Heat-stress-responsive HvHSFA2e gene regulates the heat and drought tolerance in barley through modulation of phytohormone and secondary metabolic pathways.

KEY MESSAGE: The heat stress transcription factor HSFA2e regulates both temperature and drought response via hormonal and secondary metabolism alterations. High temperature and drought are the primary yield-limiting environmental constraints for staple food crops. Heat shock transcription factors (HSF) terminally regulate the plant abiotic stress responses to maintain growth and development under extreme environmental conditions. HSF genes of subclass A2 predominantly express under heat stress (HS) and activate the transcriptional cascade of defense-related genes. In this study, a highly heat-inducible HSF, HvHSFA2e was constitutively expressed in barley (Hordeum vulgare L.) to investigate its role in abiotic stress response and plant development. Transgenic barley plants displayed enhanced heat and drought tolerance in terms of increased chlorophyll content, improved membrane stability, reduced lipid peroxidation, and less accumulation of ROS in comparison to wild-type (WT) plants. Transcriptome analysis revealed that HvHSFA2e positively regulates the expression of abiotic stress-related genes encoding HSFs, HSPs, and enzymatic antioxidants, contributing to improved stress tolerance in transgenic plants. The major genes of ABA biosynthesis pathway, flavonoid, and terpene metabolism were also upregulated in transgenics. Our findings show that HvHSFA2e-mediated upregulation of heat-responsive genes, modulation in ABA and flavonoid biosynthesis pathways enhance drought and heat stress tolerance.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

An insight into Pisum sativum HSF gene family-Genome-wide identification, phylogenetic, expression, and analysis of transactivation potential of pea heat shock transcription factor.

Field pea (Pisum sativum L, 2n = 14) is a popular temperate legume with high economic value. Heat shock factors (HSFs) are the core element in the regulatory mechanism of heat stress responses. HSFs in pea (P. sativum) have not been characterized and their role remains unclear in different abiotic stresses. To address this knowledge gap, the current study aimed to characterize the HSF gene family in pea. We identified 38 PsHsf members in P. sativum, which are distributed on the seven chromosomes, and based on phylogenetic analysis, we classified them into three representative classes i.e. A, B, and C. Conserved motif and gene structure analysis confirmed a high degree of similarity among the members of the same class. Additionally, identified cis-acting regulatory elements (CAREs) related to abiotic responses, development, growth, and hormone signaling provides crucial insights into the regulatory mechanisms of PsHsfs. Our research revealed instances of gene duplication in PsHsf gene family, suggesting that this mechanism could be driving the expansion of the PsHsf gene family. Moreover, Expression analysis of PsHsfs exhibited upregulation under heat stress (HS), salt stress (SS), and drought stress (DS) showing their phenomenal role in stress conditions. PsHsfs protein interaction network suggested their involvement in stress-responsive mechanisms. Further transactivation potential was checked for spliced variant of PsHsfA2a (PsHsfA2aI, PsHsfA2aII, and PsHsfA2aIII), PsHsfA3, PsHsfA6b, PsHsfA9, PsHsfB1a, and PsHsfB2a. Overall, these findings provide valuable insight into the evolutionary relationship of PsHsf gene family and their role in abiotic stress responses.

An injectable PEG-like conjugate forms a subcutaneous depot and enables sustained delivery of a peptide drug.

Many biologics have a short plasma half-life, and their conjugation to polyethylene glycol (PEG) is commonly used to solve this problem. However, the improvement in the plasma half-life of PEGylated drugs' is at an asymptote because the development of branched PEG has only had a modest impact on pharmacokinetics and pharmacodynamics. Here, we developed an injectable PEG-like conjugate that forms a subcutaneous depot for the sustained delivery of biologics. The PEG-like conjugate consists of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) conjugated to exendin, a peptide drug used in the clinic to treat type 2 diabetes. The depot-forming exendin-POEGMA conjugate showed greater efficacy than a PEG conjugate of exendin as well as Bydureon, a clinically approved sustained-release formulation of exendin. The injectable depot-forming exendin-POEGMA conjugate did not elicit an immune response against the polymer, so that it remained effective and safe for long-term management of type 2 diabetes upon chronic administration. In contrast, the PEG conjugate induced an anti-PEG immune response, leading to early clearance and loss of efficacy upon repeat dosing. The exendin-POEGMA depot also showed superior long-term efficacy compared to Bydureon. Collectively, these results suggest that an injectable POEGMA conjugate of biologic drugs that forms a drug depot under the skin, providing favorable pharmacokinetic properties and sustained efficacy while remaining non-immunogenic, offers significant advantages over other commonly used drug delivery technologies.

Polyethylene Glycol-Like Brush Polymer Conjugate of a Protein Drug Does Not Induce an Antipolymer Immune Response and Has Enhanced Pharmacokinetics than Its Polyethylene Glycol Counterpart.

