Early Alzheimer's disease pathology in human cortex involves transient cell states.

Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the early cortical amyloid response-were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with ß-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

Slide-tags enables single-nucleus barcoding for multimodal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

The molecular cytoarchitecture of the adult mouse brain.

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

Spatial genomics enables multi-modal study of clonal heterogeneity in tissues.

The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations1-4 as well as the makeup of the tumour microenvironment5,6. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.

A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis.

A single-cell map of antisense oligonucleotide activity in the brain.

Antisense oligonucleotides (ASOs) dosed into cerebrospinal fluid (CSF) distribute broadly throughout the central nervous system (CNS). By modulating RNA, they hold the promise of targeting root molecular causes of disease and hold potential to treat myriad CNS disorders. Realization of this potential requires that ASOs must be active in the disease-relevant cells, and ideally, that monitorable biomarkers also reflect ASO activity in these cells. The biodistribution and activity of such centrally delivered ASOs have been deeply characterized in rodent and non-human primate (NHP) models, but usually only in bulk tissue, limiting our understanding of the distribution of ASO activity across individual cells and across diverse CNS cell types. Moreover, in human clinical trials, target engagement is usually monitorable only in a single compartment, CSF. We sought a deeper understanding of how individual cells and cell types contribute to bulk tissue signal in the CNS, and how these are linked to CSF biomarker outcomes. We employed single nucleus transcriptomics on tissue from mice treated with RNase H1 ASOs against Prnp and Malat1 and NHPs treated with an ASO against PRNP. Pharmacologic activity was observed in every cell type, though sometimes with substantial differences in magnitude. Single cell RNA count distributions implied target RNA suppression in every single sequenced cell, rather than intense knockdown in only some cells. Duration of action up to 12 weeks post-dose differed across cell types, being shorter in microglia than in neurons. Suppression in neurons was generally similar to, or more robust than, the bulk tissue. In macaques, PrP in CSF was lowered 40% in conjunction with PRNP knockdown across all cell types including neurons, arguing that a CSF biomarker readout is likely to reflect ASO pharmacodynamic effect in disease-relevant cells in a neuronal disorder. Our results provide a reference dataset for ASO activity distribution in the CNS and establish single nucleus sequencing as a method for evaluating cell type specificity of oligonucleotide therapeutics and other modalities.

Antisense oligonucleotide (ASO) drugs are a type of chemically modified DNA that can be injected into cerebrospinal fluid in order to enter brain cells and reduce the amount of RNA from a specific gene. The brain is a complex mixture of hundreds of billions of cells. When an ASO lowers a target gene's RNA by 50%, is that a 50% reduction in 100% of cells, or a 100% reduction in 50% of cells? Are the many different cell types of the brain affected equally? This new study uses single cell RNA sequencing to answer these questions, finding that ASOs are broadly active across cell types and individual cells, and linking reduction of target protein in cerebrospinal fluid to disease-relevant cells.

Influence of PRKCE non-synonymous variants on protein dynamics and functionality.

Novel protein kinase C (nPKC) family member, protein kinase C epsilon (PKCε) is an AGC kinase superfamily member. It is associated with neurological and metabolic diseases as well as human cancers. No study so far has been conducted to identify genetic variations and their effect on PKCε folding and functioning. The present study aimed to identify mutational hotspots in PKCε and disease-causing non-synonymous variants (nsSNPs) along with the investigation of nsSNP impact on protein dynamics. Twenty-nine in silico tools were applied to determine nsSNP deleteriousness, their impact on protein dynamics and disease association, along with the prediction of PKCε post-translational modification (PTM) sites. The present study's outcomes indicated that most nsSNPs were concentrated in the PKCε hinge region and C-terminal tail. Most pathogenic variants mapped to the kinase domain. Regulatory domain variants influenced PKCε interaction with molecular players whereas kinase domain variants were predicted to impact its phosphorylation pattern and protein-protein interactions. Most PTM sites were mapped to the hinge region. PKCε nsSNPs have an association with oncogenicity and its expression dysregulation is responsible for poor overall survival. Understanding nsSNP structural impact is a primary step necessary for delineating the relationship of genetic level differences with protein phenotype. The obtained knowledge can eventually help in disease diagnosis and therapy design.

Preparation, Optimization and In vitro Evaluation of Doxorubicin-loaded into Hyaluronic Acid Coated Niosomes Against Breast Cancer.

