Morpho-physiological studies of sandalwood-host interaction under individual and interactive water and salt stress.

To find out the possibilities of growing white sandalwood in sub-tropical regions of India where farmers facing the problem of water deficit and salinity stress, a RBD experiment was conducted. Sandalwood grown alone and with five selected hosts (Alternanthera sp., Neem, Shisham, Dek and Agarwood) on the basis of prior study under water deficit, salinity stress and combined water deficit and salinity stress. Sandalwood plants were harvested after 180 days of imposing stress treatments. Morphological traits (plant height, collar diameter, shoot fresh and dry biomass) showed significant reduction under water deficit and salinity stress, which were further aggravated under combined water deficit and salinity stress. Studied plant water traits, ionic balance and gas exchange attributes were also reduced by these stresses. While among studied host, Shisham and Dek identified as the best host species under water deficit, salinity and interactive stress by maintaining ion homeostasis, osmotic adjustments and plant water regulation. Results depicted that sandalwood plants cultivated alone were not able to survive under salinity and combined stress conditions and showed poor growth under water deficit and control conditions. Different indices were also calculated based on morpho-physiological and ionic traits and also indicated that sandalwood grown with Dalbergia sissoo and Melia dubia showed higher drought, salt and stress tolerance potential, which made sandalwood adaptable under these stresses. Therefore, the present study signifies the importance of host especially D. sissoo and M. dubia which might be excellent long-term host species for sandalwood cultivation under sub-tropical conditions to thrive under changing environments.

Dataset on antioxidant system of non-model halophytes Urochondra setulosa and Dichanthium annulatum in saline environment.

The antioxidant potential of halophytes, Dichanthium annulatum and Urochondra setulosa, was examined under the influence of high salinity. These halophytes were grown in lysimeters filled with saline soil and further irrigated with saline water to maintain different salt levels of ECe 30, 40 and 50 dS m-1 along with the one set in normal field soil without saline irrigation serving as control. The leaf samples were collected after saline irrigation and analyzed for the antioxidative enzymes i.e., Catalase (CAT), Peroxidase (POX), Superoxide dismutase (SOD), Ascorbate peroxidase (APX), Monodehydroascorbate reductase (MDHAR), Dehydroascorbate reductase (DHAR) and Glutathione reductase (GR), including the ROS metabolites such as H2O2 content, malondialdehyde content (MDA), ascorbic acid content and total glutathione content. The mechanism of scavenging the reactive oxygen species in both the halophytes was characterized.

Halophytes as new model plant species for salt tolerance strategies.

Soil salinity is becoming a growing issue nowadays, severely affecting the world's most productive agricultural landscapes. With intersecting and competitive challenges of shrinking agricultural lands and increasing demand for food, there is an emerging need to build resilience for adaptation to anticipated climate change and land degradation. This necessitates the deep decoding of a gene pool of crop plant wild relatives which can be accomplished through salt-tolerant species, such as halophytes, in order to reveal the underlying regulatory mechanisms. Halophytes are generally defined as plants able to survive and complete their life cycle in highly saline environments of at least 200-500 mM of salt solution. The primary criterion for identifying salt-tolerant grasses (STGs) includes the presence of salt glands on the leaf surface and the Na+ exclusion mechanism since the interaction and replacement of Na+ and K+ greatly determines the survivability of STGs in saline environments. During the last decades or so, various salt-tolerant grasses/halophytes have been explored for the mining of salt-tolerant genes and testing their efficacy to improve the limit of salt tolerance in crop plants. Still, the utility of halophytes is limited due to the non-availability of any model halophytic plant system as well as the lack of complete genomic information. To date, although Arabidopsis (Arabidopsis thaliana) and salt cress (Thellungiella halophila) are being used as model plants in most salt tolerance studies, these plants are short-lived and can tolerate salinity for a shorter duration only. Thus, identifying the unique genes for salt tolerance pathways in halophytes and their introgression in a related cereal genome for better tolerance to salinity is the need of the hour. Modern technologies including RNA sequencing and genome-wide mapping along with advanced bioinformatics programs have advanced the decoding of the whole genetic information of plants and the development of probable algorithms to correlate stress tolerance limit and yield potential. Hence, this article has been compiled to explore the naturally occurring halophytes as potential model plant species for abiotic stress tolerance and to further breed crop plants to enhance salt tolerance through genomic and molecular tools.

