Reduced Fasting Duration in Cynomolgus Monkeys Enhances Animal Welfare During Toxicology Studies.

During toxicology studies, fasting animals prior to clinical pathology blood collection is believed to reduce variability in some clinical chemistry analytes. However, fasting adds stress to animals that are already stressed from the administration of potentially toxic doses of the test article. The purpose of this study was to assess the impacts of different fasting durations on cynomolgus monkeys' welfare during toxicology studies. To this end, we assessed the cynomolgus monkeys traditional and ancillary clinical pathology endpoints at different fasting times. We showed that most clinical pathology endpoints were largely comparable between different fasting times suggesting that cynomolgus monkeys could be fasted for as little as 4 hours for toxicology studies, as longer fasting times (up to 20 hours) resulted in stress, dehydration, and significant decreases in blood glucose- changes that impacts animal welfare. Shorter fasting times were associated with higher triglycerides variability among individual animals. Therefore, we propose that shorter fasting time (i.e., 4 hours) should be adequate for most toxicology studies except when: (1) parameters that could be affected by non-fasting conditions are important for safety and pharmacodynamic assessments (i.e., glucose and lipids) and (2) fasting would be needed for the bioavailability of an orally administered test article.

Prioritization of Physio-Biochemical Selection Indices and Yield-Attributing Traits toward the Acquisition of Drought Tolerance in Chickpea (Cicer arietinum L.).

Chickpea is widely grown in rainfed areas of developing countries because of its nutritional abundance and adaptability. To overcome the environmental effect of drought on yield, a characteristic-linked selection strategy is proved as well-thought-out and advantageous for the development of drought-tolerant cultivars. To precisely understand the contribution of various physio-biochemical and yield-attributing traits toward drought tolerance in chickpea (Cicer arietinum L.), forty chickpea genotypes were evaluated in the years 2020-2021 and 2021-2022 under normal irrigated as well as drought-stressed conditions. Among the studied genotypes, genotype ICC4958 retained the highest chl content (0.55 mg g-1 FW), minimal electrolyte leakage, and superoxide dismutase (1.48 U/mg FW) and peroxidase (2.21 µmol/min/g FW) activities while cultivar JG11 maintained the maximum relative water content and proline accumulation. The principal-component-based biplots prioritized the physio-biochemical and yield-accrediting characteristics based on their association significance and contribution to terminal drought tolerance. Under drought stress, grain yield per plant was depicted to have a strongly positive association with canopy temperature depression, catalase, superoxide dismutase, and peroxidase activities as well as total soluble sugar, proline, and chlorophyll content, along with the numbers of pods and biological yield per plant. These identified physio-biochemical and yield-attributing traits can be further deployed to select drought-tolerant chickpea genotypes for the breeding of climate-smart chickpea genotypes.

Validating the Nutraceutical Significance of Minor Millets by Employing Nutritional-Antinutritional Profiling.

Millets are group of underutilized cereal crops with higher nutritional values. The present investigation used different classes of minor millets, including barnyard (sava), little (kutki), finger (ragi), kodo and foxtail millets, for evaluation of their nutritional parameters, i.e., the content of proteins, total amino acids, total sugars, insoluble fibers, soluble fibers, total dietary fibers, iron (Fe) and zinc (Zn), along with antinutritional and antioxidant parameters, viz., tannic acid, phytic acid, phenol, flavonoid, proline and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Alpha amylase and alpha glucosidase activity were also thought to elevate millets as a viable staple meal. Foxtail millet showed the maximum inhibition, with an IC50 value of 20.46 ± 1.80 µg mL-1 with respect to α-amylase. The coefficient of correlation between nutritional and antinutritional compositions showed that the starch content was significantly and positively correlated with insoluble fiber (r = 0.465) and dietary fiber (r = 0.487). Moreover, sugar was positively correlated with the phytic acid (r = 0.707), Fe and Zn (r = 0.681) contents. To determine the peptides responsible for anticancer activity, the foxtail protein was subjected to ultrafiltration; it was found that the 3 kDa fraction retained the greatest anticancer activity. Selected millet germplasm line(s) that have the best nutraceutical properties could be used in millet improvement programs.

