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SARS-CoV-2's global spread has instigated a critical health and economic emergency, impacting countless individuals. Understanding the virus's phosphorylation sites is vital to unravel the molecular intricacies of the infection and subsequent changes in host cellular processes. Several computational methods have been proposed to identify phosphorylation sites, typically focusing on specific residue (S/T) or Y phosphorylation sites. Unfortunately, current predictive tools perform best on these specific residues and may not extend their efficacy to other residues, emphasizing the urgent need for enhanced methodologies. In this study, we developed a novel predictor that integrated all the residues (STY) phosphorylation sites information. We extracted ten different feature descriptors, primarily derived from composition, evolutionary, and position-specific information, and assessed their discriminative power through five classifiers. Our results indicated that Light Gradient Boosting (LGB) showed superior performance, and five descriptors displayed excellent discriminative capabilities. Subsequently, we identified the top two integrated features have high discriminative capability and trained with LGB to develop the final prediction model, LGB-IPs. The proposed approach shows an excellent performance on 10-fold cross-validation with an ACC, MCC, and AUC values of 0.831, 0.662, 0.907, respectively. Notably, these performances are replicated in the independent evaluation. Consequently, our approach may provide valuable insights into the phosphorylation mechanisms in SARS-CoV-2 infection for biomedical researchers.
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COVID-19 , Biología Computacional , SARS-CoV-2 , Fosforilación , SARS-CoV-2/metabolismo , Humanos , COVID-19/virología , COVID-19/metabolismo , Biología Computacional/métodosRESUMEN
Several coumarin-containing substitute nitrogen heterocycles have recently received considerable importance due to their diverse pharmacological properties. One-pot and rapid synthesis of coumarin derivatives was achieved via reactions of acetyl-coumarin with p-chloro-benzaldehyde and malononitrile to provide compound 2-containing cyano-amine using conventional heating. Compound 2 was condensed with different carbon electrophiles triethyl orthoformate, phenyl isocyanate, carbon disulfide, benzoyl chloride, and acetyl chloride that afforded the corresponding chromene derivatives 3-17. All the newly synthesized compounds were characterized by elemental and spectroscopic evidences. All of the synthesized compounds were tested for antimicrobial activity against S. Pneumoniae, S. Epidermidis, S. Aureus, and E. coli as Gram + ve Bacteria, K. Pneumoniae, S. Paratyphi as Gram -ve Bacteria, P. Italicum, A. Fumigatus representative for Fungi. The preliminary screening results showed that most of the compounds had moderate to high activity against all tested organisms. The most potent four compounds were subjected to further investigation against E. Coli DNA gyrase and topoisomerase IV inhibitory activity, and the results showed that all of these derivatives inhibit DNA gyrase and thus cell division. Also, in silico studies were done for the most active compounds which showed good results.
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Antiinfecciosos , Inhibidores de Topoisomerasa II , Antibacterianos/química , Antiinfecciosos/farmacología , Cumarinas/química , Girasa de ADN/química , Escherichia coli , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Staphylococcus aureus , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Stimuli-responsive emulsifiers have emerged as a class of smart agents that can permit regulated stabilization and destabilization of emulsions, which is essential for food, cosmetic, pharmaceutical, and petroleum industries. Here, we report the synthesis of novel "smart" hydroxyapatite (HaP) magnetic nanoparticles and their corresponding stimuli-responsive Pickering emulsions and explore their movement under confined spaces using a microfluidic platform. Pickering emulsions prepared with our magnetic stearic acid-functionalized Fe2O3@HaP nanoparticles exhibited pronounced pH-responsive behavior. We observed that the diameter of emulsion droplets decreases with an increase in pH. Swift demulsification was achieved by lowering the pH, whereas the reformation of emulsions was achieved by increasing the pH; this emulsification-demulsification cycling was successful for at least ten cycles. We used a microfluidic platform to test the stability of the emulsions under flowing conditions and their response to a magnetic field. We observed that the emulsion stability was diminished and droplet coalescence was enhanced by the application of the magnetic field. The smart nanoparticles we developed and their HaP-based emulsions present promising materials for pharmaceutical and petroleum industries, where responsive emulsions with controlled stabilities are required.