Doxorubicin (DOX) is widely used against solid tumors. Niosomes are self-assembled nanocarriers of non-ionic surfactants. DOX loaded into cationic niosomes (DOX-Nio) was prepared via thin film hydration method. DOX-Nio was then decorated with a hyaluronic acid (DOX-HA-Nio) via electrostatic interaction. DOX-Nio and DOX-HA-Nio displayed a particle size of 120.0±1.02 and 182.9±2.3 nm, and charge of + 35.5±0.15 and -15.6±0.25 mV, respectively, with PDI < 0.3. DOX-HA-Nio showed a good stability regarding size and charge over 4 weeks at 4 °C and maintain their integrity after lyophilization. HPLC results showed a 94.1±4.2 % encapsulation efficiency of DOX with good entrapment and slow, prolonged DOX release even after 48 hrs. Cell viability assay showed an IC50 of 14.26 nM for the DOX-HA-Nio against MCF-7 cell line with micromolar IC50 results against CD-44 negative cell lines (NIH/3T3). DOX-HA-Nio was proven to be an effective, targeted nanocarrier for DOX against MCF-7 cell line.

Use of wheat dilution to improve digestive function in broilers: application in low protein diets.

1. This study evaluated the effect of wheat dilution increasing in particle size in low crude protein diets on growth performance, digestive tract, nitrogen efficiency and litter quality in broiler chickens.2. Ross 308 male broiler chicks (n = 336) were allocated to one of four dietary treatments (each with 7 pens, 12 chicks per pen); Control (CON; commercial pellet diet with standard crude protein, CP: 22.50%), W578 (CON +20% wheat of geometric mean diameter (GMD) of 578 µm; CP: 20.25%), W1326 (CON +20% wheat of GMD 1326 µm; CP: 20.25%) and WW (CON +20% whole wheat, CP: 20.25%), from d 0 to 21 of age.3. Body weight gain was increased (P < 0.05) for birds fed CON compared to the low crude protein diets. However, WW increased (P < 0.05) body weight gain compared to W578 and W1326, while feed intake and feed conversion ratio on CON and WW were similar (P > 0.05). Birds fed W1326 showed the poorest (P < 0.05) FCR compared to CON, W578 and WW.4. Gizzard relative weight (g/kg body weight) was increased (P < 0.05) on WW compared to CON on d 14 and 21, whereas gastric isthmus diameter was significantly reduced on W1326 and WW compared with CON and W578.5. There were no differences (P > 0.05) in the depth of gizzard mucosa of lamina propria between CON and WW at d 14 and 21. Birds fed WW increased (P < 0.05) gizzard tensile strength compared to W578 and W1326, whereas no difference was observed between WW and CON on d 14.6. No significant differences were seen for ileum villus height and mucosal layer between CON and WW on d 21, however, feeding CON increased the extent of the mucosal layer compared to W578 and W1326.7. Nitrogen excretion (g/kg BWG) was significantly lower (P > 0.05) on W1326 and WW compared with CON and W578. Litter nitrogen, moisture, and footpad scores significantly decreased (P < 0.05) for birds fed WW compared with CON.8. Diluting dietary protein content from 22.50 to 20.25% resulted in lower body weight gain in broilers. However, dilution with whole wheat resulted in comparable FCR, reduced nitrogen excretion, litter moisture and footpad dermatitis compared with a standard protein diet.

Efficacy of maize differing in particle size in low-density protein diets fed to broilers from day 1 to 21 of age.

1. This study evaluated the efficacy of maize differing in particle size in low-density protein diets on performance, the digestive tract and litter characteristics in broilers. Four dietary treatments; control commercial diet with a typical crude protein content 22.50% (CON); CON + 20% maize with particle size 350 µm (M350), crude protein 19.90%; CON + 20% maize with particle size 2600 µm (M2600), crude protein 19.90%); CON + 20% whole maize (WM), crude protein 19.90%, were fed ad libitum to broiler chicks up to 21 d of age.2. No differences in body weight gain, feed intake and FCR were found between the WM and CON. WM increased body weight gain compared to M350 and M2600. M350, M2600 and WM increased (p = 0.004) gizzard relative weight compared with CON on day 14. Both WM and M2600 reduced (p = 0.001) gastric isthmus diameter on d 14 and 21 compared with CON and M350.3. No differences were seen in mucosa for the Lamina propria and the extent of Tunica muscularis of gizzard on d 21 and ileum mucosal depth on d 14 between WM and CON diets. However, WM reduced villus-to-crypt ratio compared with CON on d 21. The M350 reduced (p < 0.05) gizzard digesta particle size compared with CON, M2600 and WM on d 14.4. Both WM and M350 decreased (p < 0.05) nitrogen excretion compared to birds fed CON. Feeding WM increased nitrogen efficiency compared with M350 and M2600 diets, but was similar to birds fed CON. Feeding M350, M2600 or WM decreased (p < 0.05) litter moisture and footpad dermatitis (FPD) scores compared with results from birds fed CON.5. Overall, diluting the protein level in broiler diets with whole maize appeared better than fine or coarse maize in terms of growth performance, digestive tract development, nitrogen excretion and litter parameters. This may lead to economic benefits by reducing grinding costs and dependence on rich protein resources contributing to sustainable meat production and food security.

