Small millets are nutri-rich, climate-resilient food and fodder crops. They include finger millet, proso millet, foxtail millet, little millet, kodo millet, browntop millet, and barnyard millet. They are self-pollinated crops and belong to the family Poaceae. Hence, to widen the genetic base, the creation of variation through artificial hybridization is a prerequisite. Floral morphology, size, and anthesis behavior cause major hindrances in recombination breeding through hybridization. Manual emasculation of florets is practically very difficult; therefore, the contact method of hybridization is widely followed. However, the success rate of obtaining true F1s is 2% to 3%. In finger millet, hot water treatment (52°C) for 3 to 5 min causes temporal male sterility. Chemicals such as maleic hydrazide, gibberellic acid, and ethrel at different concentrations aid in inducing male sterility in finger millet. Partial-sterile (PS) lines developed at the Project Coordinating Unit, Small Millets, Bengaluru are also in use. The percent seed set in crosses derived from PS lines ranged from 27.4 to 49.4, with an average of 40.10%. In proso millet, little millet, and browntop millet, apart from contact method, hot water treatment, hand emasculation, and the USSR method of hybridization are also followed. A newly developed modified crossing method known as the Small Millets University of Agricultural Sciences Bengaluru (SMUASB) method in proso and little millets has a success rate of 56% to 60% in obtaining true hybrids. Hand emasculation and pollination under the greenhouse and growth chamber in foxtail millet with a success rate of 75% seed set is suggested. In barnyard millet, hot water treatment (48°C to 52°C) for 5 min followed by the contact method is often practiced. Kodo millet being cleistogamous, mutation breeding is widely followed to create variation. Most commonly, hot water treatment is followed in finger millet and barnyard millet, SMUASB in proso, and little millet. Although no specific method is suitable for all small millets, it is essential to identify a trouble-free technique that produces maximum crossed seeds in all the small millets.

Human Coronaviruses (hCoVs) belongs to the enormous and dissimilar family of positive-sense, non-segmented, single-stranded RNA viruses. The RNA viruses are prone to high rates of mutational recombination resulting in emergence of evolutionary variant to alter various features including transmissibility and severity. The evolutionary changes affect the immune escape and reduce effectiveness of diagnostic and therapeutic measures by becoming undetectable by the currently available diagnostics and refractory to therapeutics and vaccines. Whole genome sequencing studies from various countries have adequately reported mosaic recombination between different lineage strain of SARS-CoV-2 whereby RNA dependent RNA polymerase (RdRp) gene reconnects with a homologous RNA strand at diverse position. This all lead to evolutionary emergence of new variant/ lineage as evident with the emergence of XBB in India at the time of writing this review. The continuous periodical genomic surveillance is utmost required for understanding the various lineages involved in recombination to emerge into hybrid variant. This may further help in assessing virus transmission dynamics, virulence and severity factor to help health authorities take appropriate timely action for prevention and control of any future COVID-19 outbreak.

Generating functional protein variants with novel or improved characteristics has been a goal of the biotechnology industry and life sciences, for decades. Rational design and directed evolution are two major pathways to achieve the desired ends. While rational protein design approach has made substantial progress, the idea of using a method based on cycles of mutagenesis and natural selection to develop novel binding proteins, enzymes and structures has attracted great attention. Laboratory evolution of proteins/enzymes requires new tools and analytical approaches to create genetic diversity and identifying variants with desired traits. In this pursuit, construction of sufficiently large libraries of target molecules to search for improved variants and the need for new protocols to alter the properties of target molecules has been a continuing challenge in the directed evolution experiments. This review will discuss the in vivo and in vitro gene diversification tools, library screening or selection approaches, and artificial intelligence/machine-learning-based strategies to mutagenesis developed in the last 40 years to accelerate the natural process of evolution in creating new functional protein variants, optimization of microbial strains, and transformation of enzymes into industrial machines. Analyzing patent position over these techniques and mechanisms also constitutes an integral and distinctive part of this review. The aim is to provide an up-to-date resource/technology toolbox for research-based and pharmaceutical companies to discover the boundaries of competitor\'s intellectual property (IP) portfolio, their freedom-to-operate in the relevant IP landscape, and the need for patent due diligence analysis to rule out whether use of a particular patented mutagenesis method, library screening/selection technique falls outside the safe harbor of experimental use exemption. While so doing, we have referred to some recent cases that emphasize the significance of selecting a suitable gene diversification strategy in directed evolution experiments.

Spider silk, as one of the hardest natural and biocompatible substances with extraordinary strength and flexibility, have become an ideal option in various areas of science and have made their path onto the biomedical industry. Despite its growing popularity, the difficulties in the extraction of silks from spiders and farming them have made it unaffordable and almost impossible for industrial scale. Biotechnology helped production of spider silks recombinantly in different hosts and obtaining diverse morphologies out of them based on different processing and assembly procedures. Herein, the characteristics of these morphologies and their advantages and disadvantages are summarized. A detailed view about applications of recombinant silks in skin regeneration and cartilage, tendon, bone, teeth, cardiovascular, and neural tissues engineering are brought out, where there is a need for strong scaffolds to support cell growth. Likewise, spider silk proteins have applications as conduit constructs, medical sutures, and 3D printer bioinks. Other characteristics of spider silks, such as low immunogenicity, hydrophobicity, homogeneity, and adjustability, have attracted much attention in drug and gene delivery. Finally, the challenges and obstacles ahead for industrializing the production of spider silk proteins in sufficient quantities in biomedicine, along with solutions to overcome these barriers, are discussed.

