these medications can no longer cure infections. Furthermore, the emergence of multidrug-resistant (MDR), totally drug-resistant (TDR), and extensively drug-resistant (XDR) tuberculosis has increased the urgency of discovering new therapeutic leads with unique modes of action. Some natural peptides derived from actinomycetes, such as Cyclomarin A, Lassomycin, Rufomycin I, and Ecumicin, have potent and specific bactericidal activity against Mycobacterium tuberculosis, with the specificity owing to the fact that these peptides target the ClpC1 ATPase, an essential enzyme in mycobacteria, and inhibit/activate the proteolytic activity of the ClpC1/P1/P2 complex that participates in protein homeostasis. Here, we report the high-resolution crystal structure of the N-terminal domain of ClpC1 (ClpC1 NTD) in complex with Lassomycin, showing the specific binding mode of Lassomycin. In addition, the work also compares the Lassomycin complex structure with the previously known structures of ClpC1 NTD in complex with other natural peptides such as Cyclomarin A, Rufomycin I, and Ecumicin.

Chitosan: An Autocidal Molecule of Plant Pathogenic Fungus.

The rise in the world's food demand with the increasing population threatens the existence of civilization with two equally valuable concerns: increase in global food production and sustainability in the ecosystem. Furthermore, biotic and abiotic stresses are adversely affecting agricultural production. Among them, losses caused by insect pests and pathogens have been shown to be more destructive to agricultural production. However, for winning the battle against the abundance of insect pests and pathogens and their nature of resistance development, the team of researchers is searching for an alternative way to minimize losses caused by them. Chitosan, a natural biopolymer, coupled with a proper application method and effective dose could be an integral part of sustainable alternatives in the safer agricultural sector. In this review, we have integrated the insight knowledge of chitin-chitosan interaction, successful and efficient use of chitosan, recommended and practical methods of use with well-defined doses, and last but not least the dual but contrast mode of action of the chitosan in hosts and as well as in pathogens.

MBZM-N-IBT, a Novel Small Molecule, Restricts Chikungunya Virus Infection by Targeting nsP2 Protease Activity In Vitro, In Vivo, and Ex Vivo.

The increase in disease incidences and persistent Chikungunya virus (CHIKV)-induced arthritis have been a huge burden on public health globally. In the absence of specific antivirals or vaccines, it is essential to continue efforts to develop effective anti-CHIKV strategies. Our previous study showing the in vitro anti-CHIKV potential of a novel molecule 1-[(2-methylbenzimidazol-1-yl) methyl]-2-oxo-indolin-3-ylidene] amino] thiourea (MBZM-N-IBT) encouraged us to further validate its efficacy. Here, the effect of MBZM-N-IBT was evaluated in vitro in RAW 264.7 cells, in vivo in C57BL/6 mice, and ex vivo in human peripheral blood mononuclear cells (hPBMCs). The study demonstrated that CHIKV infection was efficiently abrogated in RAW 264.7 cells (IC50 = 22.34 µM) with significant inhibition in viral proteins. The inhibition was effective in the postentry step, and MBZM-N-IBT predominately interfered in the early stages of CHIKV life cycle. It was further supported when the protease activity of CHIKV-nsP2 was hindered by the compound. Moreover, it diminished the CHIKV-induced inflammatory responses in vitro through significant downregulation of all the major mitogen-activated protein kinases (MAPKs), NF-κB, cyclooxygenase (COX)-2, and cytokines. Furthermore, MBZM-N-IBT restricted CHIKV infection and inflammation in vivo, leading to reduced clinical scores and complete survival of C57BL/6 mice. Additionally, it has been noticed that the CHIKV infection was reduced remarkably in hPBMC-derived monocyte-macrophage populations ex vivo by the compound. In conclusion, it can be suggested that this novel compound MBZM-N-IBT has been demonstrated to be a potential anti-CHIKV molecule in vitro, in vivo, and ex vivo and fulfilled all the criteria to investigate further for successful treatment of CHIKV infection.

Genome-Wide Analysis and Characterization of the Proline-Rich Extensin-like Receptor Kinases (PERKs) Gene Family Reveals Their Role in Different Developmental Stages and Stress Conditions in Wheat (Triticum aestivum L.).