Deciphering trait associated morpho-physiological responses in pearlmillet hybrids and inbred lines under salt stress.

Pearl millet is a staple food for more than 90 million people residing in highly vulnerable hot arid and semi-arid regions of Africa and Asia. These regions are more prone to detrimental effects of soil salinity on crop performance in terms of reduced biomass and crop yields. We investigated the physiological mechanisms of salt tolerance to irrigation induced salinity stress (ECiw ~3, 6 & 9 dSm-1) and their confounding effects on plant growth and yield in pearl millet inbred lines and hybrids. On average, nearly 30% reduction in above ground plant biomass was observed at ECiw ~6 dSm-1 which stretched to 56% at ECiw ~9 dSm-1 in comparison to best available water. With increasing salinity stress, the crop performance of test hybrids was better in comparison to inbred lines; exhibiting relatively higher stomatal conductance (gS; 16%), accumulated lower proline (Pro; -12%) and shoot Na+/K+(-31%), synthesized more protein (SP; 2%) and sugars (TSS; 32%) compensating in lower biomass (AGB; -22%) and grain yield (GY: -14%) reductions at highest salinity stress of ECiw ~9 dSm-1. Physiological traits modeling underpinning plant salt tolerance and adaptation mechanism illustrated the key role of 7 traits (AGB, Pro, SS, gS, SPAD, Pn, and SP) in hybrids and 8 traits (AGB, Pro, PH, Na+, K+, Na+/K+, SPAD, and gS) in inbred lines towards anticipated grain yield variations in salinity stressed pearl millet. Most importantly, the AGB alone, explained >91% of yield variation among evaluated hybrids and inbreed lines at ECiw ~9 dSm-1. Cumulatively, the better morpho-physiological adaptation and lesser yield reduction with increasing salinity stress in pearl millet hybrids (HHB 146, HHB 272, and HHB 234) and inbred lines (H77/833-2-202, ICMA 94555 and ICMA 843-22) substantially complemented in increased plant salt tolerance and yield stability over a broad range of salinity stress. The information generated herein will help address in deciphering the trait associated physiological alterations to irrigation induced salt stress, and developing potential hybrids in pearl millet using these parents with special characteristics.

Getting to the roots of Cicer arietinum L. (chickpea) to study the effect of salinity on morpho-physiological, biochemical and molecular traits.

The effect of saline irrigation (ECiw 6 dS m-1 and 9 dS m-1) on the roots of Cicer arietinum L. genotypes was examined at morpho-physiological, biochemical and molecular levels. Reduction in root growth due to salinity was observed, but less effect was seen on the roots of genotypes KWR 108, ICCV 10, CSG 8962, and S7 as compared to the other genotypes. Cell turgor was maintained in tolerant genotypes through optimum water relations and osmoprotectants (proline and total soluble sugars) than the sensitive cultivars. Salinity caused oxidative stress as increased hydrogen peroxide and malondialdehyde were noticed, where low accumulation was observed in tolerant genotypes due to the higher activity of enzymatic antioxidants (superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and peroxidase). Na+/K+ ratio increased, but more increment was reported in sensitive cultivars. Gene expression studies depicted that genes encoding pyrroline-5-carboxylate synthetase and pyrroline-5-carboxylate reductase got upregulated and that of proline dehydrogenase was downregulated and more fold change with respect to control was in the salt tolerant check CSG 8962 and the genotype KWR 108. Higher expression of the genes encoding reactive oxygen species scavenging enzymes namely, superoxide dismutase, catalase, peroxidase, and those involved in the ascorbate-glutathione cycle was noticed in KWR 108 and CSG 8962 than ICC 4463. Enhanced expression of sodium transporter HKT1 due to salinity can be correlated with ion homeostasis maintenance. Cumulative effects of osmolytes, enzymatic antioxidants and maintaining ion homeostasis in root enable chickpea plants to survive in saline environments.