Investigating Machine Learning Techniques for Predicting Risk of Asthma Exacerbations: A Systematic Review.

Asthma, a common chronic respiratory disease among children and adults, affects more than 200 million people worldwide and causes about 450,000 deaths each year. Machine learning is increasingly applied in healthcare to assist health practitioners in decision-making. In asthma management, machine learning excels in performing well-defined tasks, such as diagnosis, prediction, medication, and management. However, there remain uncertainties about how machine learning can be applied to predict asthma exacerbation. This study aimed to systematically review recent applications of machine learning techniques in predicting the risk of asthma attacks to assist asthma control and management. A total of 860 studies were initially identified from five databases. After the screening and full-text review, 20 studies were selected for inclusion in this review. The review considered recent studies published from January 2010 to February 2023. The 20 studies used machine learning techniques to support future asthma risk prediction by using various data sources such as clinical, medical, biological, and socio-demographic data sources, as well as environmental and meteorological data. While some studies considered prediction as a category, other studies predicted the probability of exacerbation. Only a group of studies applied prediction windows. The paper proposes a conceptual model to summarise how machine learning and available data sources can be leveraged to produce effective models for the early detection of asthma attacks. The review also generated a list of data sources that other researchers may use in similar work. Furthermore, we present opportunities for further research and the limitations of the preceding studies.

Silicon dioxide nanoparticles suppress copper toxicity in Mentha arvensis L. by adjusting ROS homeostasis and antioxidant defense system and improving essential oil production.

Copper (Cu) is an essential micronutrient for plants; however, the excessive accumulation of Cu due to various anthropogenic activities generates progressive pollution of agricultural land and that causes a major constraint for crop production. Excess Cu (80 mg kg-1) in the soil diminished growth and biomass, photosynthetic efficiency and essential oil (EO) content in Mentha arvensis L., while amplifying the antioxidant enzyme's function and reactive oxygen species (ROS) production. Therefore, there is a pressing need to explore effective approaches to overcome Cu toxicity in M. arvensis plants. Thus, the present study unveils the potential of foliar supplementation of two distinct forms of silicon dioxide nanoparticles (SiO2 NPs) i.e., Aerosil 200F and Aerosil 300 to confer Cu stress tolerance attributes to M. arvensis. The experiment demonstrated that applied forms of SiO2 NPs (120 mg L-1), enhanced plants' growth and augmented the photosynthetic efficiency along with the activities of CA (carbonic anhydrase) and NR (nitrate reductase), however, the effects were more accentuated by Aerosil 200F application. Supplementation of SiO2 NPs also exhibited a beneficial effect on the antioxidant machinery of Cu-disturbed plants by raising the level of proline and total phenol as well as the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and glutathione reductase (GR), thereby lowering ROS and electrolytic leakage (EL). Interestingly, SiO2 NPs supplementation upscaled EO production in Cu-stressed plants with more pronounced effects received in the case of Aerosil 200F over Aerosil 300. We concluded that the nano form (Aerosil 200F) of SiO2 proved to be the best in improving the Cu-stress tolerance in plants.

Multi-Class Skin Lesions Classification Using Deep Features.

Skin cancer classification is a complex and time-consuming task. Existing approaches use segmentation to improve accuracy and efficiency, but due to different sizes and shapes of lesions, segmentation is not a suitable approach. In this research study, we proposed an improved automated system based on hybrid and optimal feature selections. Firstly, we balanced our dataset by applying three different transformation techniques, which include brightness, sharpening, and contrast enhancement. Secondly, we retrained two CNNs, Darknet53 and Inception V3, using transfer learning. Thirdly, the retrained models were used to extract deep features from the dataset. Lastly, optimal features were selected using moth flame optimization (MFO) to overcome the curse of dimensionality. This helped us in improving accuracy and efficiency of our model. We achieved 95.9%, 95.0%, and 95.8% on cubic SVM, quadratic SVM, and ensemble subspace discriminants, respectively. We compared our technique with state-of-the-art approach.

Soil-applied Nickel Generates Differential Responses in Growth, Physiology and Oxidative Metabolism of Fenugreek (Trigonella foenum-graecum L.) Varieties.