Protein therapeutics, except for antibodies, have a short plasma half-life and poor stability in circulation. Covalent coupling of polyethylene glycol (PEG) to protein drugs addresses this limitation. However, unlike previously thought, PEG is immunogenic. In addition to induced PEG antibodies, ≈70% of the US population has pre-existing anti-PEG antibodies. Both induced and preexisting anti-PEG antibodies result in accelerated drug clearance, reduced clinical efficacy, and severe hypersensitivity reactions that have limited the clinical utility of uricase, an enzyme drug for treatment for refractory gout that is decorated with a PEG corona. Here, the authors synthesize a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) conjugate of uricase that decorates the protein with multiple polymer chains to create a corona to solve these problems. The resulting uricase-POEGMA is well-defined, has high bioactivity, and outperforms its PEG counterparts in its pharmacokinetics (PK). Furthermore, the conjugate does not induce anti-POEGMA antibodies and is not recognized by anti-PEG antibodies. These findings suggest that POEGMA conjugation may provide a solution to the immunogenicity and antigenicity limitations of PEG while improving upon its PK benefits. These results transcend uricase and can be applied to other PEGylated therapeutics and the broader class of biologics with suboptimal PK.

Transcriptome based identification and validation of heat stress transcription factors in wheat progenitor species Aegilops speltoides.

Wheat, one of the major cereal crops worldwide, get adversely affected by rising global temperature. We have identified the diploid B genome progenitor of wheat, Aegilops speltoides (SS), as a potential donor for heat stress tolerance. Therefore, the present work was planned to study the total transcriptome profile of heat stress-tolerant Ae. speltoides accession pau3809 (AS3809) and compare with that of tetraploid and hexaploid wheat cultivars PDW274 and PBW725, respectively. The comparative transcriptome was utilized to identify and validate heat stress transcription factors (HSFs), the key genes involved in imparting heat stress tolerance. Transcriptome analysis led to the identification of a total of 74 K, 68 K, and 76 K genes in AS3809, PDW274, and PBW725, respectively. There was a high uniformity of GO profiles under the biological, molecular, and cellular functions across the three wheat transcriptomes, suggesting the conservation of gene function. Twelve HSFs having the highest FPKM value were identified in the AS3809 transcriptome data, while six of these HSFs namely HSFA3, HSFA5, HSFA9, HSFB2a, HSFB2b, and HSFC1b, were validated with qRT PCR. These six HSFs were identified as an important component of thermotolerance in AS3809 as evident from their comparative higher expression under heat stress.

The enhanced phosphorus use efficiency in phosphate-deficient and mycorrhiza-inoculated barley seedlings involves activation of different sets of PHT1 transporters in roots.

MAIN CONCLUSION: Transcriptional activation of subfamily II PHT1 members in roots is associated with the enhanced phosphorus use efficiency and growth promotion of barley seedlings inoculated with Glomus species. The arbuscular mycorrhizal (AM) fungi symbiotic associations in cereal crops are known to regulate growth in cultivar-specific manner and induce phosphate (Pi) transporters (PHT1) in roots. In the present study, we observed that both AM colonization of roots by Glomus species and phosphate starvation enhanced phosphorus use efficiency (PUE) in barley seedlings. Our search for the full complement of PHT1 members in the recently sequenced barley genome identified six additional genes, totaling their number to 17. Both AM colonization and Pi starvation triggered activation of common as well as different PHT1s. Pi starvation led to the robust upregulation of HvPHT1;6.2/6.3 at 7d and weak activation of HvPHT1;1 in shoots at 3d time-point. In roots, only HvPHT1;1, HvPHT1;6.2/6.3, HvPHT1;7, HvPHT1;8, HvPHT1;11.2 and HvPHT12 were induced at least one of the time-points. AM colonization specifically upregulated HvPHT1;11, HvPHT1;11.2, HvPHT1;12 and HvPHT1;13.1/13.2, members belonging to subfamily II, in roots. Sucrose availability seems to be obligatory for the robust activation of HvPHT1;1 as unavailability of this metabolite generally weakened its upregulation under Pi starvation. Intriguingly, lack of sucrose supply also led to induction of HvPHT1;5, HvPHT1;8, and HvPHT1;11.2 in either roots or shoot or both. The mRNA levels of HvPHT1;5 and HvPHT1;11.2 were not severely affected under combined deficiency of Pi and sucrose. Taken together, this study not only identify additional PHT1 members in barley, but also ascertain their AM, Pi and sucrose-specific transcript accumulation. The beneficial role of AM fungi in the promotion of PUE and barley seedlings' growth is also demonstrated.

Overexpression of wheat transcription factor (TaHsfA6b) provides thermotolerance in barley.