Effect of storage age and containers on the physicochemical degradation of guggul oleo-resin.

Guggul is a gum oleo-resin, tapped from a data deficient plant- Commiphora wightii (Arnott.) Bhandari in India. It is extensively used in ayurvedic drugs and formulations since ages. Natural plant-based products; especially aromatic ones like guggul gum oleo-resin deteriorates, qualitatively during its storage and transits before reaching the industry for its value addition. This economical and ecological loss can be avoided if it is stored in proper containers. Physico-chemical degradation of guggul samples stored were analysed by scanned electron microscopy, fourier transformed infra red, thermogravimatric, Powdered X-ray diffraction and elemental analysis for carbon, hydrogen, nitrogen and sulphur. Physico-chemical degradation of guggul oleo-resin occurs with the age of storage and the type of storage containers used. Among the four storage containers (earthen pot, plastic jar, polythene bag, jute bag) evaluated, earthen pot was found to be the best in checking the qualitative loss of guggul even upto 24 months. The qualitative information generated in the study on guggul storage may be useful to the drug industry and guggul traders. It may encourage them practice storing guggul in earthen pots against current practice of using jute bags and polythene bags, to store it.

Molecular Advances to Combat Different Biotic and Abiotic Stresses in Linseed (Linum usitatissimum L.): A Comprehensive Review.

Flax, or linseed, is considered a "superfood", which means that it is a food with diverse health benefits and potentially useful bioactive ingredients. It is a multi-purpose crop that is prized for its seed oil, fibre, nutraceutical, and probiotic qualities. It is suited to various habitats and agro-ecological conditions. Numerous abiotic and biotic stressors that can either have a direct or indirect impact on plant health are experienced by flax plants as a result of changing environmental circumstances. Research on the impact of various stresses and their possible ameliorators is prompted by such expectations. By inducing the loss of specific alleles and using a limited number of selected varieties, modern breeding techniques have decreased the overall genetic variability required for climate-smart agriculture. However, gene banks have well-managed collectionns of landraces, wild linseed accessions, and auxiliary Linum species that serve as an important source of novel alleles. In the past, flax-breeding techniques were prioritised, preserving high yield with other essential traits. Applications of molecular markers in modern breeding have made it easy to identify quantitative trait loci (QTLs) for various agronomic characteristics. The genetic diversity of linseed species and the evaluation of their tolerance to abiotic stresses, including drought, salinity, heavy metal tolerance, and temperature, as well as resistance to biotic stress factors, viz., rust, wilt, powdery mildew, and alternaria blight, despite addressing various morphotypes and the value of linseed as a supplement, are the primary topics of this review.

Genome Editing and Improvement of Abiotic Stress Tolerance in Crop Plants.

Genome editing aims to revolutionise plant breeding and could assist in safeguarding the global food supply. The inclusion of a 12-40 bp recognition site makes mega nucleases the first tools utilized for genome editing and first generation gene-editing tools. Zinc finger nucleases (ZFNs) are the second gene-editing technique, and because they create double-stranded breaks, they are more dependable and effective. ZFNs were the original designed nuclease-based approach of genome editing. The Cys2-His2 zinc finger domain's discovery made this technique possible. Clustered regularly interspaced short palindromic repeats (CRISPR) are utilized to improve genetics, boost biomass production, increase nutrient usage efficiency, and develop disease resistance. Plant genomes can be effectively modified using genome-editing technologies to enhance characteristics without introducing foreign DNA into the genome. Next-generation plant breeding will soon be defined by these exact breeding methods. There is abroad promise that genome-edited crops will be essential in the years to come for improving the sustainability and climate-change resilience of food systems. This method also has great potential for enhancing crops' resistance to various abiotic stressors. In this review paper, we summarize the most recent findings about the mechanism of abiotic stress response in crop plants and the use of the CRISPR/Cas mediated gene-editing systems to improve tolerance to stresses including drought, salinity, cold, heat, and heavy metals.