This work aims to evaluate the potential of nickel (Ni), an essential micronutrient, as an oxidative stress inducer along with associated morphological and biochemical responses in different varieties of fenugreek (Trigonella foenum-graecum L.), a chief economically cultivated crop of India. Varietal differences in crop performance upon exposure to 0, 20, 40, 60 and 80 mg Ni kg- 1 soil reflects that Ni applied at 20 mg Ni kg- 1 soil offers growth-promoting effects, improved photosynthesis attributes, carbonic and nitrate reductase activities more profound in PEB followed by AFg2, AFg1 and UM185 variety. This study observed a dose-dependent reduction in all the above parameters. Maximum toxic effects were noticed at 80 mg kg- 1 Ni, manifested in the form of enhanced H2O2 and MDA contents, which were efficiently counteracted by augmentation in proline content, SOD, POX, CAT and APX activities in PEB over other varieties, suggesting that the Ni tolerance in fenugreek varieties can be organized as PEB > AFg2 > AFg1 > UM185.

Prevalence of Ruminant Paramphistomosis and Comparative Histopathology of the Infected Rumens in Narowal District, Punjab, Pakistan.

The present study reports the prevalence of Paramphistomum spp. in small and large ruminants and their association with the histopathology of the infected rumens. A total of 384 animals were screened for Paramphistomum spp. The animals found positive for Paramphistomum spp. were divided into three groups according to the worm load/5 cm2 (G1: 10 - 20 worms/5 cm2 = Low, G2: 20 - 40 worms/5 cm2 = Medium, and G3: >41 worms/5 cm2 = High). Tissue slides were prepared from samples of the rumen (1 cm2) taken from animals positive for ruminal fluke to determine the histological parameters, including epithelial length or thickness, length and width of the ruminal papilla, and thickness of tunica submucosa and mucularis externae. The overall prevalence of Paramphistomum spp. in the ruminant population of district Narowal was 56.25 % with a significant (P < 0.05) variation among different species of ruminants. The highest prevalence was in cattle, followed in order by buffalo, goat, and sheep. Epithelium thickness was significantly correlated with parasite load in large ruminants and the most significant (P < 0.05) decrease in epithelium thickness was in Group B (31.12 ± 1.82 µm) and Group C (31.07 ± 1.68 µm) and a same trend was recorded in small ruminants. Histopathological changes due to Paramphistomum spp. are reported for the first time, which explained the histomorphological and physiological changes in Paramphistomum-infected rumens which might be associated with lowered feed efficiency and productivity in ruminants.

Enhancing artemisinin content in and delivery from Artemisia annua: a review of alternative, classical, and transgenic approaches.

MAIN CONCLUSION: This review analyses the most recent scientific research conducted for the purpose of enhancing artemisinin production. It may help to devise better artemisinin enhancement strategies, so that its production becomes cost effective and becomes available to masses. Malaria is a major threat to world population, particularly in South-East Asia and Africa, due to dearth of effective anti-malarial compounds, emergence of quinine resistant malarial strains, and lack of advanced healthcare facilities. Artemisinin, a sesquiterpene lactone obtained from Artemisia annua L., is the most potent drug against malaria and used in the formulation of artemisinin combination therapies (ACTs). Artemisinin is also effective against various types of cancers, many other microbes including viruses, parasites and bacteria. However, this specialty metabolite and its derivatives generally occur in low amounts in the source plant leading to its production scarcity. Considering the importance of this drug, researchers have been working worldwide to develop novel strategies to augment its production both in vivo and in vitro. Due to complex chemical structure, its chemical synthesis is quite expensive, so researchers need to devise synthetic protocols that are economically viable and also work on increasing the in-planta production of artemisinin by using various strategies like use of phytohormones, stress signals, bioinoculants, breeding and transgenic approaches. The focus of this review is to discuss these artemisinin enhancement strategies, understand mechanisms modulating its biosynthesis, and evaluate if roots play any role in artemisinin production. Furthermore, we also have a critical analysis of various assays used for artemisinin measurement. This may help to develop better artemisinin enhancement strategies which lead to decreased price of ACTs and increased profit to farmers.

Effects of Bacillus sp. MR-1/2 and magnetite nanoparticles on yield improvement of rice by urea fertilizer under different watering regimes.