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Mesenchymal stem cells (MSCs) are emerging as a potential therapeutic intervention for brain injury due to their neuroprotective effects and safe profile. However, the homing ability of MSCs to injury sites still needs to be improved. Fibroblast Growth Factor 21 (FGF21) was recently reported to enhance cells migration in different cells type. In this study, we investigated whether MSCs that overexpressing FGF21 (MSC-FGF21) could exhibit enhanced homing efficacy in brain injury. We used novel Molday IONEverGreen™ (MIEG) as cell labeling probe that enables a non-invasive, high-sensitive and real-time MRI tracking. Using a mouse model of traumatic brain injury (TBI), MIEG labeled MSCs were transplanted into the contralateral lateral ventricle followed by real-time MRI tracking. FGF21 retained MSC abilities of proliferation and morphology. MSC-FGF21 showed significantly greater migration in transwell assay compared to control MSC. MIEG labeling showed no effects on MSCs' viability, proliferation and differentiation. Magnetic resonance imaging (MRI) revealed that FGF21 significantly enhances the homing of MSC toward injury site. Histological analysis further confirmed the MRI findings. Taken together, these results show that FGF21 overexpression and MIEG labeling of MSC enhances their homing abilities and enables non-invasive real time tracking of the transplanted cells, provides a promising approach for MSC based therapy and tracking in TBI.
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Lesiones Traumáticas del Encéfalo/terapia , Movimiento Celular , Factores de Crecimiento de Fibroblastos/genética , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/fisiología , Animales , Células Cultivadas , Factores de Crecimiento de Fibroblastos/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are commonly used as the first-line treatment for advanced NSCLC; however, the efficacy of drug delivery remains unknown. Hence, we successfully developed erlotinib-conjugated iron oxide nanoparticles (FeDC-E NPs) as theranostic probe that can potentially provide a new avenue for monitoring drug delivering through noninvasive magnetic resonance imaging. MRI ΔR2* relaxivity measurements offer an opportunity to quantitatively evaluate the uptake of FeDC-E NPs at cellular and tumoral levels. Additionally, NF-κB reporter gene system provides NF-κB activation status monitoring to validate the therapeutic efficiency of FeDC-E NPs. FeDC-E NPs not only inhibit the tumor growth and NF-κB-modulated antiapoptotic mechanism but also trigger extrinsic and intrinsic apoptotic pathways. Taken together, dual functional FeDC-E NPs offer diagnostic and therapeutic benefits against lung cancers, indicating that our presented probe could be applied in clinical.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Sistemas de Liberación de Medicamentos , Clorhidrato de Erlotinib/farmacología , Neoplasias Pulmonares/patología , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/administración & dosificación , FN-kappa B/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Clorhidrato de Erlotinib/administración & dosificación , Clorhidrato de Erlotinib/química , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Procesamiento de Imagen Asistido por Computador , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Nanopartículas de Magnetita/química , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/genética , Fantasmas de Imagen , Células Tumorales CultivadasRESUMEN
BACKGROUND: Calotropis procera is a wild plant species in the family Apocynaceae that is able to grow in harsh, arid and heat stressed conditions. Understanding how this highly adapted plant persists in harsh environments should inform future efforts to improve the hardiness of crop and forage plant species. To study the plant response to droµght and osmotic stress, we treated plants with polyethylene glycol and NaCl and carried out transcriptomic and metabolomics measurements across a time-course of five days. RESULTS: We identified a highly dynamic transcriptional response across the time-course including dramatic changes in inositol signaling, stress response genes and cytokinins. The resulting metabolome changes also involved sharp increases of myo-inositol, a key signaling molecule and elevated amino acid metabolites at later times. CONCLUSIONS: The data generated here provide a first glimpse at the expressed genome of C. procera, a plant that is exceptionally well adapted to arid environments. We demonstrate, through transcriptome and metabolome analysis that myo-inositol signaling is strongly induced in response to drought and salt stress and that there is elevation of amino acid concentrations after prolonged osmotic stress. This work should lay the foundations of future studies in adaptation to arid environments.