Screening and Selection of Drought-Tolerant High-Yielding Chickpea Genotypes Based on Physio-Biochemical Selection Indices and Yield Trials.

Chickpea production is seriously hampered by drought stress, which could be a great threat in the future for food security in developing countries. The present investigation aimed to screen the drought-tolerant response of forty desi chickpea genotypes against drought stress through various physio-biochemical selection indices and yield-attributing traits. Principle component-based biplot analysis recognized PG205, JG2016-44, JG63, and JG24 as tolerant genotypes based on physiological selection indices. These genotypes retained higher relative water content, stomatal conductance, internal CO2 concentration, and photosynthetic rate. ICC4958, JG11, JAKI9218, JG16, JG63, and PG205 were selected as tolerant genotypes based on biochemical selection indices. These genotypes sustained higher chlorophyll, sugar and proline content with enhanced antioxidant enzyme activities. With respect to yield trials, JAKI9218, JG11, JG16, and ICC4958 had higher seed yield per plant, numbers of pods, and biological yield per plant. Finally, JG11, JAKI9218, ICC4958, JG16, JG63, and PG205 were selected as tolerant genotypes based on cumulative physio-biochemical selection indices and yield response. These identified drought-tolerant genotypes may be further employed in climate-smart chickpea breeding programs for sustainable production under a changing climate scenario.

Breeding and Genomic Approaches towards Development of Fusarium Wilt Resistance in Chickpea.

Chickpea is an important leguminous crop with potential to provide dietary proteins to both humans and animals. It also ameliorates soil nitrogen through biological nitrogen fixation. The crop is affected by an array of biotic and abiotic factors. Among different biotic stresses, a major fungal disease called Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceris (FOC), is responsible for low productivity in chickpea. To date, eight pathogenic races of FOC (race 0, 1A, and 1B/C, 2-6) have been reported worldwide. The development of resistant cultivars using different conventional breeding methods is very time consuming and depends upon the environment. Modern technologies can improve conventional methods to solve these major constraints. Understanding the molecular response of chickpea to Fusarium wilt can help to provide effective management strategies. The identification of molecular markers closely linked to genes/QTLs has provided great potential for chickpea improvement programs. Moreover, omics approaches, including transcriptomics, metabolomics, and proteomics give scientists a vast viewpoint of functional genomics. In this review, we will discuss the integration of all available strategies and provide comprehensive knowledge about chickpea plant defense against Fusarium wilt.

Molecular Breeding and Drought Tolerance in Chickpea.

Cicer arietinum L. is the third greatest widely planted imperative pulse crop worldwide, and it belongs to the Leguminosae family. Drought is the utmost common abiotic factor on plants, distressing their water status and limiting their growth and development. Chickpea genotypes have the natural ability to fight drought stress using certain strategies viz., escape, avoidance and tolerance. Assorted breeding methods, including hybridization, mutation, and marker-aided breeding, genome sequencing along with omics approaches, could be used to improve the chickpea germplasm lines(s) against drought stress. Root features, for instance depth and root biomass, have been recognized as the greatest beneficial morphological factors for managing terminal drought tolerance in the chickpea. Marker-aided selection, for example, is a genomics-assisted breeding (GAB) strategy that can considerably increase crop breeding accuracy and competence. These breeding technologies, notably marker-assisted breeding, omics, and plant physiology knowledge, underlined the importance of chickpea breeding and can be used in future crop improvement programmes to generate drought-tolerant cultivars(s).

Molecular Breeding to Overcome Biotic Stresses in Soybean: Update.

