Chemical profiling of bioactive compounds in the methanolic extract of wild leaf and callus of Vitex negundo using gas chromatographymass spectrometry

BACKGROUND The investigation of plant-based therapeutic agents in medicinal plants has revealed their presence in the extracts and provides the vision to formulate novel techniques for drug therapy.Vitex negundo(V.negundo),a perennial herb belonging to the Varbanaceae family,is extensively used in conventional medication.AIM To determine the existence of therapeutic components in leaf and callus extracts from wild V.negundo plants using gas chromatography-mass spectrometry(GCMS).METHODS In this study,we conducted GC-MS on wild plant leaf extracts and correlated the presence of constituents with those in callus extracts.Various growth regulators such as 6-benzylaminopurine(BAP),2,4-dichlorophenoxyacetic acid(2,4-D),α-naphthylacetic acid(NAA),and di-phenylurea(DPU)were added to plant leaves and in-vitro callus and grown on MS medium.RESULTS The results clearly indicated that the addition of BAP(2.0 mg/L),2,4-D(0.2 mg/mL),DPU(2.0 mg/L)and 2,4-D(0.2 mg/mL)in MS medium resulted in rapid callus development.The plant profile of Vitex extracts by GC-MS analysis showed that 24,10,and 14 bioactive constituents were detected in the methanolic extract of leaf,green callus and the methanolic extract of white loose callus,respectively.CONCLUSION Octadecadienoic acid,hexadecanoic acid and methyl ester were the major constituents in the leaf and callus methanolic extract.Octadecadienoic acid was the most common constituent in all samples.The maximum concentration of octadecadienoic acid in leaves,green callus and white loose callus was 21.93%,47.79%and 40.38%,respectively.These findings demonstrate that the concentration of octadecadienoic acid doubles in-vitro compared to in-vivo.In addition to octadecadienoic acid;butyric acid,benzene,1-methoxy-4-(1-propenyl),dospan,tridecanedialdehyde,methylcyclohexenylbutanol,chlorpyrifos,n-secondary terpene diester,anflunine and other important active compounds were also detected.All these components were only available in callus formed in-vitro.This study showed that the callus contained additional botanical characteristics compared with wild plants.Due to the presence of numerous bioactive compounds,the medical use of Vitex for various diseases has been accepted and the plant is considered an important source of therapeutics for research and development.

Prospects for 3D bioprinting of organoids

Three-dimensional(3D)organoids derived from pluripotent or adult tissue stem cells seem to possess excellent potential for studying development and disease mechanisms alongside having a myriad of applications in regenerative therapies.However,lack of precise architectures and large-scale tissue sizes are some of the key limitations of current organoid technologies.3D bioprinting of organoids has recently emerged to address some of these impediments.In this review,we discuss 3D bioprinting with respect to the use of bioinks and bioprinting methods and highlight recent studies that have shown success in bioprinting of stem cells and organoids.We also summarize the use of several vascularization strategies for the bioprinted organoids,that are critical for a complex tissue organization.To fully realize the translational applications of organoids in disease modeling and regenerative medicine,these areas in 3D bioprinting need to be appropriately harnessed and channelized.

Oxidative Stress Responsive <i>SERK1</i>Gene Directs the Progression of Somatic Embryogenesis in Cotton (<i>Gossypium hirsutum</i>L. cv. Coker 310)

Somatic embryogenesis (SE) is a prominent mode of regeneration in plants. The acquisition of SE is predominantly invoked by the oxidative stress which plays an important role in signal transduction and cellular redox. Since balanced generation of oxidants is important to cellular differentiation, modulation in cell redox could be responsive to genotypic refinement for SE. To study the dynamics of cellular redox during SE, we conducted comparative expression analyses of cotton (Gossypium hirsutum), using two independently purified near-isogenic lines for the trait of SE. We interrogated expression changes in cell-signaling factor Somatic Embryogenesis Receptor Kinase (SERK) and activity of antioxidant Glutathione in different developmental stages including cotyledonary leaf, calli from different stages of regeneration of fully-regenerating (FR) and non-regenerating (NR) lines of Coker310 cultivar. At evolutionary scale, the cotton SERKs showed high sequence similarity in receptor kinase domain with diverse systems. Exclusively, SERK1 responsible for potential signaling processes during SE revealed significant expression up-regulation in the embryogenic calli of FR line. Similarly, activity of antioxidant glutathione was substantially up-regulated in embryogenic calli of FR line in comparison to its counterpart form. In contrast, calli from early-stages of regeneration of both FR and NR lines had no significant influences on the regulation of SERK and glutathione levels prior to the acquisition of embryogenesis. These results highlight that in vitro purification of FR line in cotton for enhanced regeneration potential (through SE) resulted in signaling and metabolic transformations of the manner in which cellular redox levels have become modulated.