Comparative Flower Transcriptome Network Analysis Reveals DEGs Involved in Chickpea Reproductive Success during Salinity.

Salinity is increasingly becoming a significant problem for the most important yet intrinsically salt-sensitive grain legume chickpea. Chickpea is extremely sensitive to salinity during the reproductive phase. Therefore, it is essential to understand the molecular mechanisms by comparing the transcriptomic dynamics between the two contrasting genotypes in response to salt stress. Chickpea exhibits considerable genetic variation amongst improved cultivars, which show better yields in saline conditions but still need to be enhanced for sustainable crop production. Based on previous extensive multi-location physiological screening, two identified genotypes, JG11 (salt-tolerant) and ICCV2 (salt-sensitive), were subjected to salt stress to evaluate their phenological and transcriptional responses. RNA-Sequencing is a revolutionary tool that allows for comprehensive transcriptome profiling to identify genes and alleles associated with stress tolerance and sensitivity. After the first flowering, the whole flower from stress-tolerant and sensitive genotypes was collected. A total of ~300 million RNA-Seq reads were sequenced, resulting in 2022 differentially expressed genes (DEGs) in response to salt stress. Genes involved in flowering time such as FLOWERING LOCUS T (FT) and pollen development such as ABORTED MICROSPORES (AMS), rho-GTPase, and pollen-receptor kinase were significantly differentially regulated, suggesting their role in salt tolerance. In addition to this, we identify a suite of essential genes such as MYB proteins, MADS-box, and chloride ion channel genes, which are crucial regulators of transcriptional responses to salinity tolerance. The gene set enrichment analysis and functional annotation of these genes in flower development suggest that they can be potential candidates for chickpea crop improvement for salt tolerance.

De novo transcriptomic data of salt tolerant halophytes Dichnathium annulatum (Forssk.) stapf and Urochondra setulosa (Trin.) C.E.Hubb.

Two halophytes, Dichanthium annulatum (moderately salt tolerant) and Urochondra setulosa (highly salt tolerant) were selected to generate transcriptome at different salinity levels. Sequencing of RNA samples was done on Illumina-Hi-Seq platform for de novo transcriptome assembly from the leaf tissues of D. annulatum at salinity of ECe ∼30 dS/m and of U. setulosa at three salt levels (i.e. ECe ∼30, ∼40 and ∼50 dS/m). DESeq was used for identification of differentially expressed transcripts and a total of 267,196 and 384,442 transcripts were assembled through Trinity in both the plants respectively. A total of 32,246 and 25,479 SSRs were identified respectively in both the plants using MISA perl script with mono and tri-nucleotide repeats as most common motif.

Quantitative spatial and temporal assessment of regulatory element activity in zebrafish.

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

Effect of saline irrigation on plant water traits, photosynthesis and ionic balance in durum wheat genotypes.

In the era of climate change, decreased precipitation and increased evapo-transpiration hampers the yield of several cereal crops along with the soil salinity and poor ground water resource. Wheat being the moderately tolerant crop face many challenges in the arid and semi-arid regions under irrigated agriculture. In view of this, the study was planned to explore the potential of durum wheat genotypes under salinity on the basis of physiological traits. Experiment was designed as RBD in three replications to evaluate 15 wheat genotypes with moderate saline irrigation (ECiw - 6 dS m-1) and extreme saline irrigation (ECiw - 10 dS m-1) along with one set of control (Best available water). Different physiological traits such as water potential (ψp), osmotic potential (ψs), relative water content (RWC), Na+ and K+ content were recorded in roots as well as shoots at the reproductive stage whereas photosynthetic rate and chlorophyll content were measured in the flag leaves. A significant variability (p < 0.001) was noted among the genotypes under different stress environments and it was observed that durum genotype HI 8728 and HI 8737 showed less reduction in plant water traits (RWC, ψp and ψs) than the salinity tolerant checks of bread wheat KRL 99 and KRL 3-4. HD 4728 and HI 8708 maintained higher photosynthetic rate as well as higher chlorophyll content under the extreme salinity level of ECiw - 10 dSm-1. No significant differences were found in root Na+ in genotypes KRL 99 (3.17g), KRL 3-4 (3.34g) and HI 8737 (3.41g) while in shoots, lowest accumulation was seen in KRL 99, MACS 3949 and KRL 3-4 at ECiw - 10 dSm-1. The mean range of K+ content was 7.60-9.74% in roots and 4.21-6.61% in shoots under control environment which decreased to 50.77% in roots and 46.05% in shoots under extreme salinity condition of ECiw - 10 dSm-1. At ECiw - 10 dSm-1, KRL 99 maintained highest K+/Na+ in both root and shoot followed by KRL 3-4, HI 8737, MACS 3949, HD 4728 in roots and MACS 3949, KRL 3-4, MACS 4020, HD 4758, MACS 3972 and HI 8713 in shoots. The differential response of durum wheat genotypes under salinity particularly for physiological traits, confer their adaptability towards stress environments and exhibit their potential as genetic sources in breeding programs for improving salt stress tolerance.