Proline-rich extensin-like receptor kinases (PERKs) are a class of receptor kinases implicated in multiple cellular processes in plants. However, there is a lack of information on the PERK gene family in wheat. Therefore, we identified 37 PERK genes in wheat to understand their role in various developmental processes and stress conditions. Phylogenetic analysis of PERK genes from Arabidopsis thaliana, Oryza sativa, Glycine max, and T. aestivum grouped them into eight well-defined classes. Furthermore, synteny analysis revealed 275 orthologous gene pairs in B. distachyon, Ae. tauschii, T. dicoccoides, O. sativa and A. thaliana. Ka/Ks values showed that most TaPERK genes, except TaPERK1, TaPERK2, TaPERK17, and TaPERK26, underwent strong purifying selection during evolutionary processes. Several cis-acting regulatory elements, essential for plant growth and development and the response to light, phytohormones, and diverse biotic and abiotic stresses, were predicted in the promoter regions of TaPERK genes. In addition, the expression profile of the TaPERK gene family revealed differential expression of TaPERK genes in various tissues and developmental stages. Furthermore, TaPERK gene expression was induced by various biotic and abiotic stresses. The RT-qPCR analysis also revealed similar results with slight variation. Therefore, this study's outcome provides valuable information for elucidating the precise functions of TaPERK in developmental processes and diverse stress conditions in wheat.

Effect of heat stress during flowering and pod formation in pea (Pisum sativum L.).

Heat stress is a major constraint of yield in grain legumes including peas. Increasing global warming and human population now urge to develop climate resilient varieties. The present experiment was conducted over 2 years to evaluate the heat tolerance of 211 pea genotypes. In the present study, the field pea genotypes showed a wide variation for reproductive stage heat stress (RSHS) quantitative traits. Significant positive correlations were found between no. of seeds per plant and no. of pods per plant; seed diameter (mm) and 25-seed weight (g) in heat tolerant as well as heat susceptible genotypes. Principal component analysis revealed two major principal components contributed approximately 91% of total variations and heat tolerant and susceptible genotypes separately formed two major clusters. Stepwise multiple regression analysis revealed that no. of seeds per plant was the best predictor for no. of pods per plant. On the basis of four RSHS traits, the most prominent heat tolerant pea genotypes identified in the present study JP-625, IARI-2877, PMR-38 II, EC-318760, EC-328758 and IARI-2904 would better combat RSHS and provide yield stability under changing climatic conditions.

Crystal structures reveal N-terminal Domain of Arabidopsis thaliana ClpD to be highly divergent from that of ClpC1.

The caseinolytic protease machinery associated chaperone protein ClpC is known to be present in bacteria, plants and other eukaryotes, whereas ClpD is unique to plants. Plant ClpC and ClpD proteins get localized into chloroplast stroma. Herein, we report high resolution crystal structures of the N-terminal domain of Arabidopsis thaliana ClpC1 and ClpD. Surprisingly, AtClpD, but not AtClpC1, deviates from the typical N-terminal repeat domain organization of known Clp chaperones and have only seven α-helices, instead of eight. In addition, the loop connecting the two halves of AtClpD NTD is longer and covers the region which in case of AtClpC1 is thought to contribute to adaptor protein interaction. Taken together, the N-terminal domain of AtClpD has a divergent structural organization compared to any known Clp chaperones which hints towards its specific role during plant stress conditions, as opposed to that in the maintenance of chloroplastic homeostasis by AtClpC1. Conservation of residues in the NTD that are responsible for the binding of the cyclic peptide activator - Cyclomarin A, as reported for mycobacterial ClpC1 suggests that the peptide could be used as an activator to both AtClpC1 and AtClpD, which could be useful in their detailed in vitro functional characterization.

Histo-chemical and biochemical analysis reveals association of er1 mediated powdery mildew resistance and redox balance in pea.

