3Bs of CRISPR-Cas mediated genome editing in plants: exploring the basics, bioinformatics and biosafety landscape.

The recent thrust in research has projected the type II clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) system as an avant-garde plant genome editing tool. It facilitates the induction of site-specific double-stranded DNA cleavage by the RNA-guided DNA endonuclease (RGEN), Cas9. Elimination, addition, or alteration of sections in DNA sequence besides the creation of a knockout genotype (CRISPRko) is aided by the CRISPR-Cas9 system in its wild form (wtCas9). The inactivation of the nuclease domain generates a dead Cas9 (dCas9), which is capable of targeting genomic DNA without scissoring it. The dCas9 system can be engineered by fusing it with different effectors to facilitate transcriptional activation (CRISPRa) and transcriptional interference (CRISPRi). CRISPR-Cas thus holds tremendous prospects as a genome-manipulating stratagem for a wide gamut of crops. In this article, we present a brief on the fundamentals and the general workflow of the CRISPR-Cas system followed by an overview of the prospects of bioinformatics in propelling CRISPR-Cas research with a special thrust on the available databases and algorithms/web-accessible applications that have aided in increasing the usage and efficiency of editing. The article also provides an update on the current regulatory landscape in different countries on the CRISPR-Cas edited plants to emphasize the far-reaching impact of the genomic editing technology. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01397-3.

Single nucleotide polymorphisms of leptin gene in five Ethiopian indigenous cattle breeds and the Korean Hanwoo breed.

Considering the escalating number of scientific reports on the association between the leptin gene and diverse physiological traits and performance of cattle populations, this study was directed towards identifying SNPs in the leptin gene among five indigenous cattle breeds of Ethiopia. DNA samples were extracted from the nasal swabs of the Ethiopian indigenous cattle breeds: Arsi (n = 18), Horro (n = 20), Begait (n = 21), Boran (n = 19), and Fogera (n = 17) and the Korean Hanwoo (a representative taurine breed) (n = 20), followed by PCR amplification of exon 2 and exon 3 regions of the leptin gene and sequence analysis of the PCR products. Five SNPs, two (generating missense mutations) on exon 2 and three (generating silent mutations) on exon 3 regions, were explicated in this study. Allele frequency and genotype frequency distribution pertaining to the SNPs were recorded for the studied cattle breeds besides the minor allele frequency and deviation from the Hardy-Weinberg equilibrium. Positive FIS index values were recorded for all the markers except SNP2, illustrative of heterozygote deficiency. MEGA X software-based evolutionary divergence analysis of the phylogenetic tree based on the SNP data revealed that the large-sized breeds, Hanwoo, Begait, Boran, and Fogera, were more closely clustered compared to the small-sized Arsi breed. Among the seven haplotypes documented from the various breeds, sequence analysis was suggestive of haplotypes 1 and 2 to be ancestral haplotypes for the leptin gene. This study is envisaged to accelerate molecular breeding programs for the genetic improvement of the Ethiopian cattle breeds.

Optimization of chromium(VI) removal by indigenous microalga (Chlamydomonas sp.)-based biosorbent using response surface methodology.

Phycoremediation of heavy metals has garnered considerable recent research interest. In this study, an indigenous microalga (Chlamydomonas sp.)-based biosorbent was employed for biosorption of Cr(VI) dissolved solids (Cr(VI)-DS), optimized using response surface methodology (RSM). The effects of microalga concentration, pH, and contact time were studied with 250 mg Cr(VI)-DS L-1 . The biosorption of Cr(VI)-DS was higher at acidic pH (94.17% at pH 4) than at alkaline conditions (68.53% at pH 10). The interaction of pH and microalga concentration exerted significant (p < 0.05) influence on the biosorption. Under the optimized parameters of 1.5 g microalga L-1 , pH 4, and contact time of 30 min, a predicted biosorption of 91.31% and biosorption capacity of 152 mg Cr(VI)-DS g-1 biomass were documented. FTIR analysis attested the electronegative surface functional groups of the microalgae biomass, bracketed together with its high biosorption potency. The study evinced the potential of the indigenous microalga for remediation of hexavalent chromium. PRACTITIONER POINTS: Indigenous Ethiopian microalga (Chlamydomonas sp.) exhibited 94% Cr(VI) abatement with biosorption capacity of 152 mg Cr(VI) g-1 . FTIR analysis of the biosorbent divulged the presence of electronegative functional groups (amino, carboxyl, hydroxyl, and carbonyl groups). Higher biosorption of Cr(VI)-DS under acidic pH (94.17% at pH 4) than alkaline pH (68.53% at pH 10). Significant (p < 0.05) interaction effect of pH and biomass concentration on the biosorption, evinced in RSM optimization 91% Cr(VI) removal achieved under optimal conditions of 1.5 g biosorbent L-1 , 30 min of contact time, and pH 4.