Understanding the Phylomorphological Implications of Pollinia from <i>Dendrobium</i>(Orchidaceae)

We aim to evaluate the evolutionary trends of pollen ultra-structures and microstructures in the light of molecular phylogeny, and to test whether pollen micromorphology is a predictor of interspecific phylogenetic relationship or a consequence of ecological influences. Using scanning electron microscopy pollinia micromorphologies of 18 species of the genus Dendrobium were comprehensively examined. A phylogenetic tree from ribosomal-ITS2 sequences was constructed reflecting molecular phylogeny. In result, two major clusters were identified representing the sections Aporum, Formoase Dendrobium and Holochrysa. The section Dendrobium could be further classified, for the first time, into two clades sharing the same root of origin. Variations in the shape of pollinia were distinct and consistent constituting six novel groups. Other qualitative/quantitative keys to pollinia showed congruence with molecular phylogeny and supported the correlated evolution of these traits. Concurrently pollen sculpturing also showed large variation in exine morphology (rugular/psilate to psilate-scabrate/rugulate-scabrate) providing explicit clues for phylogenetic trend of exine evolution. For the characteristic sculpturing on the pollinia surface, a simple version of pre-pattern model is also hypothesized for deeper insight into pattern formation in other biological systems. Results indicated that distinct groups of Dendrobium species have parallel and independent evolution accompanied with genetic changes occurred during species diversification. Thus pollen micromorphology is a good candidate for inferring the genetic relationship at inter-specific level having phylogenetic implications even using abundant palynological records.

Role of Plant Somatic Embryogenesis Receptor Kinases (SERKs) in Cell-to-Embryo Transitional Activity: Key at Novel Assorted Structural Subunits

Somatic Embryogenesis Receptor Kinase (SERK) family of receptor kinases is functionally diverse, involved in cell-to-embryo transition and controlling a number of other fundamental aspects of plant development. The morphological transformation of somatic to embryonic cells has drawn scientific attention utmost due to remarkable genetic-switch system evolved across species. Receptor kinases having direct role in somatic embryogenesis (SE) and involved in other functions are designated as “SERK” and “SERK-like” genes, respectively. We aim for phylogenetic reconstruction to reveal major SERK groups across plant species (angiosperm to gymnosperm) for their functional diversification. Data indicate that the development of SERK proteins occurred prior to the divergence of monocots and eudicots. Also, the SERK orthology is not directly proportional to their functions. Structure prediction results identified novel transmembrane topologies, short linear motifs and O-glycosylation sites exclusively in SERK proteins than SERK-like proteins. Comparative temporal expression analyses of SERK and SERK-like genes provided significant accordance with their physiological function. The identification of intrinsic disordered regions (IDRs) exclusively in SERK proteins was assumed to perceive external stress-induced signals that may lead to rapid protein folding. In a result it switches-on the precise cellular signals essential for the acquisition of SE. Moreover, the regulatory sequences of SERK genes are evolved with unique cellular fate deciding AP2-like ethylene responsive transcription factor AINTEGUMENTA binding sites for their spatial expression in SE. Based on these analyses we suggest future avenues of research that may be imperative for elucidating the importance of SERK gene evolution in SE.

Natural compounds as potential inhibitors of SARS-CoV-2 main protease: An insilico study

Objective:To explore natural compounds as potential inhibitors against main protease(Mpro)of SARS-CoV-2.Methods:In the current study,systematic molecular docking analysis was conducted using AutoDock 4.2 to determine the binding affinities and interactions between natural compounds and Mpro.Selected natural compounds were further validated using a combination of molecular dynamic(MD)simulations and molecular mechanic Poisson-Boltzmann surface area(MM/PBSA)free energy c alculation s.Results:Out of twenty natural compounds,four natural metabolites namely,amentoflavone,guggulsterone,puerarin,and piperine were found to have strong interaction with Mpro of SARS-CoV-2 based on docking analysis.During MD simulations,all four natural compounds bound to Mpro at 50 ns and MM/G/P/BSA free energy calculations showed that all four shortlisted ligands had stable and favorable energies with strong binding to Mpro protein.Conclusions:Guggulsterone is a potential inhibitor of COVID-19 main protease Mpro.Further in vitro and pre-clinical studies are needed.