Soybean (Glycine max (L.) Merr.) is an important leguminous crop and biotic stresses are a global concern for soybean growers. In recent decades, significant development has been carried outtowards identification of the diseases caused by pathogens, sources of resistance and determination of loci conferring resistance to different diseases on linkage maps of soybean. Host-plant resistance is generally accepted as the bestsolution because of its role in the management of environmental and economic conditions of farmers owing to low input in terms of chemicals. The main objectives of soybean crop improvement are based on the identification of sources of resistance or tolerance against various biotic as well as abiotic stresses and utilization of these sources for further hybridization and transgenic processes for development of new cultivars for stress management. The focus of the present review is to summarize genetic aspects of various diseases caused by pathogens in soybean and molecular breeding research work conducted to date.

In Vitro Production of Somaclones with Decreased Erucic Acid Content in Indian Mustard [Brassica juncea (Linn.) Czern&Coss].

Brassica juncea is a crucial cultivated mustard species and principal oilseed crop of India and Madhya Pradesh, grown for diverse vegetables, condiments, and oilseeds. Somaclonal variation was explored as a probable source of additional variability for the manipulation of fatty acids, especially low erucic acid contents that may be valuable for this commercially important plant species. The plantlets regenerated from tissue cultures (R0), their R1 generation and respective parental lines were compared for morpho-physiological traits and fatty acid profile for the probable existence of somaclonal variations. The first putative somaclone derived from genotype CS54 contained 5.48% and 5.52% erucic acid in R0 and R1 regenerants, respectively, compared to the mother plant (41.36%). In comparison, the second somaclone acquired from PM30 exhibited a complete absence of erucic acid corresponding to its mother plant (1.07%). These putative somaclones present a source of variation for exploitation in the development of future mustard crops with low erucic acid content.

Cell Suspension Culture and In Vitro Screening for Drought Tolerance in Soybean Using Poly-Ethylene Glycol.

Soybean (Glycine max (L) Merrill) is used in India mostly as a substantial fund of protein and oil, which makes the crop significantly important. Somaclonal variation has been researched as a base of additional variability for drought in soybean. In the present experiment calli/cell clumps/embryoids rose from immature and mature embryonic axis and cotyledons explants were exposed to different concentrations of polyethylene glycol (PEG6000). A discontinuous method proved to be superior as it permitted the calli/embryoids/cell clumps to regain their regeneration competence. A total of 64 (12.21%) plantlets of genotype JS335 and 78 (13.13%) of genotype JS93-05 were regenerated after four consequent subcultures on the selection medium with an effective lethal concentration of 20% PEG6000, and proliferated calli/embryoids/cell clumps were further subcultured on Murashige and Skoog regeneration medium supplemented with 0.5 mgL-1 each of α-napthalene acetic acid (NAA), 6-benzyladenine (BA) and Kinetin (Kn), 20.0 gL-1 sucrose and 7.5 gL-1 agar. Putative drought-tolerant plantlets were acquired from genotype JS93-05 (38) in more numbers compared to genotype JS335 (26). Random decamer primers confirmed the presence of variability between mother plants and regenerated plants from both the genotypes. Since these plantlets recovered from tolerant calli/embryoids/cell clumps selected from the medium supplemented with PEG6000, the possibility exists that these plants may prove to be tolerant against drought stress.

DNA barcoding of some medicinally important plant species of Lamiaceae family in India.

Several species of the Lamiaceae family are the primary source of bioactive aromatic oils and secondary metabolites, having broader applications in the cosmetics, pharmaceuticals, food, confectionery and liquor industries. Due to the scarcity of raw materials and high costs of this family's economically vital species, its products often adulterated to cater to the market's high demand. The present study provides a DNA based approach for identifying different species of this family. Henceforth, the performance of three already proposed barcode loci (matK, trnH-psbA and trnL) was examined for their PCR amplification and species recognition efficacy on various Lamiaceae species and cultivars using three different approaches such as pairwise genetic distance method, BLASTn and phylogenetic tree based on maximum likelihood (ML) analysis. Results illustrate that among all the DNA barcoding loci, matK locus can accurately and efficiently distinguish all the studied species followed by trnH-psbA and trnL. Present investigation may help diminish the illegal trade and events of adulteration of medicinally important plants species in genus Mentha, Ocimum and Plectranthus. This investigation will also help fulfil the scarcity of sequences of barcode loci of these species in the NCBI database. Apart from providing a molecular level reference for identifying processed herbal products, this technique also offers a convenient method for species identification and germplasm conservation of the Lamiaceae family.