de novo transcriptomic profiling of differentially expressed genes in grass halophyte Urochondra setulosa under high salinity.

Soil salinity is one of the major limiting factors for crop productivity across the world. Halophytes have recently been a source of attraction for exploring the survival and tolerance mechanisms at extreme saline conditions. Urochondra setulosa is one of the obligate grass halophyte that can survive in up to 1000 mM NaCl. The de novo transcriptome of Urochondra leaves at different salt concentrations of 300-500 mM NaCl was generated on Illumina HiSeq. Approximately 352.78 million high quality reads with an average contig length of 1259 bp were assembled de novo. A total of 120,231 unigenes were identified. On an average, 65% unigenes were functionally annotated to known proteins. Approximately 35% unigenes were specific to Urochondra. Differential expression revealed significant enrichment (P < 0.05) of transcription factors, transporters and metabolites suggesting the transcriptional regulation of ion homeostasis and signalling at high salt concentrations in this grass. Also, about 143 unigenes were biologically related to salt stress responsive genes. Randomly selected genes of important pathways were validated for functional characterization. This study provides useful information to understand the gene regulation at extremely saline levels. The study offers the first comprehensive evaluation of Urochondra setulosa leaf transcriptome. Examining non-model organisms that can survive in harsh environment can provide novel insights into the stress coping mechanisms which can be useful to develop improved agricultural crops.

Physiological response of diverse halophytes to high salinity through ionic accumulation and ROS scavenging.

Salt stress induced modulations in different ionic ratios and ROS system were studied in ten halophytic species, namely Atriplex lentiformis, Tamarix aphylla, Sporobolus marginatus, Suaeda nudiflora, Urochondra setulosa, Arundo donax, Aeluropus lagopoides, Heliotropium ramossimum, Atriplex nummularia, Leptachloa fusca at salinity level of ECe ∼ 30 dSm-1 (≈300 mM NaCl) to explore their possible role in salt tolerance ability of these halophytes. These halophytes were categorized for their salt tolerance levels based on the ratios of Na+/K+, Na+/Ca2+, Na+/Cl- and Na + Cl/K + Ca. Variable responses were observed among all halophytes where Atriplex lentiformis had lowest leaf Na+/K+ (0.44) which is one of the best indicator of salt tolerance, Heliotropium ramossimum had lowest Na+/Ca2+ and Na+/Cl- (0.97 and 0.18), whereas Sporobolus marginatus had lowest Na + Cl/K + Ca (0.79). Specific enzymes activities of ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) were also assessed to get better comprehension of the ROS scavenging system under salinity in these halophytes. Urochondra setulosa showed highest APX and SOD activity followed by Atriplex lentiformis. Most efficient enzyme in degrading hydrogen peroxide i.e. CAT showed highest activity in Suaeda nudiflora followed by Atriplex nummularia and Urochondra setulosa, whereas Atriplex nummularia and Atriplex lentiformis showed higher POX activity. Significant variability in H2O2 and MDA content was also observed. These results possibly suggest higher inbuilt genetic potential of these halophytes to combat high salinity induced oxidative stress via higher antioxidant activities. Novelty statement: Halophytic plant adopt different strategies to cope up with the toxic ions and our studies show that the induction of antioxidant defense system to scavenge ROS, alongwith structural modifications in terms of lipid peroxidation and compartmentalization of toxic ions are the main strategies for tighter control of ion fluxes in the studied halophytes.