Modulations in seed micromorphology reveal signature of adaptive species-diversification in <i>Dendrobium</i>(Orchidaceae)

During speciation in flowering plants, many morphological characters were evolved in response to continuous selection pressure. Such characters are largely governed by genetic changes or get modulated according to ecological adaptations. To explore the potential of latter, a novel comparative analysis of seed micromorphology and ultrastructure was performed in twenty species in the genus Dendrobium (Orchidaceae) from well-defined altitude based phytogeographical realms i.e. temperate, subtropical and tropical regions. Seed morphological characteristics from all collected species were examined using 13 quantitative traits descriptors. Comparative seed morphology revealed that the species from temperate region have larger seed volumes and seed volume/embryo volume ratio than species from subtropical or tropical regions irrespective of their phylogenetic association. Hence, phylogenetically related species emphasized their preference for a climatic region as their prime habitat. Similar trends towards gradual increase of percentage free air space were observed in the species of tropical to subtropical and subtropical to temperate regions. The higher percentage of free air space may have an advantage for the species from temperate region with relatively low atmospheric pressure that had greater buoyancy for optimal seed dispersal. Also, phylogenetically distant species from a particular climatic region, showed characteristic features of the testa cell walls sculptured with bead-like orna-mentations or covered with cottony white substances. Based on these observations, it may be concluded that seed volume, % free air space and seed coat ornamentation in Dendrobium species are signatures of adaptation according to climatic preferences rather than their selection during speciation.

Smoking under hypoxic conditions: a potent environmental risk factor for inflammatory and autoimmune diseases

Autoimmune disease management presents a significant challenge to medical science. Environmental factors potentially increase the risk of developing inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and lupus. Among various environmental stresses, cigarette smoke and hypoxia have both been reported to lead to an enhanced risk of inflammatory and autoimmune diseases. In this review, we shed light on all reported mechanisms whereby cigarette smoke and a hypoxic environment can induce inflammatory and autoimmune diseases and discuss how hypoxic conditions influence the cigarette smoke-induced threat of inflammatory and autoimmune disease development. Cigarette smoke and hypoxia both lead to increased oxidative stress and production of reactive oxygen species and other free radicals, which have various effects including the generation of autoreactive pro-inflammatory T cells and autoantibodies, reductions in T regulatory(Treg) cell activity, and enhanced expression of pro-inflammatory mediators [e.g., interleukin-6(IL-6), interleukin-4(IL-4) and interleukin-8(IL-8)]. Accordingly, smoking and hypoxic environments may synergistically act as potent environmental risk factors for inflammatory and autoimmune diseases. To our knowledge, no studies have reported the direct association of cigarette smoke and hypoxic environments with the risk of developing inflammatory and autoimmune diseases. Future studies exploring the risk of autoimmune disease development in smokers at high altitudes, particularly military personnel and mountaineers who are not acclimatized to high-altitude regions, are required to obtain a better understanding of disease risk as well as its management.

Bacterial Surface Layer Proteins: From Moonlighting to Biomimetics: A New Horizonto Lead

The landmark discovery of moonlighting proteins embarks the significant progress in understanding the biological complexity and their closed-circuit analysis. The growing continuum in the variety of moonlighting functions paved the way for further elucidation of structural-functional aspects of protein evolution and design of proteins with novel functions. Currently, the moonlighting functions in various adhesive properties of surface layer proteins, an essential component of cell surface architecture of archaea and all phylogenetic groups of eubacteria become more prominently recognized. The remarkable credentials of surface layer proteins to self-assemble into supramolecular structures at nano-scale dimension have been exploited for the production of smart biomaterials in the form of biomimetics has been thrust area of research. The finely tuned topological features in terms of shape, size, geometry and surface chemistry of surface layer proteins are crucial for the production of biomimetics. The current developments of biomimetic lipid bilayers and composite membranes find applicability in understanding the functional dynamism of evolutionary relationship of bacterial cell envelopes and vaccine development, drug development and drug delivery. Though the development of biomimetics embraces fascination but faces with technological challenges. The plethora of literature has been available for the moonlighting aspects and nano-technological applications separately but none of the review describes towards the rhythmic transition from moonlighting functions of surface layer proteins of bacteria to biomimetics development and applications. Therefore, this review describes certain basic aspects of moonlighting functions and their mechanism of action, surface layer proteins and their moonlighting functions of commensal bacteria and their transition towards biomimetics. The recent developments of biomimetics based on surface layer proteins have been summarized and also posited different challenges and future prospects.

