Comparative transcriptome analysis of two contrasting genotypes provides new insights into the drought response mechanism in pigeon pea (Cajanus cajan L. Millsp.).

BACKGROUND: Despite plant's ability to adapt and withstand challenging environments, drought poses a severe threat to their growth and development. Although pigeon pea is already quite resistant to drought, the prolonged dehydration induced by the aberrant climate poses a serious threat to their survival and productivity. OBJECTIVE: Comparative physiological and transcriptome analyses of drought-tolerant (CO5) and drought-sensitive (CO1) pigeon pea genotypes subjected to drought stress were carried out in order to understand the molecular basis of drought tolerance in pigeon pea. METHODS: The transcriptomic analysis allowed us to examine how drought affects the gene expression of C. cajan. Using bioinformatics tools, the unigenes were de novo assembled, annotated, and functionally evaluated. Additionally, a homology-based sequence search against the droughtDB database was performed to identify the orthologs of the DEGs. RESULTS: 1102 potential drought-responsive genes were found to be differentially expressed genes (DEGs) between drought-tolerant and drought-sensitive genotypes. These included Abscisic acid insensitive 5 (ABI5), Nuclear transcription factor Y subunit A-7 (NF-YA7), WD40 repeat-containing protein 55 (WDR55), Anthocyanidin reductase (ANR) and Zinc-finger homeodomain protein 6 (ZF-HD6) and were highly expressed in the tolerant genotype. Further, GO analysis revealed that the most enriched classes belonged to biosynthetic and metabolic processes in the biological process category, binding and catalytic activity in the molecular function category and nucleus and protein-containing complex in the cellular component category. Results of KEGG pathway analysis revealed that the DEGs were significantly abundant in signalling pathways such as plant hormone signal transduction and MAPK signalling pathways. Consequently, in our investigation, we have identified and validated by qPCR a group of genes involved in signal reception and propagation, stress-specific TFs, and basal regulatory genes associated with drought response. CONCLUSION: In conclusion, our comprehensive transcriptome dataset enabled the discovery of candidate genes connected to pathways involved in pigeon pea drought response. Our research uncovered a number of unidentified genes and transcription factors that could be used to understand and improve susceptibility to drought.

Methotrexate delivering microneedle patches for improved therapeutic efficacy in treatment of rheumatoid arthritis.

Arthritis is an inflammatory disorder that leads to degeneration and swelling in the joints thereby severely affecting mobility. Till date, a complete cure for this disorder remains elusive. Administration of disease modifying anti-rheumatic drugs has not proved effective owing to poor retention of drugs at the site of inflammation in the joints. In most cases, lack of adherence to the therapeutic regimen further aggravates the condition. Localized administration of the drugs through intra-articular injections is highly invasive and painful. A possible solution to overcome these issues will be to ensure sustained release of the anti-arthritic drug at the site of inflammation through a minimally invasive method. The present work focuses on the development of a microneedle patch for localized and minimally invasive delivery of methotrexate to arthritic joints in guinea pig model. The microneedle patch was found to elicit minimal immune response and ensured sustained release of the drug that was manifested through faster restoration of mobility and a distinct reduction in inflammatory and rheumatoid markers at the joints when compared to untreated and those treated through conventional hypodermic injections. Our results demonstrate the promise of microneedle-based platform for an effective arthritic therapy.

Single step fabrication of hollow microneedles and an experimental package for controlled drug delivery.

Hollow microneedle arrays (HMNs) are an excellent choice for managing chronic diseases requiring the administration of multiple drug doses over a prolonged duration. However, HMNs have gained partial success due to limitations in their manufacturing capabilities, and cumbersome processes. In the present study, polymeric HMNs were fabricated using a novel single-step drop-casting process without needing cleanroom facilities, and sophisticated instrumentation. When drop casted on the pyramidal tip stainless steel needles, the optimized polymer solution allowed the reproducible formation of desired height HMMs on a detachable acrylic base. To enable broader applications, the base with HMNs was integrated into an experimental package built to deliver a dose of âˆ¼ 5 µL per 30° clockwise rotation of the actuator, allowing multiple metered drug dose administrations. The fabricated HMNs were optically imaged, and tested for mechanical integrity and stability. The working and functional utility of the HMNs package in delivering metered drug doses was demonstrated by delivering vitamin B12 (ex vivo) and insulin (in vivo), respectively. The optimized process can be used for the large-scale manufacturing of HMNs and the experimental package shows the potential to be further developed into a wearable device.