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Calotropis/metabolismo , Calotropis/genética , Genes de Plantas , Metaboloma , Estrés Oxidativo , Estrés Fisiológico , TranscriptomaRESUMEN
BACKGROUND: Targeted superparamagnetic iron oxide (SPIO) nanoparticles have emerged as a promising biomarker detection tool for molecular magnetic resonance (MR) image diagnosis. To identify patients who could benefit from Epidermal growth factor receptor (EGFR)-targeted therapies, we introduce lipid-encapsulated SPIO nanoparticles and hypothesized that anti-EGFR antibody cetuximab conjugated of such nanoparticles can be used to identify EGFR-positive glioblastomas in non-invasive T2 MR image assays. The newly introduced lipid-coated SPIOs, which imitate biological cell surface and thus inherited innate nonfouling property, were utilized to reduce nonspecific binding to off-targeted cells and prevent agglomeration that commonly occurs in nanoparticles. RESULTS: The synthesized targeted EGFR-antibody-conjugated SPIO (EGFR-SPIO) nanoparticles were characterized using dynamic light scattering, zeta potential assays, gel electrophoresis mobility shift assays, transmission electron microscopy (TEM) images, and cell line affinity assays, and the results showed that the conjugation was successful. The targeting efficiency of the synthesized EGFR-SPIO nanoparticles was confirmed through Prussian blue staining and TEM images by using glioblastoma cell lines with high or low EGFR expression levels. The EGFR-SPIO nanoparticles preferentially targeted U-251 cells, which have high EGFR expression, and were internalized by cells in a prolonged incubation condition. Moreover, the T2 MR relaxation time of EGFR-SPIO nanoparticles could be used for successfully identifying glioblastoma cells with elevated EGFR expression in vitro and distinguishing U-251 cells from U-87MG cells, which have low EFGR expression. CONCLUSION: These findings reveal that the lipid-encapsulated EGFR-SPIO nanoparticles can specifically target cells with elevated EGFR expression in the three tested human glioblastoma cell lines. The results of this study can be used for noninvasive molecular MR image diagnosis in the future.
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Antineoplásicos Inmunológicos/farmacología , Biomarcadores de Tumor/metabolismo , Cetuximab/farmacología , Receptores ErbB/metabolismo , Inmunoconjugados/farmacocinética , Nanopartículas de Magnetita/administración & dosificación , Neuroglía/efectos de los fármacos , Antineoplásicos Inmunológicos/química , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Cetuximab/química , Composición de Medicamentos , Receptores ErbB/genética , Expresión Génica , Humanos , Inmunoconjugados/química , Ligandos , Lípidos/química , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Neuroglía/metabolismo , Neuroglía/patología , Unión ProteicaRESUMEN
BACKGROUND: Climate change is predicted to be a serious threat to agriculture due to the need for crops to be able to tolerate increased heat stress. Desert plants have already adapted to high levels of heat stress so they make excellent systems for identifying genes involved in thermotolerance. Rhazya stricta is an evergreen shrub that is native to extremely hot regions across Western and South Asia, making it an excellent system for examining plant responses to heat stress. Transcriptomes of apical and mature leaves of R. stricta were analyzed at different temperatures during several time points of the day to detect heat response mechanisms that might confer thermotolerance and protection of the plant photosynthetic apparatus. RESULTS: Biological pathways that were crosstalking during the day involved the biosynthesis of several heat stress-related compounds, including soluble sugars, polyols, secondary metabolites, phenolics and methionine. Highly downregulated leaf transcripts at the hottest time of the day (40-42.4 °C) included genes encoding cyclin, cytochrome p450/secologanin synthase and U-box containing proteins, while upregulated, abundant transcripts included genes encoding heat shock proteins (HSPs), chaperones, UDP-glycosyltransferase, aquaporins and protein transparent testa 12. The upregulation of transcripts encoding HSPs, chaperones and UDP-glucosyltransferase and downregulation of transcripts encoding U-box containing proteins likely contributed to thermotolerance in R. stricta leaf by correcting protein folding and preventing protein degradation. Transcription factors that may regulate expression of genes encoding HSPs and chaperones under heat stress included HSFA2 to 4, AP2-EREBP and WRKY27. CONCLUSION: This study contributed new insights into the regulatory mechanisms of thermotolerance in the wild plant species R. stricta, an arid land, perennial evergreen shrub common in the Arabian Peninsula and Indian subcontinent. Enzymes from several pathways are interacting in the biosynthesis of soluble sugars, polyols, secondary metabolites, phenolics and methionine and are the primary contributors to thermotolerance in this species.