Repeated Dose Toxicity Study and Developmental and Reproductive Toxicology Studies of a Respiratory Syncytial Virus Candidate Vaccine in Rabbits and Rats.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections, and vaccines are needed to treat young children and older adults. One of GSK's candidate vaccines for RSV contains recombinant RSVPreF3 protein maintained in the prefusion conformation. The differences in immune function of young children and older adults potentially require different vaccine approaches. For young children, anti-RSV immunity can be afforded during the first months of life by vaccinating the pregnant mother during the third trimester with unadjuvanted RSVPreF3, which results in protection of the infant due to the transplacental passage of anti-RSV maternal antibodies. For older adults with a waning immune response, the approach is to adjuvant the RSVPreF3 vaccine with AS01 to elicit a more robust immune response.The local and systemic effects of biweekly intramuscular injections of the RSVPreF3 vaccine (unadjuvanted, adjuvanted with AS01, or coadministered with a diphtheria-tetanus-acellular pertussis vaccine) was tested in a repeated dose toxicity study in rabbits. After three intramuscular doses, the only changes observed were those commonly related to a vaccine-elicited inflammatory reaction. Subsequently, the effects of unadjuvanted RSVPreF3 vaccine on female fertility, embryo-fetal, and postnatal development of offspring were evaluated in rats and rabbits. There were no effects on pregnancy, delivery, lactation, or the pre- and postnatal development of offspring.In conclusion, the RSVPreF3 vaccine was well-tolerated locally and systemically and was not associated with any adverse effects on female reproductive function or on the pre- and postnatal growth and development of offspring.

Scientific Regulatory Policy Committee Points to Consider*: Nuisance Factors, Block Effects, and Batch Effects in Nonclinical Safety Assessment Studies.

Detection of test article-related effects and the determination of the adversity of those changes are the primary goals of nonclinical safety assessment studies for drugs and chemicals in development. During these studies, variables that are not of primary interest to investigators may change and influence data interpretation. These variables, often referred to as "nuisance factors," may influence other groups of data and result in "block or batch effects" that complicate data interpretation. Definitions of the terms "nuisance factors," "block effects," and "batch effects," as they apply to nonclinical safety assessment studies, are reviewed. Multiple case examples of block and batch effects in safety assessment studies are provided, and the challenges these bring to pathology data interpretation are discussed. Methods to mitigate the occurrence of block and batch effects in safety assessment studies, including statistical blocking and utilization of study designs that minimize potential confounding variables, incorporation of adequate randomization, and use of an appropriate number of animals or repeated measurement of specific parameters for increased precision, are reviewed. [Box: see text].

Atypical presentation and pathogenesis of a macaque lymphocryptoviral-associated B-cell lymphoma in a cynomolgus monkey.

We report the unique pathogenesis and presentation of a rapidly progressive B-cell lymphoma in a 3-year-old female cynomolgus monkey on day 50 of a 13-week toxicity study. Clinical pathology evaluation revealed a marked leukocytosis with bicytopenia. A serum protein electrophoresis was consistent with monoclonal gammopathy. The architecture of the lymph node, spleen, and thymus were variably effaced by neoplastic cells, which also infiltrated other tissues. Immunohistochemistry of the affected tissues confirmed a predominant population of CD20+, CD79a+, CD3-, CD68-, and CD34-neoplastic cells. The full data best support a diagnosis of Stage V lymphoma. Nextgen sequencing and negative prestudy serology results suggested a recent infection by macaque lymphocryptovirus (mLCV) with a unique transcriptional profile comparable with a rarely observed direct LCV infection model. This infection model might be associated with a temporary lack of an LCV antigen-specific cytotoxic T-cell adaptive immune response. Consistent with the established mechanisms of LCV-related lymphoproliferation, MYC and BCL2L11 gene expression were increased and decreased, respectively. While there was no overt immunosuppression, immunophenotyping revealed the index animal had a relatively low NK cell count, which further decreased by >50% on day 24 of the study. In addition to the temporary lack of adaptive immunity, the low NK cell counts were suggestive of an impaired innate immunity to control the virally-transformed cells and the subsequent unchecked lymphoproliferation. To our knowledge, this is the first report of a Stage V lymphoma with a unique pathogenesis in an otherwise immunocompetent cynomolgus monkey.