AIMS: The present research aimed to examine the use of magnetite nanoparticles (MNPs) in combination with phyto-beneficial rhizobacterium (PhBR) for improvement of applied N recovery (ANR) from urea fertilizer in rice grown under deficient and optimum watering conditions. METHODS AND RESULTS: The Bacillus sp. MR-1/2 was positive for acetylene reduction, phosphate solubilization and ACC deaminase activity at temperature ranges 35-45°C. In a pot experiment, urea, MNPs and Bacillus sp. MR-1/2 were applied either alone or in combination to rice plants grown in pots under water deficit and optimal watering conditions. Combined application of urea, MNPs and Bacillus sp. MR-1/2 increased the plant N content and ANR by 27 and 65%, respectively, over their respective control values in rice grown under optimum watering conditions, whereas these increases were 27 and 41%, respectively, in rice grown under water deficit conditions. This treatment also increased the kernel weight and plant dry matter by 36 and 60%, respectively, over control (urea alone) values in rice grown under water deficit conditions, whereas these increases were 31 and 21·8%, respectively, in rice grown under optimum watering conditions. Values of malondialdehyde (MDA) contents, ascorbate peroxidase (APX), catalase and ethylene concentration were higher in control treatment under both the watering regimes. The application of Bacillus sp. MR-1/2 either alone or in combination with MNPs and urea reduced MDA contents, APX, catalase and ethylene production in the rice plants. CONCLUSION: The combined application of MNPs+Bacillus sp. MR-1/2 reduced the N losses from applied urea, increased N uptake and ANR in rice, decreased MDA contents, APX and catalase activity and ethylene level in rice grown under deficit and optimum water conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of MNPs together with Bacillus sp. MR-1/2 may help to increase ANR and rice productivity under water deficit conditions with low cost of production.

Genotyping for Dombrock blood group alleles in Northern Pakistani blood donors.

Genotyping can be used to identify rare blood group antigens and to solve suspected blood group discrepancies, particularly when serologic methods are limited. Unfortunately, only a few such studies have been performed in Pakistan. The present study was conducted to determine the frequency of Dombrock blood group alleles by genotyping samples from blood donors from the north of Pakistan. Blood samples were taken with consent from 300 blood donors; DNA was extracted and tested for DO*01 and DO*02 alleles by sequence-specific primer polymerase chain reaction (PCR-SSP), followed by gel electrophoresis. Allele frequencies were calculated. The observed and expected genotype frequencies were compared using the χ2 test. The allele frequencies for DO*01 and DO*02 were 0.40 and 0.60, respectively. Genotype frequencies were in Hardy-Weinberg equilibrium. This study in Pakistani blood donors provides Dombrock blood group allele frequencies by PCR-SSP. This approach is efficient and economical and can be applied in developing countries. The findings can contribute to the development of in-house red blood cell panels, identification of rare blood types, and establishment of a national rare blood donor program.Genotyping can be used to identify rare blood group antigens and to solve suspected blood group discrepancies, particularly when serologic methods are limited. Unfortunately, only a few such studies have been performed in Pakistan. The present study was conducted to determine the frequency of Dombrock blood group alleles by genotyping samples from blood donors from the north of Pakistan. Blood samples were taken with consent from 300 blood donors; DNA was extracted and tested for DO*01 and DO*02 alleles by sequence-specific primer polymerase chain reaction (PCR-SSP), followed by gel electrophoresis. Allele frequencies were calculated. The observed and expected genotype frequencies were compared using the χ2 test. The allele frequencies for DO*01 and DO*02 were 0.40 and 0.60, respectively. Genotype frequencies were in Hardy-Weinberg equilibrium. This study in Pakistani blood donors provides Dombrock blood group allele frequencies by PCR-SSP. This approach is efficient and economical and can be applied in developing countries. The findings can contribute to the development of in-house red blood cell panels, identification of rare blood types, and establishment of a national rare blood donor program.

Molecular Mechanisms of Nitric Oxide (NO) Signaling and Reactive Oxygen Species (ROS) Homeostasis during Abiotic Stresses in Plants.

Abiotic stressors, such as drought, heavy metals, and high salinity, are causing huge crop losses worldwide. These abiotic stressors are expected to become more extreme, less predictable, and more widespread in the near future. With the rapidly growing human population and changing global climate conditions, it is critical to prevent global crop losses to meet the increasing demand for food and other crop products. The reactive gaseous signaling molecule nitric oxide (NO) is involved in numerous plant developmental processes as well as plant responses to various abiotic stresses through its interactions with various molecules. Together, these interactions lead to the homeostasis of reactive oxygen species (ROS), proline and glutathione biosynthesis, post-translational modifications such as S-nitrosylation, and modulation of gene and protein expression. Exogenous application of various NO donors positively mitigates the negative effects of various abiotic stressors. In view of the multidimensional role of this signaling molecule, research over the past decade has investigated its potential in alleviating the deleterious effects of various abiotic stressors, particularly in ROS homeostasis. In this review, we highlight the recent molecular and physiological advances that provide insights into the functional role of NO in mediating various abiotic stress responses in plants.