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Apocynaceae/genética , Proteínas de Plantas/genética , Transcripción Genética , Apocynaceae/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Calor , Proteínas de Plantas/fisiología , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
BACKGROUND: The ultimate goal of this work was to detect the role of transcription factors (TFs) concordantly expressed with genes related to programmed cell death (PCD) during PCD and salt stress. This work was based on the hypothesis that TFs and their driven genes likely co-express under different stimuli. The conserved superfamily ethylene responsive factor (AP2/ERF) draw attention of the present study as it participates in the response to biotic and abiotic stimuli as well as to program cell death (PCD). RESULTS: RNA-Seq analysis was done for tobacco (N. benthamiana) leaves exposed to oxalic acid (OA) at 20 mM for 0, 2, 6, 12 and 24 h to induce PCD. Genes up-regulated after 2 h of OA treatment with known function during PCD were utilized as landmarks to select TFs with concordant expression. Knockdown mutants of these TFs were generated in tobacco via virus induced gene silencing (VIGS) in order to detect their roles during PCD. Based on the results of PCD assay, knockout (KO) T-DNA insertion mutants of Arabidopsis as well as over-expression lines of two selected TFs, namely ERF109 and TFIID5, analogs to those in tobacco, were tested under salt stress (0, 100, 150 and 200 mM NaCl). CONCLUSIONS: Results of knockdown mutant tobacco cells confirmed the influence of these two TFs during PCD. Knockout insertion mutants and over-expression lines indicated the role of ERF109 in conferring salt tolerance in Arabidopsis.
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Apoptosis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Etilenos/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Represoras/metabolismo , Cloruro de Sodio/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Ácido Oxálico/metabolismo , Proteínas de Plantas/genética , Proteínas Represoras/genética , Tolerancia a la Sal , Nicotiana/citología , Nicotiana/genética , Factores de Transcripción/genéticaRESUMEN
PURPOSE: To evaluate the efficacy of intraoral drainage of isolated submandibular space abscess as a minimally invasive surgical technique compared to the standard trans-cervical approach. PATIENTS AND METHODS: This prospective study included 40 subjects with isolated submandibular space abscesses. They were randomly divided into 2 equal groups: trans-cervical surgical drainage (group A) and intra-oral surgical drainage (group B). The included data were demographics, repeated surgery requirement, postsurgical hospitalization duration, formation of scar, and complications. RESULTS: Intraoral drainage (Group B) reduced the mean operative time by 15.25 min (P < 0.001) compared with trans-cervical incision (Group A). No considerable difference was found between the 2 groups in regarding hospitalization postoperatively. No weakness in marginal mandibular nerve was found in both groups. Three patients only have a cervical scar in a group (B) who required external drainage due to recollection. No recurrence was detected in a group (A). CONCLUSION: The current study demonstrated that isolated submandibular abscesses can be successfully managed with an intraoral drainage modality, and it is a better option than the trans-cervical approach regarding better cosmetic outcome and shorter operative time.
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Background: Anterior lamellar keratoplasty (ALK) is a less invasive procedure than PK, and thus avoids many of the intraocular complications associated with PK. DALK can be performed using several different techniques, with either a manual dissection, a keratome or femtosecond-laser assisted dissection, or with a big bubble technique. To analyse the outcomes and compare the results of three deep anterior lamellar keratoplasty (DALK) techniques. Methods: This study included 105 DALK cases performed at Queen Victoria Hospital, East Grinstead, UK, in the period between January 2016 and May 2022. Cases were classified into four groups based on technique: BB-DALK, manual DALK, FS-DALK and 'converted to PK group'. Results: There was significant improvement in VA and Kmax compared to the preoperative values in all groups. There was no significant difference detected in VA and Kmax between all groups. Conclusions: Performing DALK surgery with any suitable technique (manual, big-bubble or femtosecond-assisted) is effective and causes significant improvements in VA and Kmax, even in cases where a conversion to penetrating keratoplasty is required. However, every technique has its pros and cons and should be tailored according to surgeon preference and individual case pathology.
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Calotropis procera (C. procera) was evaluated as a pharmaceutically useful plant and for its therapeutic effects in the most significant studies. Uzarigenin and Calotropagenin are significant components of this plant that have pharmacological effects on certain systems, including the digestive, immunological, and focal, and peripheral sensory systems. In this study, pathway genes are extracted from high throughput data acc.no. SRR1554320. Seven critical enzymes are involved in studying the effects of sunlight on the formation of Uzaragenin and Calotropagenin in C. procera before and after irrigation. Molecular identification and NCBI submission of six enzyme genes were successful; HSD (acc.no. OQ091761) for 3ß-hydroxystroid dehydrogenase, OR (acc.no. OQ091762) for 5beta-pregnan oxidoreductase, MO (acc.no. OQ091763) for Pregnan monooxygenase, HOX (acc.no. OQ091764) for Steroid hydroxylase, MAT (acc.no. OQ091765) for Melonyletransferase, UHOX (acc.no. OQ091766) for Uzarigenin hydroxylase. During dawn after irrigation, the Uzargenin pathway showed the highest activity, however midday after irrigation was the lowest. The most period that showed high activity for the Uzargenin pathway was dawn after irrigation, however, midday after irrigation was the lowest. This data is confirmed by chromatography analysis (UPLC) to calculate the accumulation of Uzarigenin and Calotropagenin in different periods.