Recent advances in bioprinting technologies for engineering hepatic tissue.

In the sphere of liver tissue engineering (LTE), 3D bioprinting has emerged as an effective technology to mimic the complex in vivo hepatic microenvironment, enabling the development of functional 3D constructs with potential application in the healthcare and diagnostic sector. This review gears off with a note on the liver's microscopic 3D architecture and pathologies linked to liver injury. The write-up is then directed towards unmasking recent advancements and prospects of bioprinting for recapitulating 3D hepatic structure and function. The article further introduces available stem cell opportunities and different strategies for their directed differentiation towards various hepatic stem cell types, including hepatocytes, hepatic sinusoidal endothelial cells, stellate cells, and Kupffer cells. Another thrust of the article is on understanding the dynamic interplay of different hepatic cells with various microenvironmental cues, which is crucial for controlling differentiation, maturation, and maintenance of functional hepatic cell phenotype. On a concluding note, various critical issues and future research direction towards clinical translation of bioprinted hepatic constructs are discussed.

Magnetically recyclable, antimicrobial, and catalytically enhanced polymer-assisted "green" nanosystem-immobilized Aspergillus niger amyloglucosidase.

The present work reports the integration of polymer matrix-supported nanomaterial and enzyme biotechnology for development of industrially feasible biocatalysts. Aqueous leaf extract of Mesua ferrea L. was used to prepare silver nanoparticles distributed within a narrow size range (1-12 nm). In situ oxidative technique was used to obtain poly(ethylene glycol)-supported iron oxide nanoparticles (3-5 nm). Sonication-mediated mixing of above nanoparticles generated the immobilization system comprising of polymer-supported silver-iron oxide nanoparticles (20-30 nm). A commercially important enzyme, Aspergillus niger amyloglucosidase was coupled onto the immobilization system through sonication. The immobilization enzyme registered a multi-fold increment in the specific activity (807 U/mg) over the free counterpart (69 U/mg). Considerable initial activity of the immobilized enzyme was retained even after storing the system at room temperature as well as post-repeated magnetic recycling. Evaluation of the commendable starch saccharification rate, washing performance synergy with a panel of commercial detergents, and antibacterial potency strongly forwards the immobilized enzyme as a multi-functional industrially feasible system.

Can CRISPR/Cas Technology Be a Felicitous Stratagem Against the COVID-19 Fiasco? Prospects and Hitches.

The current global debacle of COVID-19, spelled by SARS-CoV-2 needs no elaboration. With incessant and constantly clambering number of deaths across various nations, the need of the hour is to develop readily deployable, fast, affordable detection assays and kits, yielding precise and consistent results as well as timely availability of efficacious anti-SARS-CoV-2 strategies to contain it. Conventionally employed real time PCR based technique for detection of the virus suffers from a couple of handicaps. Amongst other approaches, CRISPR based technology has ushered in new hopes. Recent efforts have been directed toward developing CRISPR/Cas based low-cost, rapid detection methods as well as development of one-pot assay platforms. The plausible application of CRISPR-Cas system to counteract the viral assault has also been assessed. The write up in this article mirrors the current status, the prospects and the practical snags of CRISPR/Cas technology for the detection and inactivation of the novel corona virus, SARS-CoV-2.

Polymer-assisted iron oxide magnetic nanoparticle immobilized keratinase.

Nanotechnology holds the prospect for avant-garde changes to improve the performance of materials in various sectors. The domain of enzyme biotechnology is no exception. Immobilization of industrially important enzymes onto nanomaterials, with improved performance, would pave the way to myriad application-based commercialization. Keratinase produced by Bacillus subtilis was immobilized onto poly(ethylene glycol)-supported Fe3O4 superparamagnetic nanoparticles. The optimization process showed that the highest enzyme activity was noted when immobilized onto cyanamide-activated PEG-assisted MNP prepared under conditions of 25 degrees C and pH 7.2 of the reaction mixture before addition of H2O2 (3% w/w), 2% (w/v) PEG(6000) and 0.062:1 molar ratio of PEG to FeCl2 x 4H2O. Further statistical optimization using response surface methodology yielded an R2 value that could explain more than 94% of the sample variations. Along with the magnetization studies, the immobilization of the enzyme onto the PEG-assisted MNP was characterized by UV, XRD, FTIR and TEM. The immobilization process had resulted in an almost fourfold increase in the enzyme activity over the free enzyme. Furthermore, the immobilized enzyme exhibited a significant thermostability, storage stability and recyclability. The leather-industry-oriented application of the immobilized enzyme was tested for the dehairing of goat-skin.