Powdery mildew caused by Erysiphe pisi is one of the important diseases responsible for heavy yield losses in pea crop worldwide. The most effective method of controlling the disease is the use of resistant varieties. The resistance to powdery mildew in pea is recessive and governed by a single gene er1. The objective of present study is to investigate if er1 mediated powdery mildew resistance is associated with changes in the redox status of the pea plant. 16 pea genotypes were screened for powdery mildew resistance in field condition for two years and, also, analyzed for the presence/absence of er1 gene. Histochemical analysis with DAB and NBT staining indicates accumulation of reactive oxygen species (ROS) in surrounding area of powdery mildew infection which was higher in susceptible genotypes as compared to resistant genotypes. A biochemical study revealed that the activity of superoxide dismutase (SOD) and catalase, enzymes involved in scavenging ROS, was increased in, both, resistant and susceptible genotypes after powdery mildew infection. However, both enzymes level was always higher in resistant than susceptible genotypes throughout time course of infection. Moreover, irrespective of any treatment, the total phenol (TP) and malondialdehyde (MDA) content was significantly high and low in resistant genotypes, respectively. The powdery mildew infection elevated the MDA content but decreased the total phenol in pea genotypes. Statistical analysis showed a strong positive correlation between AUDPC and MDA; however, a negative correlation was observed between AUDPC and SOD, CAT and TP. Heritability of antioxidant was also high. The study identified few novel genotypes resistant to powdery mildew infection that carried the er1 gene and provided further clue that er1 mediated defense response utilizes antioxidant machinery to confer powdery mildew resistance in pea.

First evidence of molecular characterization of rohu carp Sox2 gene being expressed in proliferating spermatogonial cells.

Because little is known about the function of Sox2 (Sry-related box-2) in teleosts, the objective of this study was to clone and characterize Sox2 complementary DNA (cDNA) from the testis of Indian major carp, Labeo rohita (rohu). The full-length cDNA contained an open reading frame of 936 nucleotides bearing the typical structural features. Phylogenetically, Sox2 of L rohita was most closely related to freshwater counterparts than marine water. The sequence information of cDNA and genomic DNA together revealed that the Sox2 gene is encoded by an uninterrupted exon. Furthermore, comparative mRNA expression profile in various organs including proliferating spermatogonial stem cells (SSCs) suggested about the participatory role of Sox2 during fish male germ cell development and maintenance of stem cells. In support, we have also provided evidence that Sox2 protein is indeed present in rohu SSCs by Western blot analysis. The evolutionarily conserved high-mobility group box domain indicated its possible involvement in common networking pathways for stem cell maintenance and pluripotency between mammals and nonmammals. Our findings could be the first step toward the use of Sox2 as a potential biomarker for proliferating SSCs and understanding the transcriptional regulatory network involved during male germ cell development and maintenance in fish species.

Identification of promoter within the first intron of Plzf gene expressed in carp spermatogonial stem cells.

Promyelocytic leukemia zinc finger (Plzf), a transcriptional repressor, is involved in survival and maintenance of pluripotent stem cells including embryonic and spermatogonial stem cells in mammals. Its cDNA was characterized and expression in proliferating spermatogonial stem cells of rohu (Labeo rohita), a farmed carp, was documented. In teleost, the information on its promoter activity is lacking. Here, we have isolated, sequenced and performed the first characterization of regulatory elements for Plzf being expressed in proliferating spermatogonial stem cells of rohu. About 3.2 kb of 5'-flanking region, relative to ATG start codon, derived by genome walking was sequenced. The 5'-RACE (rapid amplification of cDNA ends) analysis not only mapped the transcriptional start site but also detected non-coding exons. Interestingly, computational analysis detected several putative regulatory elements including TATA-box positioned in the first intron. Luciferase reporter assay was performed for serially deleted constructs to measure their promoter activities. The region containing putative TATA- and CAAT-boxes including GC-rich motif, positioned within first intron, was identified as a potential promoter; but its full promoter activity was dependent on upstream region containing a putative Evi-1-like element. Moreover, our findings also identified a region acting as transcriptional repressor. These findings could be used as roadmap for future understandings of its regulated expression during male germ cell development in fish species.

Gene structure and identification of minimal promoter of Pou2 expressed in spermatogonial cells of rohu carp, Labeo rohita.