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Genomic studies not only help researcher not only to identify genomic features in organisms, but also facilitate understanding of evolutionary relationships. Species in the Withania genus have medicinal benefits, and one of them is Withania frutescens, which is used to treat various diseases. This report investigates the nucleotides and genic features of chloroplast genome of Withania frutescens and trying to clarify the evolutionary relationship with Withania sp and family Solanaceae. We found that the total size of Withania frutescens chloroplast genome was 153.771 kb (the smallest chloroplast genome in genus Withania). A large single-copy region (91.285 kb), a small single-copy region (18.373 kb) form the genomic region, and are distinct from each other by a large inverted repeat (22.056 kb). 137 chloroplast genes are found including 4 rRNAs, 38 tRNAs and 83 protein-coding genes. The Withania frutescens chloroplast genome as well as four closest relatives was compared for features such as structure, nucleotide composition, simple sequence repeats (SSRs) and codon bias. Compared to other Withania species, Withania frutescens has unique characteristics. It has the smallest chloroplast genome of any Withania species, isoleucine is the major amino acid, and tryptophan is the minor, In addition, there are no ycf3 and ycf4 genes, fourth, there are only fifteen replicative genes, while in most other species there are more. Using fast minimum evolution and neighbor joining, we have reconstructed the trees to confirm the relationship with other Solanacaea species. The Withania frutescens chloroplast genome is submitted under accession no. ON153173.
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Numerous studies have shown that stress in plant cells and organelles with transport electron chains is related to RNA editing. The ATP synthase complex present in mitochondria plays a crucial role in cellular respiration and consists of several subunits. Among them is the b subunit, which is encoded by the mitochondrial atp4 gene. Computing-based analysis of the effects of RNA editing of the Withania somnifera atp4 gene in mitochondria leading to alterations in the b subunit of ATP synthase. Using the CLC Genomic Workbench 3, RNA editing analysis between the control and salt stress conditions was not significantly different. Depending on RNA editing, the tertiary structure model revealed a change in the states of the b subunit, reflecting differences in the central stalk and F1-catalytic domain. The study found that polar edits in the N-terminus of the b subunit allow for efficient H + ion selectivity and introduce a new coiled-coil alpha-helical structure that may help stabilize the complex. The most noteworthy finding of this study was the strong impact of these editing events on the tertiary structure of the b subunit, which has the potential to affect the ATPase activity and indicate that the editing in this subunit aimed to restore the original active protein and not as a response to salt stress.
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Objective: Alopecia areata (AA) is a common form of potentially reversible non-scarring hair disorder characterized by limited patchy hair loss (alopecia areata), loss of all scalp hair (alopecia totalis), or all body hair (alopecia universalis). Several lines of treatment have been used with variable outcomes. We aimed to compare the efficacy of intralesional pentoxifylline (PTX) and triamcinolone acetonide (TRA) injection in the treatment of alopecia areata. Methods: Our study included 60 patients with localized AA recruited from the Dermatology Outpatient Clinics of Al-Azhar University Hospitals. Patients were divided into two groups of alopecia areata patches; Group A who received intralesional TRA injections while Group B received intralesional PTX. Results: The study showed that both modalities are effective in treating AA and each modality has its own advantages. According to the response, patients were grouped into three categories: partial response (0-33% terminal hair regrowth), moderate response (33-66% terminal hair regrowth), and high response (66-100% terminal hair regrowth). The high response after use of the PTX was found in 50 percent of patients. The high response was observed in 46.6 percent of patients treated with TRA. Limitations: Small sample size and short follow-up period. Conclusion: This study showed that intralesional injection of PTX seems to be effective and safe treatment for localized AA and could be used as a good alternative to triamcinolone acetonide.