Minipigs play an important role in biomedical research and they have also been used as donor animals for preclinical xenotransplantations. Since zoonotic microorganisms including viruses can be transmitted when pig cells, tissues or organs are transplanted, virus safety is an important feature in xenotransplantation. Whereas most porcine viruses can be eliminated from pig herds by different strategies, this is not possible for porcine endogenous retroviruses (PERVs). PERVs are integrated in the genome of pigs and some of them release infectious particles able to infect human cells. Whereas PERV-A and PERV-B are present in all pigs and can infect cells from humans and other species, PERV-C is present in most, but not all pigs and infects only pig cells. Recombinant viruses between PERV-A and PERV-C have been found in some pigs; these recombinants infect human cells and are characterized by high replication rates. PERV-A/C recombinants have been found mainly in minipigs of different origin. The possible reasons of this high prevalence of PERV-A/C in minipigs, including inbreeding and higher numbers and expression of replication-competent PERV-C in these animals, are discussed in this review. Based on these data, it is highly recommended to use only pig donors in clinical xenotransplantation that are negative for PERV-C.

Infectious bronchitis virus (IBV) was first isolated in Australia in 1962. Ongoing surveillance and characterization of Australian IBVs have shown that they have evolved separately from strains found throughout the rest of the world, resulting in the evolution of a range of unique strains and changes in the dominant wild-type strains, affecting tissue tropism, pathogenicity, antigenicity, and gene arrangement. Between 1961 and 1976 highly nephropathogenic genotype GI-5 and GI-6 strains, causing mortalities of 40% to 100%, predominated, while strains causing mainly respiratory disease, with lower mortality rates, have predominated since then. Since 1988, viruses belonging to two distinct and novel genotypes, GIII and GV, have been detected. The genome organization of the GIII strains has not been seen in any other gammacoronavirus. Mutations that emerged soon after the introduction of vaccination, incursion of strains with a novel lineage from unknown sources, recombination between IBVs from different genetic lineages, and gene translocations and deletions have contributed to an increasingly complex IBV population. These processes and the consequences of this variation for the biology of these viruses provide an insight into the evolution of endemic coronaviruses during their control by vaccination and may provide a better understanding of the potential for evolution of other coronaviruses, including SARS-CoV-2. Furthermore, the continuing capacity of attenuated IBV vaccines developed over 40 years ago to provide protection against viruses in the same genetic lineage provides some assurance that coronavirus vaccines developed to control other coronaviruses may continue to be effective for an extended period.

Porcine torovirus (PToV) is a potential enteric swine pathogen, found at especially high rates in piglets with diarrhea. It was first reported in the Netherlands in 1998 and has emerged in many countries around the world. Infections are generally asymptomatic and have not directly caused large economic losses, though co-infections with other swine pathogens and intertype recombination may lead to unpredictable outcomes. This review introduces progress in PToV research regarding its discovery, relationship with other Toroviruses, virion morphological characteristics, genetic structure and variation, recent epidemiology, diagnostic methods, and possibilities for future research.

Noroviruses are recognized as the major global cause of sporadic and epidemic non-bacterial gastroenteritis in humans. Molecular mechanisms driving norovirus evolution are the accumulation of point mutations and recombination. Intragenotypic recombination has long been postulated to be a driving force of GII.4 noroviruses, the predominant genotype circulating in humans for over two decades. Increasingly, emergence and re-emergence of different intragenotype recombinants have been reported. The number and types of norovirus recombinants remained undefined until the 2007 Journal of General Virology research article \'Norovirus recombination\' reported an assembly of 20 hitherto unclassified intergenotypic norovirus recombinant types. In the intervening decade, a host of novel recombinants has been analysed. New recombination breakpoints have been described, in vitro and in vivo studies supplement in silico analyses, and advances have been made in analysing factors driving norovirus recombination. This work presents a timely overview of these data and focuses on important aspects of norovirus recombination and its role in norovirus molecular evolution. An overview of intergenogroup, intergenotype, intragenotype and \'obligatory\' norovirus recombinants as detected via in silico methods in the field is provided, enlarging the scope of intergenotypic recombinant types to 80 in total, and notably including three intergenogroup recombinants. A recap of advances made studying norovirus recombination in the laboratory is given. Putative drivers and constraints of norovirus recombination are discussed and the potential link between recombination and norovirus zoonosis risk is examined.

The human leukocyte antigen (HLA) loci are among the most polymorphic genes in the human genome. The diversity of these genes is thought to be generated by different mechanisms including point mutation, gene conversion and crossing-over. During routine HLA typing, we discovered seven novel HLA alleles which were probably generated by different evolutionary mechanisms. HLA-B*41:21, HLA-DQB1*02:10 and HLA-DQA1*01:12 likely emerged from the common alleles of their groups by point mutations, all of which caused non-synonymous amino acid substitutions. In contrast, a deletion of one nucleotide leading to a frame shift with subsequent generation of a stop codon is responsible for the appearance of a null allele, HLA-A*01:123N. Whereas HLA-B*35:231 and HLA-B*53:31 were probably products of intralocus gene conversion between HLA-B alleles, HLA-C*07:294 presumably evolved by interlocus gene conversion between an HLA-C and an HLA-B allele. Our analysis of these novel alleles illustrates the different mechanisms which may have contributed to the evolution of HLA polymorphism.