Fluorescence Signal Enhancement by a Spray-Assisted Layer-by-Layer Technique on Aluminum Tape Devices for Biosensing Applications.

Layer-by-layer (LbL) self-assembled polyelectrolyte multilayer (PEM) films are a simple yet elegant bottom-up technology to create films at the nano-microscale. This low-cost technology has been widely used as a universal functionalization technique on a broad spectrum of substrates. Biomolecules under investigation can be incubated onto films based on complementary charge interactions between the films and biomolecules. There is a great demand for developing an ultralow-cost biosensing device, which can optimally enhance the fluorescence signal of the adsorbed biomolecules from the traditional labeled sensing platforms. In this work, we have incorporated a blend of the conventional metal enhanced fluorescence technology and the PEM as a dielectric spacer and functionalized film, coated on an aluminum paper (tape)-based substrate. These device has been found to be capable of holding biomolecules in three-dimensional PEM space. The devices fabricated by the proposed spray LbL technique provide significant fluorescence signal enhancement by holding a relatively higher mass per volume of the adsorbed biomolecules, when compared to traditional spin- and dip-coating techniques. Interestingly, our proposed device has expressed a fluorescence enhancement factor, which is 9 times higher than PEM-functionalized glass-based devices. To demonstrate the practical utility of our devices, we also compared our devices to Whatman FAST slides. Our experimental fluorescence results are almost comparable to Whatman FAST slides. Such PEM devices fabricated on top of low-cost aluminum tape using a spray LbL technique give new insights into the future development of ultralow-cost, high-throughput, and disposable lab-on-chip diagnostic applications.

Microscale engineering of hollow microneedle tips: design, manufacturing, optimization and validation.

Transdermal and intradermal drug delivery utilizing microneedles is an emerging front in painless therapeutics. Drug delivery using hollow microneedles is the most preferred method for delivering generic transdermal drugs in the clinical setup. The needle tip must be extremely short as the drug is administered to sub-millimeter depths. Also, they need to be sharp enough to pierce through the skin with minimal skin flexing. There are multiple challenges in engineering a tip profile that is short and sharp at the same time. Stainless steel (SS) hypodermic needles with the lancet tip profile are ubiquitous in subcutaneous and intramuscular injections. They have long bevel lengths that make them inappropriate as microneedles. Thus, designing a unique tip profile and developing the manufacturing technology for microneedle applications are necessary. This article presents the design and optimization of microneedle tip profiles through analytical models. Further, manufacturing strategies for reliably obtaining designed profiles are discussed. The article concludes with experimental validation of improved piercing performance of the optimized tip profile compared to other tip profiles. The article discusses about tip geometries of stainless steel needles for microneedle applications, where depth of delivery is less than 1 mm. Through series of analyses, the optimum needle tip geometry evolved from single plane bevel (SPB) to hex plane bevel (HPB) progressively improving piercing performance.

Microneedles for Extended Transdermal Therapeutics: A Route to Advanced Healthcare.

Sustained release of drugs over a pre-determined period is required to maintain an effective therapeutic dose for variety of drug delivery applications. Transdermal devices such as polymeric microneedle patches and other microneedle-based devices have been utilized for sustained release of their payload. Swift clearing of drugs can be prevented either by designing a slow-degrading polymeric matrix or by providing physiochemical triggers to different microneedle-based devices for on-demand release. These long-acting transdermal devices prevent the burst release of drugs. This review highlights the recent advances of microneedle-based devices for sustained release of vaccines, hormones, and antiretrovirals with their prospective safe clinical translation.

Impact of Bioinspired Nanotopography on the Antibacterial and Antibiofilm Efficacy of Chitosan.

Chitosan derived from chitin is one of the most abundant naturally occurring biocompatible polymers obtained from fungi and arthropods. In this work, we report the enhancement in the bactericidal efficacy of CHI in the presence of a sharp nanotopography. High-aspect ratio nanostructured surface (NSS) was fabricated using a single-step deep reactive ion etching technique (DRIE). Post fabrication, CHI coating was carried out using a layer-by-layer (LBL) dip coating process on the flat and nanostructured surfaces. Antibacterial efficacy of the flat silicon surface coated with CHI (Si_CHI) and NSS coated with CHI (NSS_CHI) was tested against both Gram-negative (G-ve) bacteria E. coli and Gram-positive (G+ve) bacteria S. aureus. NSS_CHI exhibited superior antibacterial property against G-ve and G+ve microbes as compared with Si_CHI and NSS substrates. Scanning electron microscopy (SEM) and fluorescence microscopy were used to study the morphology and viability of the bacteria on all the surfaces. Also, biofilm quantification was carried out on all the engineered surfaces for both E. coli and S. aureus using crystal violet (CV) staining. NSS_CHI was found to have the minimum biofilm formation on its surface exhibiting its superior antibacterial property. This study shows that the antibacterial and antibiofilm efficiency of CHI can be augmented by combining it with a sharp nanotopography.