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BACKGROUND: Multifocal (MFBC)/multicentric (MCBC) breast cancer is being more recognized due to the improved imaging modalities and the greater orientation with this form of breast cancer, however, optimal surgical treatment, still poses a challenge. The standard surgical treatment is mastectomy, however, breast-conserving surgeries (BCS) may be appropriate in certain situations. METHODS: A total of 464 cases of MF/MCBC out of 4798 cases of breast cancer were retrospectively analyzed from the database of the Oncology Center, Mansoura University (OCMU), between January 2008 and December 2019. RESULTS: Radiologic involvement of multiple quadrants was reported in 27.9% by ultrasonography, 19% by mammography, and 59.1% by magnetic resonance imaging. BCS was performed in 32 cases (6.9%) while 432 cases underwent a mastectomy. Postoperative pathology revealed infiltration of other quadrants grossly in 23.5%, and under the microscope in 63.6% of the examined cases. Mean disease-free and overall survival were 95.5 and 164.6 months, respectively. When compared with MFBC, MCBC showed higher pathologic tumor size (p < 0.001), higher stages (p < 0.001), higher recurrence rates (p = 0.006), and lower DFS (P = 0.009) but with similar OS (P = 0.8). CONCLUSION: Mastectomy is still the primary treatment option for MCBC with higher recurrence rates compared with MFBC. However, BCS for properly selected MFBC is considered oncologically safe, following the same rules of breast conservation for unifocal disease.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/cirugía , Mastectomía , Estudios Retrospectivos , Egipto/epidemiología , Mama/patología , Mastectomía Segmentaria/métodosRESUMEN
Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.
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Ingeniería de Proteínas/métodos , Transactivadores/genética , Roturas del ADN de Doble Cadena , División del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II , Endonucleasas/genética , Endonucleasas/metabolismo , Proteínas de Homeodominio/genética , Hojas de la Planta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Reproducibilidad de los Resultados , Nicotiana/genéticaRESUMEN
The major reports on Calotropis procera (C. procera) indicated the importance of this plant as a resource of pharmaceutically active ingredients as well as its medical advantages. ß-amyrin (BA) is a significant substance in this plant and has a pharmacological effects in some frameworks, like focal and fringe sensory system, digestive and immune systems. In this study, the impact of sunlight before and after irrigation on the BA production in C. procera is studied its pathway with involved eight key enzymes. The eight enzymes' genes were characterized and successfully submitted to NCBI; AAS (acc.no. KU997645) for α-amyrin synthase, BAS (acc.no. MW976955) for ß-amyrin synthase, SE (acc.no. MW976956) for squalene epoxidase, SS (acc.no. MW976957) for squalene synthase, GPPS, (acc.no. MW976958) for geranyl pyrophosphate synthase, FPPS (acc.no. MW976959) for farnasyl pyrophosphate synthase, CAS1, (acc.no. MZ00598) for cycloartenol synthase1 and LS (acc.no. MZ005982) for lupeol synthase. qRT-PCR analysis revealed high expression levels of GPPS, FPPS, SS, SE, and BAS genes at all times specially midday. Otherwise, CAS1, LS and BAS expression levels were very low at all daylight periods. The UPLC ß-amyrin data are in accordance with qRT-PCR results. This indicates that triterpenes biosynthetic pathway in C. procera is going to ß-amyrin accumulation with the highest level at midday.
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Hydroxyapatite nanoparticles (HAn) have been produced as biomaterial from biowaste, especially snail shells (Atactodea glabrata). It is critical to recycle the waste product in a biomedical application to overcome antibiotic resistance as well as biocompatibility with normal tissues. Moreover, EDX, TEM, and FT-IR analyses have been used to characterize snail shells and HAn. The particle size of HAn is about 15.22 nm. Furthermore, higher inhibitory activity was observed from HAn than the reference compounds against all tested organisms. The synthesized HAn has shown the lowest MIC values of about 7.8, 0.97, 3.9, 0.97, and 25 µg/mL for S. aureus, B. subtilis, K. pneumonia, C. albicans, and E. coli, respectively. In addition, the HAn displayed potent antibiofilm against S. aureus and B. subtilis. According to the MTT, snail shell and HAn had a minor influence on the viability of HFS-4 cells. Consequently, it could be concluded that some components of waste, such as snail shells, have economic value and can be recycled as a source of CaO to produce HAn, which is a promising candidate material for biomedical applications.
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The COVID-19 pandemic has strained healthcare systems. Sensitive, specific, and timely COVID-19 diagnosis is crucial for effective medical intervention and transmission control. RT-PCR is the most sensitive/specific, but requires costly equipment and trained personnel in centralized laboratories, which are inaccessible to resource-limited areas. Antigen rapid tests enable point-of-care (POC) detection but are significantly less sensitive/specific. CRISPR-Cas systems are compatible with isothermal amplification and dipstick readout, enabling sensitive/specific on-site testing. However, improvements in sensitivity and workflow complexity are needed to spur clinical adoption. We outline the mechanisms/strategies of major CRISPR-Cas systems, evaluate their on-site diagnostic capabilities, and discuss future research directions.