Comprehensive Review on Silk at Nanoscale for Regenerative Medicine and Allied Applications.

Materials at the nanoscale offer numerous avenues to be explored and exploited in diverse realms. Among others, proteinaceous biomaterials such as silk hold immense prospects in the domain of nanoengineering. Silk offers a unique combination of desirable facets like biocompatibility; extraordinary mechanical properties, such as elongation, elasticity, toughness, and modulus; and tunable biodegradability which are far better than most naturally occurring and engineered materials. Much of these properties are due to the molecular structure of the silk protein and it is self-assembly into hierarchical structures. Taking advantage of the hierarchical assembly, a large number of fabrication strategies have now emerged that allow the tailoring of silk structure of at the nanoscale. Harnessing the favorable properties of silk, such methods offer a promising direction toward producing structurally and functionally optimized silk nanomaterials. This review discusses the critical structure-property relationship in silk that occurs at the nanoscale and also aims to bring out the recent status in the approaches for fabrication, characterization, and the gamut of applications of various silk-based nanomaterials (nanoparticles, nanofibers, and nanocomposites) in the niche of translational research. Harnessing the favorable nanostructure of silk, the review also takes into account the impetus of silk in avant-garde applications such as chemo-biosensing, energy harvesting, microfluidics, and environmental applications.

Mimicking Hierarchical Complexity of the Osteochondral Interface Using Electrospun Silk-Bioactive Glass Composites.

The anatomical complexity and slow regeneration capacity of hyaline cartilage at the osteochondral interface pose a great challenge in the repair of osteochondral defects (OCD). In this study, we utilized the processing feasibility offered by the sol derived 70S bioactive glass and silk fibroin (mulberry Bombyx mori and endemic Indian non-mulberry Antheraea assama), in fabricating a well-integrated, biomimetic scaffolding matrix with a coherent interface. Differences in surface properties such as wettability and amorphousness between the two silk groups resulted in profound variations in cell attachment and extracellular matrix protein deposition. Mechanical assessment showed that the biphasic composites exhibited both an elastic region pertinent for cartilage tissue and a stiff compression resistant region simulating the bone phase. In vitro biological studies revealed that the biphasic mats presented spatial confinement for the growth and maturation of both osteoblasts and chondrocytes, marked by increased alkaline phosphatase (ALP) activity, osteopontin (OPN), sulfated glycosaminoglycan (sGAG) and collagen secretion in the cocultured mats. The non-mulberry silk based biphasic composite mats performed better than their mulberry counterpart, as evidenced by enhanced expression levels of key cartilage and bone specific marker genes. Therefore, the developed biphasic scaffold show great promise for improving the current clinical strategies for osteochondral tissue repair.

Silk-microfluidics for advanced biotechnological applications: A progressive review.

Silk based biomaterials have not only carved a unique niche in the domain of regenerative medicine but new avenues are also being explored for lab-on-a-chip applications. It is pertinent to note that biospinning of silk represents nature's signature microfluidic-maneuver. Elucidation of non-Newtonian flow of silk in the glands of spiders and silkworms has inspired researchers to fabricate devices for continuous extrusion and concentration of silk. Microfluidic channel networks within porous silk scaffolds ensure optimal nutrient and oxygen supply apart from serving as precursors for vascularization in tissue engineering applications. On the other hand, unique topographical features and surface wettability of natural silk fibers have inspired development of a number of simple and cost-effective devices for applications like blood typing and chemical sensing. This review mirrors the recent progress and challenges in the domain of silk-microfluidics for prospective avant-garde applications in the realm of biotechnology.

Comparative analysis of codon usage bias in Crenarchaea and Euryarchaea genome reveals differential preference of synonymous codons to encode highly expressed ribosomal and RNA polymerase proteins.

The present study was undertaken to investigate the pattern of optimal codon usage in Archaea. Comparative analysis was executed to understand the pattern of codon usage bias between the high expression genes (HEG) and the whole genomes in two Archaeal phyla, Crenarchaea and Euryarchaea. The G+C% of the HEG was found to be less in comparison to the genome G+C% in Crenarchaea, whereas reverse was the case in Euryarchaea. The preponderance of U/A ending codons that code for HEG in Crenarchaea was in sharp contrast to the C/G ended ones in Euryarchaea. The analysis revealed prevalence of Uending codons even within theWWY(nucleotide ambiguity code) families in Crenarchaea vis-à-vis Euryarchaea, bacteria and Eukarya. No plausible interpretation of the observed disparity could be made either in the context of tRNA gene composition or genome G+C%. The results in this study attested that the preferential biasness for codons in HEG of Crenarchaea might be different from Euryarchaea. The main highlights are (i) varied CUB in the HEG and in the whole genomes in Euryarchaea and Crenarchaea. (ii) Crenarchaea was found to have some unusual optimal codons (OCs) compared to other organisms. (iii) G+C% (and GC3) of the HEG were different from the genome G+C% in the two phyla. (iv) Genome G+C% and tRNA gene number failed to explain CUB in Crenarchaea. (v) Translational selection is possibly responsible for A+T rich OCs in Crenarchaea.

Sonication assisted assemblage of exotic polymer supported nanostructured bio-hybrid system and prospective application.

This work was focused on sonication mediated immobilization of porcine pancreatic lipase (PPL) onto poly(ethylene glycol) supported silver-iron oxide hybrid nanoparticles (PEG-Ag/IONPs). Selected process parameters of sonication were optimized using response surface methodology. Sonication assisted assemblage of spherical PEG-Ag/IONPs and consequent evolution of nanorods post PPL immobilization were documented. The efficacy of the reported immobilization strategy was attested by the increased thermostability, storage stability and enhanced activity of the biocatalyst, suggestive of plausible structural modulations post immobilization. The commercial prospect of the antibacterial and magnetically recyclable system was vouched by its excellent compatibility with some commercial detergents for oil de-staining.

Bio-based hyperbranched poly(ester amide)-MWCNT nanocomposites: multimodalities at the biointerface.

There has been a growing interest in the use of nanomaterials featuring potent biocompatibility and biodegradability together with the added facet of antibacterial activity, particularly against drug-resistant bacterial species. Addressing these three features at the biointerface, we report the fabrication of multimodal bio-based hyperbranched poly(ester amide) (HBPEA)-microwave functionalized multiwalled carbon nanotube (f-MWCNT) nanocomposites by incorporation of various weight percentages (1, 2.5, and 5 wt%) of the f-MWCNTs into HBPEA by using an ex situ polymerization technique. Fourier transform infrared spectroscopy confirmed the structural changes upon interaction of the f-MWCNTs with HBPEA. The formation of thermosetting nanocomposites resulted in an acceptable improvement of the desired properties including their mechanical properties (∼170%), instrumental for providing mechanical integrity in cultured cells. The nanocomposite films were found to be biocompatible substrates for the in vitro adhesion and proliferation of peripheral blood mononuclear cells (PBMC) with enhanced cell viability correlating with the increase of the f-MWCNT content. The antibacterial results, monitored by a CFU count and the protein concentration, demonstrated that the prepared nanocomposites were more toxic towards Gram positive bacteria and Mycobacterium smegmatis than the Gram negative ones. The damage of bacterial cells upon interaction with the nanocomposites was validated by UV-visible spectroscopy and a SEM study. The antibacterial and biocompatibility studies suggested that these microporous nanocomposite films (3D interconnected porous structures with pore diameters of 5-105 µm and a porosity of 39.90%) possess concurrent long-term lethal activity against the bacterial cells and biocompatibility with PBMC. Thus, the prepared nanocomposites may find potential bio-medical applications, particularly as antimicrobial dressing materials for infected burn wounds.

Electrospun cellulose acetate nanofibers: the present status and gamut of biotechnological applications.

Cellulose acetate (CA) has been a material of choice for spectrum of utilities across different domains ranging from high absorbing diapers to membrane filters. Electrospinning has conferred a whole new perspective to polymeric materials including CA in the context of multifarious applications across myriad of niches. In the present review, we try to bring out the recent trend (focused over last five years' progress) of research on electrospun CA fibers of nanoscale regime in the context of developmental strategies of their blends and nanocomposites for advanced applications. In the realm of biotechnology, electrospun CA fibers have found applications in biomolecule immobilization, tissue engineering, bio-sensing, nutraceutical delivery, bioseparation, crop protection, bioremediation and in the development of anti-counterfeiting and pH sensitive material, photocatalytic self-cleaning textile, temperature-adaptable fabric, and antimicrobial mats, amongst others. The present review discusses these diverse applications of electrospun CA nanofibers.

Isolation and immobilization of aroid polyphenol on magnetic nanoparticles: enhancement of potency on surface immobilization.

Five polyphenolic compounds were isolated from four aroid species [using High Performance Liquid Chromatography (HPLC)] and identified using Fourier Transform Infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance (NMR) analysis. The compounds benzoic acid, caffeic acid, coumaric acid, ferulic acid and syringic acids were immobilized on magnetic nanoparticles (MNPs) using poly(ethylene glycol) (PEG) as linker polymer. The obtained PEG-MNP-polyphenol trios were characterized using transmission electron microscopy (TEM) and FTIR. Their biochemical characterization were done to find out the DPPH scavenging capacity and amount of polyphenol loaded (galic acid equivalent) in each type of MNP-polyphenol trio. To know the antioxidant activity the MNP-polyphenol trios were subjected to H(2)O(2) induced haemolysis prevention assay and syringic acid trio was found to most potent. To ensure that the MNP-polyphenol trios were magnetically active the hysteresis loop analysis was also done.

Diameter-tuning of electrospun cellulose acetate fibers: a Box-Behnken design (BBD) study.

This work focuses on the use of statistical approach in optimizing shape-size accord of electrospun cellulose acetate (CA) mats - an apt material for biomedical and industrial applications. Modulation of three processing parameters, namely potential difference, distance between tip-to-collector and feed rate led to myriad of fiber-morphology (beaded, bead free, branched and ribbon) with diverse size-spectrum. Response surface methodology using Box-Behnken design technique indicated significant linear and quadratic influence of the chosen parameters. Fibers with minimal diameter of ~139 nm (with a mean coherency co-efficient of 0.5192) were predicted for 30 kV (voltage), 15 cm (tip-to-collector distance) and 2 mL/h (feed rate). Reasonable agreement existed between the predicted R-squared value (0.9565) and adjusted R-squared value (0.9824) with similar observation for the experimental and model values over the entire factor space. The developed model may serve as a base model for understanding process - parametric influence on electrospinning CA and related polymers.

Non-hazardous anticancerous and antibacterial colloidal 'green' silver nanoparticles.

Poly(ethylene glycol) stabilized colloidal silver nanoparticles were prepared using the reductive potency of the aqueous extract of Thuja occidentalis leaves under ambient conditions. The nanoparticles were well dispersed within a narrow size spectrum (7-14 nm) and displayed characteristic surface plasmon resonance peak at around 420 nm and Bragg's reflection planes of fcc structure. MTT assay revealed the dose-dependent cytocompatibility and toxicity of the nanoparticles with the L929 normal cell line. On the other hand, the antiproliferative action of the nanoparticles was evaluated on HeLa cell (cancerous cells) line. Fluorescence and phase contrast microscopic imaging indicated the appearance of multinucleate stages with aggregation and nuclear membrane disruption of the HeLa cells post treatment with the nanoparticles. The interaction at the prokaryotic level was also assessed via differential antibacterial efficacy against Staphylococcus aureus (MTCC 3160) and Escherichia coli (MTCC 40). Under these perspectives, it is also necessary to observe the environmental impact of the prepared silver nanoparticles. Hence, the dose dependent toxicity of silver nanoparticles was evaluated upon the earthworm species Eisenia fetida. Neither the survival nor the reproduction was affected by the addition of silver nanoparticles up to 1000 ppm. Thus these 'green' silver nanoparticles have promising potential as future materials.

Ultrasonication--a complementary 'green chemistry' tool to biocatalysis: a laboratory-scale study of lycopene extraction.

Lycopene is bequeathed with multiple bio-protective roles, primarily attributed to its unique molecular structure. The concomitant exploitation of two of the green chemistry tools viz., sonication and biocatalysis is reported here for the laboratory scale extraction of lycopene from tomato peel. The coupled system improved the extraction by 662%, 225% and 150% times over the unaided, only cellulase 'Onozuka R-10' treated and only sonication treated samples respectively. The sonication parameters (duration, cycle and amplitude) during the coupled operation were optimized using response surface methodology (RSM). Derivative UV-visible spectra (i.e., dA/dλ and d(2)A/dλ(2) against λ), FTIR analysis, and DPPH scavenging test suggested that the reported extraction protocol did not affect the molecular structure and bioactivity of the extracted lycopene. The influence of sonication on the probable structural modulation (through UV-visible spectral analysis) and activity of the enzyme were also analyzed. A plausible mechanism is proposed for the enhanced extraction achieved via the coupled system.