Bacterial Surface Layer Proteins: A Promising Nano-Technological Tool for Bio-Sensing Applications

The phenomenal rise in the demand of biosensors accelerated their rapid development and immersive applications in the myriads of fields. The essential requirement of developing efficient bio-sensing platform is to find stable well organized interfacial architecture that can serve as an excellent matrix for binding and recognizing biomolecules. In this context, the enormous potential has been envisaged in surface layer proteins that represented themselves as most primitive and simplest self-assembled system with repetitive physicochemical properties for the molecular functionalization of surfaces and various interfaces. The prominence of S-layer proteins has been broadened by integrating genetic engineering approaches for the fine tuning of functional groups and protein domains in geometrically well-defined manner. The efficient and stable binding of various nanomaterials with S-layers in regular arrays has led to paradigmatic shift in their nano-biotechnological sensing applications. More recently, functional S-layer supported lipid membranes have been generated through covalent binding of lipid molecules either with native or recombinant S-layer proteins at nano-scale dimensions serving as “proof of concept” for the development of bio-sensing platform. Thus, in the light of benefits conferred by surface layer proteins for the development of highly efficient biosensors, an exciting path has been opened for broadening their translational applications in drug delivery, disease diagnosis, vaccines development, lab-on-chip devices etc. Therefore, this review intends to describe about the importance of surface layer proteins in the development of biosensors.

A Botanist’s Cognitive View on Plant Growth: Cross-Talk between Developmental and Sensitivity Networks

An alteration in plant phenotypes assisted by their responses to the environmental stimuli (=tropism) has been fundamental to understand the “plant sensitivity ” that plays a crucial role in plants’ adaptive success. Plants succeed through the deployment of moderators controlling polar auxin-transport determining organ bending. Stimulus-specific effectors can be synthesized by the outer peripheral cells at the bending sites where they target highly conserved cellular processes and potentially persuade the plant sensitivity at large. Remarkably, the peripheral cells require different time-intervals to achieve the threshold expression-levels of stimulus-specific molecular responders. After stimulus perception, tropic curvatures (especially at growing root-apices) are duly coordinated via integrated chemical and electrical signalling which is the key to cellular communications. Thus, the acquired phenotypic alterations are the perplexed outcome of plant’s developmental pace, complemented by the sensitivity. A novel aspect of this study is to advance our understanding of plant developmental-programming and the extent of plant-sensitivity, determining the plant growth and their future applications.

To Bloom or Not to Bloom： Role of MicroRNAs in Plant Flowering

During the course of their life cycles, plants undergo various morphological and physiological changes un- derlying juvenile-to-adult and adult-to-flowering phase transitions. To flower or not to flower is a key step of plasticity of a plant toward the start of its new life cycle. In addition to the previously revealed intrinsic genetic programs, exogenous cues, and endogenous cues, a class of small non-coding RNAs, microRNAs （miRNAs）, plays a key role in plants making the decision to flower by integrating into the known flowering pathways. This review highlights the age-dependent flowering pathway with a focus on a number of timing miRNAs in determining such a key process. The contributions of other miRNAs which exist mainly outside the age pathway are also discussed. Approaches to study the flowering-determining miRNAs, their inter- actions, and applications are presented.

Visualizing the Transport of Porcine Reproductive and Respiratory Syndrome Virus in Live Cells by Quantum Dots-Based Single Virus Tracking

Quantum dots(QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time.The porcine reproductive and respiratory syndrome virus(PRRSV)is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide.A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies.In this study,we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells.Our results indicated that the labeling method had negligible effect on viral infectivity.We also observed that prior to the entry,PRRSV vibrated on the plasma membrane,and entered the cells via endosome mediated cell entry pathway.Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell.Our results also showed that once inside the cell,PRRSV moved along the microtubule,microfilament and vimentin cytoskeletal elements.During the transport process,virus particles also made contacts with non-muscle myosin heavy chainⅡ-A(NMHCⅡ-A),visualized as small spheres in cytoplasm.This study can facilitate the application of QDs in virus infection imaging,especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism.