Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.

COVID-19 Infection Percentage Estimation from Computed Tomography Scans: Results and Insights from the International Per-COVID-19 Challenge.

COVID-19 analysis from medical imaging is an important task that has been intensively studied in the last years due to the spread of the COVID-19 pandemic. In fact, medical imaging has often been used as a complementary or main tool to recognize the infected persons. On the other hand, medical imaging has the ability to provide more details about COVID-19 infection, including its severity and spread, which makes it possible to evaluate the infection and follow-up the patient's state. CT scans are the most informative tool for COVID-19 infection, where the evaluation of COVID-19 infection is usually performed through infection segmentation. However, segmentation is a tedious task that requires much effort and time from expert radiologists. To deal with this limitation, an efficient framework for estimating COVID-19 infection as a regression task is proposed. The goal of the Per-COVID-19 challenge is to test the efficiency of modern deep learning methods on COVID-19 infection percentage estimation (CIPE) from CT scans. Participants had to develop an efficient deep learning approach that can learn from noisy data. In addition, participants had to cope with many challenges, including those related to COVID-19 infection complexity and crossdataset scenarios. This paper provides an overview of the COVID-19 infection percentage estimation challenge (Per-COVID-19) held at MIA-COVID-2022. Details of the competition data, challenges, and evaluation metrics are presented. The best performing approaches and their results are described and discussed.

Characterization of sulfur-oxidizing bacteria isolated from mustard (Brassica juncea L.) rhizosphere having the capability of improving sulfur and nitrogen uptake.

AIMS: The present investigation was carried out to isolate, screen and characterize potential sulfur-oxidizing bacteria (SOB) isolated from mustard field's soil. METHODS AND RESULTS: A total of 130 bacteria were isolated and after screening five maximum sulfate-producing isolates were optimized for culture conditions. The incubation time of 48 h was found optimum for all bacterial isolates and 30°C was the best temperature for the growth of SSD11, SSR1 and SSG8 whereas 35°C for SSF17. The pH 8 was found best for all four isolates except SSF17 (6 pH). Media having glucose as a carbon source and ammonium sulphate as an N-source were producing maximum sulphate. The isolates SSF17, SSR1 and SSG8 were identified as Burkholderia cepacia (accession no. MT559819), Enterobacter cloacae (accession no. MT559820) and Klebsiella oxytoca (accession no. MT372097), respectively, on the basis of morphological, biochemical and molecular characterization. The isolates were also found to increase N and S uptake efficiently in both wheat and mustard crops. CONCLUSION: This study strongly concludes that SOB isolated from the mustard field can oxidize sulfur in vitro and in vivo conditions. The three best isolates come out of the study were identified as Burkholderia, Enterobacter and Klebsiella strains. Also, inoculation of SOB increased the uptake of S and N nutrient in mustard and wheat crops and thus may be proved as an important plant growth-promoting bacteria having the biofertilization capability. SIGNIFICANCE AND IMPACT OF THE STUDY: As we know, our soil is continuously deteriorating day by day due to excessive utilization and immoderate use of chemical fertilizers. The SOB could minimize the application of chemical fertilizers thus reducing environmental deterioration by improving soil health in sustainable agricultural practices.

ß-Cleavage of the prion protein in the human eye: Implications for the spread of infectious prions and human ocular disorders.

Recently, we reported ß-cleavage of the prion protein (PrPC) in human ocular tissues. Here, we explored whether this is unique to the human eye, and its functional implications. A comparison of the cleavage pattern of PrPC in human ocular tissues with common nocturnal and diurnal animals revealed mainly ß-cleavage in humans, and mostly full-length PrPC in animal retinas. Soluble FL PrPC and N-terminal fragment (N2) released from ß-cleavage was observed in the aqueous and vitreous humor (AH & VH). Expression of human PrPC in ARPE-19 cells, a human retinal pigmented epithelial cell line, also showed ß-cleaved PrPC. Surprisingly, ß-cleavage was not altered by a variety of insults, including oxidative stress, suggesting a unique role of this cleavage in the human eye. It is likely that ß-cleaved C- or N-terminal fragments of PrPC protect from various insults unique to the human eye. On the contrary, ß-cleaved C-terminus of PrPC is susceptible to conversion to the pathological PrP-scrapie form, and includes the binding sites for ß1-integrin and amyloid-ß, molecules implicated in several ocular disorders. Considering the species and tissue-specific cleavage of PrPC, our data suggest re-evaluation of prion infectivity and other ocular disorders of the human eye conducted in mouse models.

It Is Time for an Oil Change: Polyunsaturated Fatty Acids and Human Health.

Polyunsaturated fatty acids (PUFAs) such as docosahexaneoic acid (DHA) and eicosapentaneoic acid (EPA), play a critical role in a variety of neuronal functions, including facilitating neuronal growth and differentiation, increasing the density of the neuritic network, modulating cell membrane fluidity, regulating intracellular signaling and gene expression, and exhibiting antioxidant characteristics. Dietary DHA is selectively enriched and actively retained in the central nervous system, mainly in synaptic membranes, dendrites, and photoreceptors. In this review, we highlight the myriad roles of PUFAs in brain function and human health. Diets rich in DHA are inversely proportional to cognitive decline and incidence of neurodegenerative disorders. Conversely, diets deficient in DHA impair the proper development of brain and the visual system in children and increase risk of brain disorders in the elderly. Finally, DHA and EPA have been shown to reduce inflammation and may prove to be beneficial in reducing the severity of the SARS-COVID infection.

Prions and prion diseases: Insights from the eye.

Prion diseases are invariably fatal neurodegenerative disorders that have gained much publicity due to their transmissible nature. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common human prion disorder, with an incidence of 1 in a million. Inherited prion disorders are relatively rare, and associated with mutations in the prion protein gene. More than 50 different point mutations, deletions, and insertions have been identified so far. Most are autosomal dominant and fully penetrant. Prion disorders also occur in animals, and are of major concern because of the potential for spreading to humans. The principal pathogenic event underlying all prion disorders is a change in the conformation of prion protein (PrPC) from a mainly α-helical to a ß-sheet rich isoform, PrP-scrapie (PrPSc). Accumulation of PrPSc in the brain parenchyma is the major cause of neuronal degeneration. The mechanism by which PrPSc is transmitted, propagates, and causes neurodegenerative changes has been investigated over the years, and several clues have emerged. Efforts are also ongoing for identifying specific and sensitive diagnostic tests for sCJD and animal prion disorders, but success has been limited. The eye is suitable for these evaluations because it shares several anatomical and physiological features with the brain, and can be observed in vivo during disease progression. The retina, considered an extension of the central nervous system, is involved extensively in prion disorders. Accordingly, Optical Coherence Tomography and electroretinogram have shown some promise as pre-mortem diagnostic tests for human and animal prion disorders. However, a complete understanding of the physiology of PrPC and pathobiology of PrPSc in the eye is essential for developing specific and sensitive tests. Below, we summarize recent progress in ocular physiology and pathology in prion disorders, and the eye as an anatomically accessible site to diagnose, monitor disease progression, and test therapeutic options.

Local synthesis of hepcidin in the anterior segment of the eye: A novel observation with physiological and pathological implications.

PURPOSE: The avascular cornea, trabecular meshwork (TM), and lens obtain iron, an essential biometal, from the aqueous humor (AH). The mechanism by which this exchange is regulated, however, is unclear. Recently we reported that non-pigmented ciliary epithelial cells express ferroportin (Fpn) (Ashok, 2018b), an iron export protein modulated by hepcidin, the master regulator of iron homeostasis secreted mainly by the liver. Here, we explored whether ciliary epithelial and other cells in the anterior segment synthesize hepcidin, suggesting local regulation of iron exchange at this site. METHODS: Human and bovine eyes were dissected to isolate the ciliary body (CB), corneal endothelial (CE), TM, lens epithelial (LE), and outer epithelial cell layer of the iris. Total mRNA and protein lysates were processed to evaluate the synthesis and expression of hepcidin, the iron regulatory peptide hormone, Fpn, the only known iron export protein, ceruloplasmin (Cp), a ferroxidase necessary for iron export, transferrin receptor (TfR), a major iron uptake protein, and ferritin, a major iron storage protein. A combination of techniques including reverse transcription polymerase chain reaction (RT-PCR) of total mRNA, Western blotting of protein lysates, and immunofluorescence of fixed tissue sections were used to accomplish these goals. RESULTS: RT-PCR of isolated tissue samples revealed hepcidin-specific mRNA in the CB, TM, CE, and LE of the bovine eye. Western blotting of protein lysates from these tissues showed reactivity for hepcidin, Fpn, ferritin, and TfR. Western blotting and immunohistochemistry of similar tissues isolated from cadaveric human eyes showed expression of hepcidin, Fpn, and Cp in these samples. Notably, Fpn and Cp were expressed on the basolateral membrane of non-pigmented ciliary epithelial cells, facing the AH. CONCLUSIONS: Synthesis and expression of hepcidin and Fpn in the ciliary epithelium suggests local regulation of iron transport from choroidal plexus in the ciliary body to the AH across the blood-aqueous barrier. Expression of hepcidin and Fpn in CE, TM, and LE cells indicates additional regulation of iron exchange between the AH and cornea, TM, and lens, suggesting autonomous regulation of iron homeostasis in the anterior segment. Physiological and pathological implications of these observations are discussed.

Retinal Degeneration and Alzheimer's Disease: An Evolving Link.

Age-related macular degeneration (AMD) and glaucoma are degenerative conditions of the retina and a significant cause of irreversible blindness in developed countries. Alzheimer's disease (AD), the most common dementia of the elderly, is often associated with AMD and glaucoma. The cardinal features of AD include extracellular accumulation of amyloid ß (Aß) and intracellular deposits of hyper-phosphorylated tau (p-tau). Neuroinflammation and brain iron dyshomeostasis accompany Aß and p-tau deposits and, together, lead to progressive neuronal death and dementia. The accumulation of Aß and iron in drusen, the hallmark of AMD, and Aß and p-tau in retinal ganglion cells (RGC), the main retinal cell type implicated in glaucoma, and accompanying inflammation suggest overlapping pathology. Visual abnormalities are prominent in AD and are believed to develop before cognitive decline. Some are caused by degeneration of the visual cortex, while others are due to RGC loss or AMD-associated retinal degeneration. Here, we review recent information on Aß, p-tau, chronic inflammation, and iron dyshomeostasis as common pathogenic mechanisms linking the three degenerative conditions, and iron chelation as a common therapeutic option for these disorders. Additionally discussed is the role of prion protein, infamous for prion disorders, in Aß-mediated toxicity and, paradoxically, in neuroprotection.

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer.

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5ß1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell-matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer.

Identification of new biomarkers for Acute Respiratory Distress Syndrome by expression-based genome-wide association study.

BACKGROUND: Accumulated to-date gene microarray data on Acute Respiratory Distress Syndrome (ARDS) in the Gene Expression Omnibus (GEO) represent a rich source for identifying new unsuspected targets and mechanisms of ARDS. The recently developed expression-based genome-wide association study (eGWAS) for analysis of GEO data was successfully used for analysis of gene expression of comparatively noncomplex adipose tissue, 75 % of which is represented by adipocytes. Although lung tissue is more heterogenic and does not possess a prevalent cell type for driving gene expression patterns, we hypothesized that eGWAS of ARDS samples will generate biologically meaningful results. METHODS: The eGWAS was conducted according to (Proc Natl Acad Sci U S A 109:7049-7054, 2012) and genes were ranked according to p values of chi-square test. RESULTS: The search of GEO retrieved 487 ARDS related entries. These entries were filtered for multiple qualitative and quantitative conditions and 219 samples were selected: mouse n sham/ARDS = 67/92, rat n = 13/13, human cells n = 11/11, canine n = 6/6 with the following ARDS model distributions: mechanical ventilation (MV)/cyclic stretch n = 11; endotoxin (LPS) treatment n = 8; MV + LPS n = 3; distant organ injury induced ARDS n = 3; chemically induced ARDS n = 2; Staphylococcus aureus induced ARDS n = 2; and one experiment each for radiation and shock induced ARDS. The eGWAS of this dataset identified 42 significant (Bonferroni threshold P < 1.55 × 10(-6)) genes. 66.6 % of these genes, were associated previously with lung injury and include the well known ARDS genes such as IL1R2 (P = 4.42 × 10(-19)), IL1ß (P = 3.38 × 10(-17)), PAI1 (P = 9.59 × 10(-14)), IL6 (P = 3.57 × 10(-12)), SOCS3 (P = 1.05 × 10(-10)), and THBS1 (P = 2.01 × 10(-9)). The remaining genes were new ARDS candidates. Expression of the most prominently upregulated genes, CLEC4E (P = 4.46 × 10(-14)) and CD300LF (P = 2.31 × 10(-16)), was confirmed by real time PCR. The former was also validated by in silico pathway analysis and the latter by Western blot analysis. CONCLUSIONS: Our first in the field application of eGWAS in ARDS and utilization of more than 120 publicly available microarray samples of ARDS not only justified applicability of eGWAS to complex lung tissue, but also discovered 14 new candidate genes which associated with ARDS. Detailed studies of these new candidates might lead to identification of unsuspected evolutionarily conserved mechanisms triggered by ARDS.

Induced Pluripotent Stem Cells in the Era of Precise Genome Editing.

Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.

Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production.

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 µM) and in vitro culture (IVC) medium (0, 0.1 µM). Supplementation of IVM medium with 0.1 µM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 µM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 µM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.

Remediation plan of nano/microplastic toxicity in food.

Microplastic pollution is causing a stir globally due to its persistent and ubiquitous nature. The scientific collaboration is diligently working on improved, effective, sustainable, and cleaner measures to control the nano/microplastic load in the environment especially wrecking the aquatic habitat. This chapter discusses the challenges encountered in nano/microplastic control and improved technologies like density separation, continuous flow centrifugation, oil extraction protocol, electrostatic separation to extract and quantify the same. Although it is still in the early stages of research, biobased control measures, like meal worms and microbes to degrade microplastics in the environment have been proven effective. Besides the control measures, practical alternatives to microplastics can be developed like core-shell powder, mineral powder, and biobased food packaging systems like edible films and coatings developed using various nanotechnological tools. Lastly, the existing and ideal stage of global regulations is compared, and key research areas are pinpointed. This holistic coverage would enable manufacturers and consumers to reconsider their production and purchase decisions for sustainable development goals.

Microbes-mediated sulphur cycling in soil: Impact on soil fertility, crop production and environmental sustainability.