Scientific and Regulatory Policy Committee Points to Consider*: Approaches to the Conduct and Interpretation of Vaccine Safety Studies for Clinical and Anatomic Pathologists.

The design and execution of toxicology studies supporting vaccine development have some unique considerations relative to those supporting traditional small molecules and biologics. A working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee conducted a review of the scientific, technical, and regulatory considerations for veterinary pathologists and toxicologists related to the design and evaluation of regulatory toxicology studies supporting vaccine clinical trials. Much of the information in this document focuses on the development of prophylactic vaccines for infectious agents. Many of these considerations also apply to therapeutic vaccine development (such as vaccines directed against cancer epitopes); important differences will be identified in various sections as appropriate. The topics addressed in this Points to Consider article include regulatory guidelines for nonclinical vaccine studies, study design (including species selection), technical considerations in dosing and injection site collection, study end point evaluation, and data interpretation. The intent of this publication is to share learnings related to nonclinical studies to support vaccine development to help others as they move into this therapeutic area. [Box: see text].

Scientific and Regulatory Policy Committee Points to Consider*: The Toxicologic Pathologist's Role in the 3Rs.

Pathologists are trained medical professionals with special expertise in diagnostics, research, and pathophysiology. In these roles, pathologists are well qualified and positioned to engage in conversations about animal use replacement, reduction, and refinement (3Rs), thereby championing the guiding principles of the 3Rs. In particular, toxicology or nonclinical safety assessment is an important area where the discipline of toxicologic pathology can have a critical role in adopting 3Rs principles. As such, a working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee was formed to investigate and summarize some of the areas where veterinary pathologists working in the field of toxicology can increase involvement and impact on 3Rs. This "Points to Consider" publication provides an overview of areas within toxicology where the veterinary pathologist's perspective may maximize animal value, including refinement of study design, optimizing sample collection, the development of 3Rs focused regulatory policy, and humane end point determination.[Box: see text].

Interpretative considerations for clinical pathology findings in nonclinical toxicology studies.

The interpretation of clinical pathology results from nonclinical safety studies is a fundamental component in hazard identification of new drug candidates. The ever-increasing complexity of nonclinical safety studies and sophistication of modern analytical methods have made the interpretation of clinical pathology information by a highly trained subject matter expert imperative. Certain interpretive techniques are particularly effective in the identification and characterization of clinical pathology effects. The purpose of this manuscript is to provide an overview of contemporary interpretive practices for clinical pathology results and to provide nonbinding recommendations aimed at improving consistency, quality, and overall value of clinical pathology interpretations generated in support of nonclinical toxicology studies.

Overview and considerations for the reporting of clinical pathology interpretations in nonclinical toxicology studies.

Clinical pathology reporting practices are diverse among individuals and organizations involved in nonclinical toxicology studies. Clear, informative, and consistent reporting of clinical pathology results increases their value and avoids misinterpretation, resulting in decreased drug development costs. In recent years, certain common practices in clinical pathology reporting have been embraced by industry leaders and more consistently utilized across the pharmaceutical industry. The purpose of this manuscript is to review current clinical pathology reporting practices and to provide nonbinding suggestions to improve consistency, quality, and value of clinical pathology reports generated in support of nonclinical toxicology studies.