TRAIL mediated apoptosis ruling and anticancer trigger by fine-tuned nano spheres of Fagonia cretica methanolic extracts as novel cancer regime.

Fagonia cretica L. is a tropical plant of family Zygophyllaceae with wide range of medicinally important secondary metabolites. The low cellular uptake of the polar compounds in the extract of the plant limits its biological application. In present study efficacy of F. cretica modified bioactive nano-formulations for in vitro modulation of TRAIL mediated extrinsic apoptotic pathway as cancer therapy was investigated. F. cretica methanolic extracts were formulated at nano-scale for green synthesis of silver nanoparticles, albumin conjugation and liposomes encapsulation to enhance targeted bioactivity against cancer. Physical characterization of the synthesized nanoparticles was done by SEM, EDX and Zeta potential analyzer. In vitro cell viability assay MTT was done for MCF-7, Hep-2, HUH-7 and HCEC cell lines. Relative expression variation of the apoptotic pathway-associated genes was done by qRT-PCR. SEM revealed spherical shape of 56.62 ± 8.04, 143 ± 11.54 and 83.36 ± 38.73 nm size and zeta potential - 18.6, - 15.5 and - 18.3 mV for liposomes, silver and albumin nanoparticles. Silver nanoparticles showed highest anticancer activity in vitro than albumin and liposomes nanoparticles with IC50 0.101 ± 0.004, 0.177 ± 0.03 and 0.434 ± 0.022 mg/mL in MCF-7, Hep-2 and HUH-7 respectively. F. cretica albumin and silver nanoparticles upregulated the in vitro TRAIL, DR4, DR5 and FADD gene expression at statistically significant levels in Hep-2 cell lines. Nano-formulations of F. cretica proved therapeutically important biomolecules in vitro. The hypothesized modulation of extrinsic apoptosis pathway genes through the plant nanoparticles proved novel medicinal options for effective treatment of cancer and enhancing the bioavailability of the active plant metabolites.

Corrigendum: CRISPR/Cas9-mediated targeting of susceptibility factor eIF4E-enhanced resistance against Potato virus Y.

[This corrects the article DOI: 10.3389/fgene.2022.922019.].

CRISPR/Cas9-Mediated Targeting of Susceptibility Factor eIF4E-Enhanced Resistance Against Potato Virus Y.

Potato (Solanum tuberosum L.) is an important staple food around the world, and potato virus Y (PVY) is a major constraint for potato production. The VPg protein of PVY interacts with the translation initiation factor eIF4E of the host that works as a susceptibility factor during infection. The interaction between eIF4E and VPg was disrupted by CRISPR/Cas9. The homozygous conserved region of eIF4E of the potato variety "Kruda" was mutated by CRISPR/Cas9. Tracking of insertion, deletion, and conversion events was performed by Sanger sequencing with ∼15% editing efficiency. Truncated and mutated eIF4E proteins were unable to interact with VPg, and the virus was not able to exploit the host machinery for replication and systemic spreading. Mutated eIF4E lines showed enhanced resistance to PVYO strain. DAS-ELISA and RT-PCR were used for validation of the observed resistance. PVY resistance in tetraploid lines via CRISPR/Cas9 provides a route to develop novel resistant potato cultivars.

Broad-spectrum resistance against multiple PVY-strains by CRSIPR/Cas13 system in Solanum tuberosum crop.

Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.

Natural plant extracts mediated expression regulation of TGF-ß receptors and SMAD genes in human cancer cell lines.

BACKGROUND: Transforming growth factor beta (TGF-ß) superfamily has key role in cell proliferation which leads to tumor promoting activities at metastatic stage of cancer. Inhibition of transforming growth factor beta receptor (TGFßR) signaling pathway can provide better therapeutic strategy to control cancer. Natural products are best known for their safety, less toxic nature, antioxidant characteristics making them a promising candidate to inhibit TGFßR signaling pathway. METHODS AND RESULTS: Crude methanolic extracts (CMEs) of 16 selected plants were prepared by using maceration method and subjected to phytochemical assays for identification of major phytometabolites particularly cancer chemopreventive antioxidant constituents. Total flavonoid content of all plants CME was > 0.6 mg/ml exhibiting the Cichorium intybus contains comparatively highest amount of total flavonoid content (0.53 mg/ml). Scanvenging activity of all plants was determined having IC50 ranges between 2 and 88 (µg/ml) while Moringa oleifera revealed the maximum scavenging activity (IC50 2.03 µg/ml). Comparative cytotoxicity of plant extracts was evaluated in HUH and MCF-7 cell lines using 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) colorimetric assay. The nine active plant extracts i.e. Fagonia cretica, Argemone Mexicana, Rubus fruticosus, M. oleifera, Punica granatum, Cichorium intybus, Xanthium strumarium, Carissa opaca, Cyperus rotundus were identified based on their high antiproliferative activity > 50% against cancer cell lines and subjected to relative expression studies. Modulation of TGFß signaling molecules (i.e.TGFßR1, 2 & 3, SMAD3, SMAD5) and ubiquitous proteins i.e. SMURF1 and SMURF2 genetic expression by potent extracts was determined by RT-PCR using GAPDH (housekeeping gene) as gene of reference. CONCLUSIONS: This present study revealed that CME of Fagonia cretica and Argemone mexicana significantly inhibit TGF beta mediated signaling cascade by downregulating the gene expression fold change > 1 of TGFßR 1, 2 & 3 and receptor associated complex protein SMAD3 as compared to control.