Reduction in soil fertility and depletion of natural resources due to current intensive agricultural practices along with climate changes are the major constraints for crop productivity and global food security. Diverse microbial populations' inhabiting the soil and rhizosphere participate in biogeochemical cycling of nutrients and thereby, improve soil fertility and plant health, and reduce the adverse impact of synthetic fertilizers on the environment. Sulphur is 4th most common crucial macronutrient required by all organisms including plants, animals, humans and microorganisms. Effective strategies are required to enhance sulphur content in crops for minimizing adverse effects of sulphur deficiency on plants and humans. Various microorganisms are involved in sulphur cycling in soil through oxidation, reduction, mineralization, and immobilization, and volatalization processes of diverse sulphur compounds. Some microorganisms possess the unique ability to oxidize sulphur compounds into plant utilizable sulphate (SO42-) form. Considering the importance of sulphur as a nutrient for crops, many bacteria and fungi involved in sulphur cycling have been characterized from soil and rhizosphere. Some of these microbes have been found to positively affect plant growth and crop yield through multiple mechanisms including the enhanced mobilization of nutrients in soils (i.e., sulphate, phosphorus and nitrogen), production of growth-promoting hormones, inhibition of phytopathogens, protection against oxidative damage and mitigation of abiotic stresses. Application of these beneficial microbes as biofertilizers may reduce the conventional fertilizer application in soils. However, large-scale, well-designed, and long-term field trials are necessary to recommend the use of these microbes for increasing nutrient availability for growth and yield of crop plants. This review discusses the current knowledge regarding sulphur deficiency symptoms in plants, biogeochemical cycling of sulphur and inoculation effects of sulphur oxidizing microbes in improving plant biomass and crop yield in different crops.

Role of Oxidative Stress in Ocular Diseases: A Balancing Act.

Redox homeostasis is a delicate balancing act of maintaining appropriate levels of antioxidant defense mechanisms and reactive oxidizing oxygen and nitrogen species. Any disruption of this balance leads to oxidative stress, which is a key pathogenic factor in several ocular diseases. In this review, we present the current evidence for oxidative stress and mitochondrial dysfunction in conditions affecting both the anterior segment (e.g., dry eye disease, keratoconus, cataract) and posterior segment (age-related macular degeneration, proliferative vitreoretinopathy, diabetic retinopathy, glaucoma) of the human eye. We posit that further development of therapeutic interventions to promote pro-regenerative responses and maintenance of the redox balance may delay or prevent the progression of these major ocular pathologies. Continued efforts in this field will not only yield a better understanding of the molecular mechanisms underlying the pathogenesis of ocular diseases but also enable the identification of novel druggable redox targets and antioxidant therapies.

Mitochondria-targeted antioxidant MitoQ ameliorates ROS production and improves cell viability in cryopreserved buffalo fibroblasts.

Cryopreservation commonly decreases the cellular functionality and post-thaw viability of cells. Reactive oxygen species (ROS) generated during cryopreservation degrade mitochondrial activity and promote the release of cytochrome C which activates caspases required for apoptosis. Antioxidants have the potential to improve the recovery efficiency of cells by reducing ROS production and maintaining mitochondrial membrane potential (MMP). The present study was conducted to explore the role of MitoQ, a derivative of coenzyme Q10 on cryopreserved fibroblasts derived from buffalo skin. To achieve our goal, buffalo skin fibroblasts were treated with varying concentrations of MitoQ (0, 0.1, 0.5, 1, 2, and 10 µM) for 24, 48, and 72 h. The MMP, ROS generation, cell viability was measured by flow cytometry. Furthermore, expression of genes related to mitochondrial oxidative stress (NRF2, GPX, and SOD), apoptosis (BAK and caspase 3) and cell proliferation (AKT) were also assessed. The results showed that over a period of 72 h lower concentrations of MitoQ (0.1-0.5 µM) decrease the ROS production, improves MMP and cell viability whilst the high concentration of MitoQ (2-10 µM) increased the oxidative damage to the cells. Taken together, our study provide important insights into the novel role of MitoQ in cryopreserved buffalo skin fibroblasts. In conclusion, we demonstrated the dose-dependent functional role of MitoQ on cryopreserved fibroblasts for improving post-thaw cell viability and cellular function.

Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes.