Polyelectrolyte multilayers for bio-applications: recent advancements.

The synergistic relationship between structure and the bulk properties of polyelectrolyte multilayer (PEM) films has generated tremendous interest in their application for loading and release of bioactive species. Layer-by-layer assembly is the simplest, cost effective process for fabrication of such PEMs films, leading to one of the most widely accepted platforms for incorporating biological molecules with nanometre precision. The bulk reservoir properties of PEM films render them a potential candidate for applications such as biosensing, drug delivery and tissue engineering. Various biomolecules such as proteins, DNA, RNA or other desired molecules can be incorporated into the PEM stack via electrostatic interactions and various other secondary interactions such as hydrophobic interactions. The location and availability of the biological molecules within the PEM stack mediates its applicability in various fields of biomedical engineering such as programmed drug delivery. The development of advanced technologies for biomedical applications using PEM films has seen rapid progress recently. This review briefly summarises the recent successes of PEM being utilised for diverse bio-applications.

Subdiffraction-Resolution Optical Measurements of Molecular Transport in Thin Polymer Films.

The measurement of molecular transport within polymer films yields information about the internal structural organization of the films and is useful in applications such as the design of polymeric capsules for drug delivery. Layer-by-layer assembly of polyelectrolyte multilayer films has been widely used in such applications where the multilayer structure often exhibits anisotropic transport resulting in different diffusivities in the lateral (parallel to the film) and transverse (normal to the film) directions. Although lateral transport can be probed using techniques such as fluorescence recovery after photobleaching (FRAP), it cannot be applied to probing transverse diffusivity in polymer films smaller than the diffraction limit of light. Here we present a technique to probe the transport of molecules tagged with fluorphores in polymer films thinner than the optical diffraction limit using the modulation of fluorescence emission depending on the distance of the tagged molecules from a metal surface. We have used this technique to probe the diffusion of proteins biotin and bovine serum albumin (BSA) in polyelectrolyte multilayer films. We also studied the interdiffusion of chains in multilayer films using this technique. We observed a 3 order of magnitude increase in interdiffusion as a function of the ionic strength of the medium. This technique, along with FRAP, will be useful in studying anisotropic transport in polymer films, even those thinner than the diffraction limit, because the signal in this technique arises only from transverse and not lateral transport. Finally, this technique is also applicable to studying the diffusion of chromophore-labeled species within a polymer film. We demonstrate this aspect by measuring the transverse diffusion of methylene blue in the PAH-PAA multilayer system.

A dual electrochrome of poly-(3,4-ethylenedioxythiophene) doped by N,N'-bis(3-sulfonatopropyl)-4-4'-bipyridinium--redox chemistry and electrochromism in flexible devices.

An electrochromic zwitterionic viologen, N,N'-bis(3-sulfonatopropyl)-4-4'-bipyridinium, has been used for the first time for doping poly (3,4-ethylenedioxythiopene) (PEDOT) films during electropolymerization. Slow and fast diffusional rates for the monomer at deposition potentials of +1.2 and +1.8 V, respectively yielded the viologen-doped PEDOT films with granular morphology and with dendrite-like shapes. The dual electrochrome formed at +1.8 V, showed enhanced coloration efficiency, larger electrochemical charge storage capacity, and superior redox activity in comparison to its analogue grown at +1.2 V, thus demonstrating the role of dendritic shapes in amplifying electrochromism. Flexible electrochromic devices fabricated with the viologen-doped PEDOT film grown at +1.8 V and Prussian blue with an ionic liquid-based gel electrolyte film showed reversible coloration between pale and dark purple with maximum coloration efficiency of 187 cm2C(-1) at lambda=693 nm. The diffusional impedance parameters and switching kinetics of the device showed the suitability of this dual electrochrome formed as a single layer for practical electrochromic cells.