Study of Guillain-Barre syndrome etiology in Pakistani patients.

OBJECTIVE: To examine clinical features, biochemical markers, demographic features, antecedent infections, frequency and treatment strategies related to Guillain-Barré syndrome. METHODS: The case-control study was conducted at the Pakistan Institute of Medical Sciences, Islamabad, Pakistan, and the District Headquarters Hospital, Rawalpindi, Pakistan, from 2018 to 2020, and comprised Guillain-Barré syndrome patients in group A and healthy controls in group B. The patients were diagnosed on the basis of clinical presentation, nerve conduction study, electromyography, cerebrospinal fluid analysis and biochemical profile. Data was analysed using SPSS 23. RESULTS: Of the 167 subjects, 90(54%) were in group A and 77(46%) were in group B. The mean age of group A was 40.20±14.90 years, while there were 61(67.7%) males and 29(32.2%) females compared to 50 (64.93%) males and 27 (35.06%) females with mean age 38.40±12.34 years in group B. Acute inflammatory demyelinating polyneuropathy was the most common electrophysiological variant of Guillain-Barré syndrome 41(46%). There was significant difference in mean interleukin-17 levels between group A 23.12±3.41 pg/ml and group B 8.82±2.49 (p<0.05). Gastrointestinal infection was the most common preceding infection 51(56.66%). The mean cerebrospinal fluid protein was 100.83±51.32g/dl and albumiocytologic dissociation was found in all the four variants (p= 0.005). CONCLUSION: Guillain-Barré syndrome affected patients regardless of age, while males were more affected than females. Majority of the patients had an antecedent infection before disease onset. Increased levels of interleukin-17 showed involvement of autoimmunity. Albuminocytologic dissociation differentiated it from poliomyelitis.

Biosynthesis and characterization of titanium dioxide nanoparticles and its effects along with calcium phosphate on physicochemical attributes of wheat under drought stress.

Drought stress is reducing the production of crops globally. This research was designed to evaluate the role of titanium dioxide (TiO2 NPs) nanoparticles and calcium phosphate on wheat facing drought stress. TiO2 NPs were prepared by green synthesis and their characterization (by UV-visible spectroscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX)) was also done. The results showed that TiO2 NPs worked efficiently and improved plant growth under drought. However, the best results were obtained from combined applications of 40 ppm TiO2 NPs and 40 ppm calcium phosphate on plants. They increased root length (33%), shoot length (53%), fresh weight (48%), and dry weight (44%) of wheat as compared to control. The physiological parameters including chlorophyll content, relative water content, membrane stability index, and osmolyte content (proline and sugar) were also improved. The increase in superoxide dismutase, peroxidase and, catalase activity by the combined application of TiO2 NPs and calcium phosphate was 83% and 78%, 74% and 52%, 81%, and 67% in Pakistan-13 and Zincol-16 respectively, as compared to untreated drought exposed plants. They also enhanced the nutrients uptake (including potassium, phosphorus, and nitrogen) that ultimately improved plant biomass. They also maintained the level of growth hormones in plants. These hormones regulate cellular processes and are responsible for germination, development, and plant reaction in drought stress. The increase in the yield was also significant, hence it is recommended that the 40 ppm concentration of TiO2 NPs along with calcium phosphate improves the productivity of wheat under drought stress.

Correlation of C-reactive protein levels, gene polymorphism and platelets count in Dengue infection.

OBJECTIVE: To determine the correlation of polymorphism in C-reactive protein gene with variation in serum levels in dengue patients. METHODS: The cross-sectional study was conducted at Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan, from October 2017 to October 2018, and comprised blood samples from dengue patients which were used to measure the serum levels of C-reactive protein. Deoxyribonucleic acid extraction followed by tetra amplification-refractory mutation system polymerase chain reaction was used to analyse the genotype variation T>G for single nucleotide polymorphism rs199953854 using allele-specific primers. Correlation of serum C-reactive protein levels with the C-reactive protein polymorphism in dengue patients was explored. Data was analysed using SPSS 21. RESULTS: Of the 200 patients, 108(54%) had very high C-reactive protein levels, 48(24%) had levels slightly higher than the upper limit, 14(7%) had low and 30(15%) had normal levels. Also, 162(81%) patients had low platelets count. Amplification of only T alleles was noted. CONCLUSIONS: C-reactive protein levels were found to be increased with suppressed platelets count in dengue patients. Single nucleotide polymorphism rs199953854 appeared to have no polymorphism.

First detection and molecular characterization of avian polyomavirus in young parrots in Pakistan.

Avian polyomavirus (APV) infection, also called as budgerigar fledgling disease (BFD) causes various health problems in many psittacine species which may cause untimely death. The aims of this study were to investigate, for the first time, the detection, molecular characterization and phylogenetic analysis of avian polyomavirus (APV) in Pakistani psittacine birds. In an aviary a disease similar to APV was found and 90% of the nestlings died within a few weeks. Seven to ten-day-old parrot nestlings (n = 3) from the aviary were presented with feather abnormalities, plumage defect and were clinically depressed. Birds died at 11th, 14th and 16th day of age. Samples of hearts, livers, spleen, feathers and kidneys were collected from the dead birds. Samples were analyzed for the presence of APV DNA by using PCR. APV VP1 gene was partially sequenced, and phylogenetic analysis was performed. The APV strain was similar to those previously reported in other areas of the world. The results of this investigation indicate presence of a high frequency of APV infections in psittacine birds in Pakistan.