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for the introduction of mutations using CRISPR-Cas9 electroporation of zygote (CRISPR-EP) in buffalo. The goal of the study was to determine the optimal conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced buffalo zygotes by electroporation. Electroporation was performed using different combinations of voltage, pulse and time, and we observed that the electroporation in buffalo zygote at 20 V/mm, 5 pulses, 3 msec at 10 h post insemination (hpi) resulted in increased membrane permeability and higher knockout efficiency without altering embryonic developmental potential. Using the above parameters, we targeted buffalo POU5F1 gene as a proof of concept and found no variations in embryonic developmental competence at cleavage or blastocyst formation rate between control, POU5F1-KO, and electroporated control (EC) embryos. To elucidate the effect of POU5F1-KO on other pluripotent genes, we determined the relative expression of SOX2, NANOG, and GATA2 in the control (POU5F1 intact) and POU5F1-KO-confirmed blastocyst. POU5F1-KO significantly (p ≤ 0.05) altered the expression of SOX2, NANOG, and GATA2 in blastocyst stage embryos. In conclusion, we standardized an easy and straightforward protocol CRISPR-EP method that could be served as a useful method for studying the functional genomics of buffalo embryos.

MS-ResNet: disease-specific survival prediction using longitudinal CT images and clinical data.

PURPOSE: Medical imaging data of lung cancer in different stages contain a large amount of time information related to its evolution (emergence, development, or extinction). We try to explore the evolution process of lung images in time dimension to improve the prediction of lung cancer survival by using longitudinal CT images and clinical data jointly. METHODS: In this paper, we propose an innovative multi-branch spatiotemporal residual network (MS-ResNet) for disease-specific survival (DSS) prediction by integrating the longitudinal computed tomography (CT) images at different times and clinical data. Specifically, we first extract the deep features from the multi-period CT images by an improved residual network. Then, the feature selection algorithm is used to select the most relevant feature subset from the clinical data. Finally, we integrate the deep features and feature subsets to take full advantage of the complementarity between the two types of data to generate the final prediction results. RESULTS: The experimental results demonstrate that our MS-ResNet model is superior to other methods, achieving a promising 86.78% accuracy in the classification of short-survivor, med-survivor, and long-survivor. CONCLUSION: In computer-aided prognostic analysis of cancer, the time dimension features of the course of disease and the integration of patient clinical data and CT data can effectively improve the prediction accuracy.

A multitask dual-stream attention network for the identification of KRAS mutation in colorectal cancer.

PURPOSE: It is of great significance to accurately identify the KRAS gene mutation status for patients in tumor prognosis and personalized treatment. Although the computer-aided diagnosis system based on deep learning has gotten all-round development, its performance still cannot meet the current clinical application requirements due to the inherent limitations of small-scale medical image data set and inaccurate lesion feature extraction. Therefore, our aim is to propose a deep learning model based on T2 MRI of colorectal cancer (CRC) patients to identify whether KRAS gene is mutated. METHODS: In this research, a multitask attentive model is proposed to identify KRAS gene mutations in patients, which is mainly composed of a segmentation subnetwork and an identification subnetwork. Specifically, at first, the features extracted by the encoder of segmentation model are used as guidance information to guide the two attention modules in the identification network for precise activation of the lesion area. Then the original image of the lesion and the segmentation result are concatenated for feature extraction. Finally, features extracted from the second step are combined with features activated by the attention modules to identify the gene mutation status. In this process, we introduce the interlayer loss function to encourage the similarity of the two subnetwork parameters and ensure that the key features are fully extracted to alleviate the overfitting problem caused by small data set to some extent. RESULTS: The proposed identification model is benchmarked primarily using 15-fold cross validation. Three hundred and eighty-two images from 36 clinical cases were used to test the model. For the identification of KRAS mutation status, the average accuracy is 89.95 ± 1.23%, the average sensitivity is 89.29 ± 1.79%, the average specificity is 90.53 ± 2.45%, and the average area under the curve (AUC) is 95.73 ± 0.52%. For segmentation of lesions, the average dice is 88.11 ± 0.86%. CONCLUSIONS: We developed a novel deep learning-based model to identify the KRAS status in CRC. We demonstrated the excellent properties of the proposed identification through comparison with ground truth gene mutation status of 36 clinical cases. And all these results show that the novel method has great potential for clinical application.