Genetic Dissection and Identification of Candidate Genes for Salinity Tolerance Using Axiom®CicerSNP Array in Chickpea.

Globally, chickpea production is severely affected by salinity stress. Understanding the genetic basis for salinity tolerance is important to develop salinity tolerant chickpeas. A recombinant inbred line (RIL) population developed using parental lines ICCV 10 (salt-tolerant) and DCP 92-3 (salt-sensitive) was screened under field conditions to collect information on agronomy, yield components, and stress tolerance indices. Genotyping data generated using Axiom®CicerSNP array was used to construct a linkage map comprising 1856 SNP markers spanning a distance of 1106.3 cM across eight chickpea chromosomes. Extensive analysis of the phenotyping and genotyping data identified 28 quantitative trait loci (QTLs) explaining up to 28.40% of the phenotypic variance in the population. We identified QTL clusters on CaLG03 and CaLG06, each harboring major QTLs for yield and yield component traits under salinity stress. The main-effect QTLs identified in these two clusters were associated with key genes such as calcium-dependent protein kinases, histidine kinases, cation proton antiporter, and WRKY and MYB transcription factors, which are known to impart salinity stress tolerance in crop plants. Molecular markers/genes associated with these major QTLs, after validation, will be useful to undertake marker-assisted breeding for developing better varieties with salinity tolerance.

Zebrafish: A Powerful Model for Understanding the Functional Relevance of Noncoding Region Mutations in Human Genetic Diseases.

Determining aetiology of genetic disorders caused by damaging mutations in protein-coding genes is well established. However, understanding how mutations in the vast stretches of the noncoding genome contribute to genetic abnormalities remains a huge challenge. Cis-regulatory elements (CREs) or enhancers are an important class of noncoding elements. CREs function as the primary determinants of precise spatial and temporal regulation of their target genes during development by serving as docking sites for tissue-specific transcription factors. Although a large number of potential disease-associated CRE mutations are being identified in patients, lack of robust methods for mechanistically linking these mutations to disease phenotype is currently hampering the understanding of their roles in disease aetiology. Here, we have described the various systems available for testing the CRE potential of stretches of noncoding regions harbouring mutations implicated in human disease. We highlight advances in the field leading to the establishment of zebrafish as a powerful system for robust and cost-effective functional assays of CRE activity, enabling rapid identification of causal variants in regulatory regions and the validation of their role in disruption of appropriate gene expression.

Linear short histidine and cysteine modified arginine peptides constitute a potential class of DNA delivery agents.

The success of gene therapy relies on the development of safe and efficient multifunctional carriers of nucleic acids that can overcome extra- and intracellular barriers, protect the nucleic acid and mediate its release at the desired site allowing gene expression. Peptides bear unique properties that are indispensable for any carrier, e.g., they can mediate DNA condensation, cellular targeting, membrane translocation, endosomal escape and nuclear localization. In an effort to design a multifunctional peptide, we have modified an arginine homopeptide R16 by replacement of seven arginines with histidines and addition of one cysteine at each end respectively to impart endosomal escape property while maintaining the DNA condensation and release balance. Addition of histidines imparts endosomal escape property to arginine homopeptide, but their arrangement with respect to arginines is more critical in controlling DNA condensation, release and transfection efficiency. Intriguingly, R5H7R4 peptide where charge/arginine is distributed in blocks is preferred for strong condensation while more efficient transfection is seen in the variants R9H7 and H4R9H3, which exhibit weak condensation and strong release. Addition of cysteine to each of these peptides further fine-tuned the condensation-release balance without application of any oxidative procedure unlike other similar systems reported in the literature. This resulted in a large increase in the transfection efficiency in all of the histidine modified peptides irrespective of the arginine and histidine positions. This series of multifunctional peptides shows comparable transfection efficiency to commercially available transfection reagent Lipofectamine 2000 at low charge ratios, with simple preparative procedure and exhibits much less toxicity.