Mammalian Pou5f1 is a known transcriptional regulator involving maintenance of embryonic and spermatogonial stem cells. Little is known about teleost Pou2, an ortholog of mammalian Pou5f1. Evidences of discrepancy in expression pattern between fish species were documented. To better understand, we have cloned and characterized Pou2 gene of farmed rohu carp, Labeo rohita. It contained five exons with an open reading frame of 1419 bp long, translatable to 472 aa. A bipartite DNA binding domain termed POU domain, comprising of POU-specific and POU-homeo sub-domains, was identified. Rohu Pou2 is highly conserved with zebrafish counterpart, as evidenced by 92% overall sequence identity of deduced protein. The POU domain remained highly conserved (showing more than 90% identities) within fish species. Even though there is a divergence between Pou2 and Pou5f1, the common POU-specific domain remained conserved throughout eukaryotes indicating their possible involvements in common trans-activation pathway(s) between mammals and non-mammals. In support, we have provided evidence that Pou2 is indeed abundantly expressed in proliferating rohu spermatogonial cells and hence participates in stem cell maintenance. Its mRNA accumulation in the ovary supported about its maternal transmission with possible regulatory roles during embryogenesis. The 5'-flanking region (~2.7 kb) of rohu Pou2 was sequenced and computational analysis detected several putative regulatory elements. These elements have been conserved among fish species analysed. Luciferase assay identified a mammalian-type 'TATA-less promoter' capable of driving Pou2 gene transcription. These findings will help for future studies in elucidating participatory role of fish Pou2 in male germ cell development.

Identification and characterization of differentially expressed transcripts in the gills of freshwater prawn (Macrobrachium rosenbergii) under salt stress.

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important species. It is a euryhaline shrimp, surviving in wide-range salinity conditions. A change in gene expression has been suggested as an important component for stress management. To better understand the osmoregulatory mechanisms mediated by the gill, a subtractive and suppressive hybridization (SSH) tool was used to identify expressed transcripts linked to adaptations in saline water. A total of 117 transcripts represented potentially expressed under salinity conditions. BLAST analysis identified 22% as known genes, 9% as uncharacterized showing homologous to unannotated ESTs, and 69% as unknown sequences. All the identified known genes representing broad spectrum of biological pathways were particularly linked to stress tolerance including salinity tolerance. Expression analysis of 10 known genes and 7 unknown/uncharacterized genes suggested their upregulation in the gills of prawn exposed to saline water as compared to control indicating that these are likely to be associated with salinity acclimation. Rapid amplification of cDNA ends (RACE) was used for obtaining full-length cDNA of MRSW-40 clone that was highly upregulated during salt exposure. The sequenced ESTs presented here will have potential implications for future understanding about salinity acclimation and/or tolerance of the prawn.

Cloning of cDNA and prediction of peptide structure of Plzf expressed in the spermatogonial cells of Labeo rohita.

The promyelocytic leukemia zinc finger (Plzf) gene containing an evolutionary conserved BTB (bric-a-brac/tramtrack/broad complex) domain plays a key role in self-renewal of mammalian spermatogonial stem cells (SSCs) via recruiting transcriptional co-repressors. Little is known about the function of Plzf in vertebrate, especially in fish species. To gain better understanding of its role in fishes, we have cloned Plzf from the testis of Labeo rohita (rohu), a commercially important freshwater carp. The full-length cDNA contains an open reading frame (ORF) of 2004bp translatable to 667 amino acids (aa) containing a conserved N-terminal BTB domain and C-terminal C(2)H(2)-zinc finger motifs. L. rohita Plzf, which is phylogenetically related to Danio rerio counterpart, abundantly expressed in spermatogonial stem cells (SSCs). A three-dimensional (3D) model of BTB domain of Plzf protein was constructed by homology modeling approach. Molecular docking on this 3D structure established a homo-dimer between two BTB domains creating a charged pocket containing conserved aa residues: L33, C34, D35 and R49. Thus, Plzf of SSC is structurally and possibly functionally conserved. The conserved aa residues in the cleft resulting from Plzf BTB self-association are likely to be the binding platform for interaction with recruited co-repressor peptides. The identified Plzf could be the first step towards exploring its role in rohu SSC behavior.