MAIN CONCLUSION: Overexpressing a heat shock factor gene (TaHsfA6bT) from wheat provides thermotolerance in barley by constitutive expression of heat and other abiotic stress-response genes. Temperature is one of the most crucial abiotic factors defining the yield potential of temperate cereal crops, such as barley. The regulators of heat shock response (HSR), heat stress transcription factors (Hsfs), modulate the transcription level of heat-responsive genes to protect the plants from heat stress. In this study, an Hsf from wheat (TaHsfA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHsfA6b showed improvement in thermotolerance. The constitutive overexpression of a TaHsfA6b gene upregulated the expression of major heat shock proteins and other abiotic stress-responsive genes. RNA-seq and qRT-PCR analysis confirmed the upregulation of Hsps, chaperonins, DNAJ, LEA protein genes, and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of reactive oxygen species (ROS) occurred in wild-type (WT) plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms, showing lesser ROS accumulation under high temperature. No negative phenotypic changes were observed in overexpression lines. These results suggest that TaHsfA6b is a regulator of HSR and its overexpression altered the expression patterns of some main stress-related genes and enhanced the thermotolerance of this cereal crop.

Updated inventory, evolutionary and expression analyses of G (PDR) type ABC transporter genes of rice.

ABC transporters constitute the largest family of transporter proteins in living organisms and divided into eight subfamilies, from A-H. ABCG members, specific to plants and fungi, belong to subfamily G. In this study, we provide updated inventory, detailed account of phylogeny, gene structure characteristics, and expression profiling during reproductive development, abiotic and biotic stresses of members of ABCG gene family in rice along with reannotation and cloning of FL-cDNA of OsABCG50/PDR23. We observed that of the 22 ABCGs/PDRs, four genes evolved as a result of gene duplication events and their expression pattern changed after duplication. Analysis of expression revealed seed and developmental stage preferential expression of five ABCG/PDR members. Transcript levels of eight ABCGs/PDRs were affected by abiotic and biotic stresses. Expression of seven ABCG/PDR genes was also altered by hormonal elicitors. The modulated expression is nicely correlated with the presence of tissue/stress specific cis-acting elements present in putative promoter region.

Red Blood Cells-Derived Vesicles for Delivery of Lipophilic Drug Camptothecin.

Recently, cell membrane-derived nanoparticles, particularly of RBCs, have been explored for delivery of hydrophilic solutes of varied size and complexities. So far, these naturally derived nanoparticles show a significant overlap with liposomes in terms of stability, solute encapsulation, and release. Unlike hydrophilic molecules, which are loaded inside the aqueous core, hydrophobic moieties largely partition inside the lipophilic shell, hence fate of these nanocarriers may be different. Since vesicles have more complex membrane architecture (due to natural lipids and additional proteins and glycoproteins), ease of loading hydrophobic drug, its release pattern, and overall particle stability cannot be compared to those of synthetic lipid-based carriers. Therefore, we derived nanovesicles (NVEs) from RBC membrane, loaded with hydrophobic drug camptothecin (CPT) and labeled noncovalently with amphiphilic fluorophore (CM-DiI). Although both CPT and CM-DiI are known to partition inside the membrane, the overall stability of NVEs and composition of membrane proteins, particularly CD47, "marker of self", did not change. Additionally, the developed NVEs were found to be nonphagocytic even in the presence of serum and showed minimal stimulation of macrophages to release cytokines. Further, this system showed slow release but strong retention of CPT and CM-DiI, respectively, over 24 h, hence appropriate for theranostic applications. Also, NVEs were internalized by lung carcinoma cells and possessed slightly higher toxicity than free CPT. When injected intravenously in balb/c mice, these nanovesicles showed higher retention in blood over 48 h and insignificant accumulation in vital organs like heart and kidneys, thus suggesting its potential for in vivo application. We believe that this system has superior stealth and comparable physicochemical properties to synthetic lipid-based nanocarriers; hence, it can be further developed as personalized medicine.

Nanoparticle-based drug delivery systems: promising approaches against infections

Despite the fact that many new drugs and technologies have been developed to combat the infectious diseases, these have continued to be global health challenges. The use of conventional antimicrobial agents against these infections is always associated with problems such as the development of multiple drug resistance and adverse side effects. In addition, the inefficient traditional drug delivery system results in inadequate therapeutic index, low bioavailability of drugs and many other limitations. In this regard, antimicrobial nanoparticles and nanosized drug delivery carriers have emerged as potent effective agents against the infections. Nanoparticles have unique properties owing to their ultra small and controllable size such as high surface area, enhanced reactivity, and functionalizable structure. This review focused on different classes of antimicrobial nanoparticles, including metal, metal oxide and others along with their mechanism of action and their potential use against the infections. The review also focused on the development of nanoparticle systems for antimicrobial drug delivery and use of these systems for delivery of various antimicrobial agents, giving an overview about modern nanoparticle based therapeutic strategies against the infections.