Engineered reversal of drug resistance in cancer cells--metastases suppressor factors as change agents.

Building molecular correlates of drug resistance in cancer and exploiting them for therapeutic intervention remains a pressing clinical need. To identify factors that impact drug resistance herein we built a model that couples inherent cell-based response toward drugs with transcriptomes of resistant/sensitive cells. To test this model, we focused on a group of genes called metastasis suppressor genes (MSGs) that influence aggressiveness and metastatic potential of cancers. Interestingly, modeling of 84 000 drug response transcriptome combinations predicted multiple MSGs to be associated with resistance of different cell types and drugs. As a case study, on inducing MSG levels in a drug resistant breast cancer line resistance to anticancer drugs caerulomycin, camptothecin and topotecan decreased by more than 50-60%, in both culture conditions and also in tumors generated in mice, in contrast to control un-induced cells. To our knowledge, this is the first demonstration of engineered reversal of drug resistance in cancer cells based on a model that exploits inherent cellular response profiles.

Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells.

Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton's jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4-6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro.

Structural rearrangements and chemical modifications in known cell penetrating peptide strongly enhance DNA delivery efficiency.

Amphipathic peptides with unusual cellular translocation properties have been used as carriers of different biomolecules. However, the parameters which control the delivery efficiency of a particular cargo by a peptide and the selectivity of cargo delivery are not very well understood. In this work, we have used the known cell penetrating peptide pVEC (derived from VE-cadherin) and systematically changed its amphipathicity (from primary to secondary) as well as the total charge and studied whether these changes influence the plasmid DNA condensation ability, cellular uptake of the peptide-DNA complexes and in turn the efficiency of DNA delivery of the peptide. Our results show that although the efficiency of DNA delivery of pVEC is poor, modification of the same peptide to create a combination of nine arginines along with secondary amphipathicity improves its plasmid DNA delivery efficiency, particularly in presence of an endosomotropic agent like chloroquine. In addition, presence of histidines along with 9 arginines and secondary amphipathicity shows efficient DNA delivery with low toxicity even in absence of chloroquine in multiple cell lines. We attribute these enhancements in transfection efficiency to the differences in the mechanism of complex formation by the different variants of the parent peptide which in turn are related to the chemical nature of the peptide itself. These results exhibit the importance of understanding the physicochemical parameters of the carrier and complex in modulating gene delivery efficiency. Such studies can be helpful in improving peptide design for delivery of different cargo molecules.

Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies.

Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

Exogenous and cell surface glycosaminoglycans alter DNA delivery efficiency of arginine and lysine homopeptides in distinctly different ways.

Glycosaminoglycans (GAGs) expressed ubiquitously on the cell surface are known to interact with a variety of ligands to mediate different cellular processes. However, their role in the internalization of cationic gene delivery vectors such as liposomes, polymers, and peptides is still ambiguous and seems to be controlled by multiple factors. In this report, taking peptides as model systems, we show that peptide chemistry is one of the key factors that determine the dependence on cell surface glycosaminoglycans for cellular internalization and gene delivery. Arginine peptides and their complexes with plasmid DNA show efficient uptake and functional gene transfer independent of the cell surface GAGs. On the other hand, lysine peptides and complexes primarily enter through a GAG-dependent pathway. The peptide-DNA complexes also show differential interaction with soluble GAGs. In the presence of exogenous GAGs under certain conditions, arginine peptide-DNA complexes show increased transfection efficiency that is not observed with lysine. This is attributed to a change in the complex nature that ensures better protection of the compacted DNA in the case of arginine complexes, whereas the lysine complexes get destabilized under these conditions. The presence of a GAG coating also ensures better cell association of arginine complexes, resulting in increased uptake. Our results indicate that the role of both the cell surface and exogenous glycosaminoglycans in gene delivery is controlled by the nature of the peptide and its complex with DNA.

Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose.

The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z(+/-)). The nanoparticles at Z(+/-)12 are spherical with 70+/-7 nm diameter in AFM and displayed positive surface charge and similar sizes (83+/-8 nm) by Zetasizer. The nanoparticles of Z(+/-) 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, approximately 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability.