Phytoconstituents of Artemisia Annua as potential inhibitors of SARS CoV2 main protease: an in silico study.

BACKGROUND: In November 2019, the world faced a pandemic called SARS-CoV-2, which became a major threat to humans and continues to be. To overcome this, many plants were explored to find a cure. METHODS: Therefore, this research was planned to screen out the active constituents from Artemisia annua that can work against the viral main protease Mpro as this non-structural protein is responsible for the cleavage of replicating enzymes of the virus. Twenty-five biocompounds belonging to different classes namely alpha-pinene, beta-pinene, carvone, myrtenol, quinic acid, caffeic acid, quercetin, rutin, apigenin, chrysoplenetin, arteannunin b, artemisinin, scopoletin, scoparone, artemisinic acid, deoxyartemisnin, artemetin, casticin, sitogluside, beta-sitosterol, dihydroartemisinin, scopolin, artemether, artemotil, artesunate were selected. Virtual screening of these ligands was carried out against drug target Mpro by CB dock. RESULTS: Quercetin, rutin, casticin, chrysoplenetin, apigenin, artemetin, artesunate, sopolin and sito-gluside were found as hit compounds. Further, ADMET screening was conducted which represented Chrysoplenetin as a lead compound. Azithromycin was used as a standard drug. The interactions were studied by PyMol and visualized in LigPlot. Furthermore, the RMSD graph shows fluctuations at various points at the start of simulation in Top1 (Azithromycin) complex system due to structural changes in the helix-coil-helix and beta-turn-beta changes at specific points resulting in increased RMSD with a time frame of 50 ns. But this change remains stable after the extension of simulation time intervals till 100 ns. On other side, the Top2 complex system remains highly stable throughout the time scale. No such structural dynamics were observed bu the ligand attached to the active site residues binds strongly. CONCLUSION: This study facilitates researchers to develop and discover more effective and specific therapeutic agents against SARS-CoV-2 and other viral infections. Finally, chrysoplenetin was identified as a more potent drug candidate to act against the viral main protease, which in the future can be helpful.

Transforming Diagnosis and Therapeutics Using Cancer Genomics.

In past quarter of the century, much has been understood about the genetic variation and abnormal genes that activate cancer in humans. All the cancers somehow possess alterations in the DNA sequence of cancer cell's genome. In present, we are heading toward the era where it is possible to obtain complete genome of the cancer cells for their better diagnosis, categorization and to explore treatment options.

Quinovic acid purified from medicinal plant Fagonia indica mediates anticancer effects via death receptor 5.

Plants are major source for discovery and development of anticancer drugs. Several plant-based anticancer drugs are currently in clinical use. Fagonia indica is a plant of medicinal value in the South Asian countries. Using mass spectrometry and NMR spectroscopy, several compounds were purified from the F. indica extract. We have used one of the purified compounds quinovic acid (QA) and found that QA strongly suppressed the growth and viability of human breast and lung cancer cells. QA did not inhibit growth and viability of non-tumorigenic breast cells. QA mediated its anticancer effects by inducing cell death. QA-induced cell death was associated with biochemical features of apoptosis such as activation of caspases 3 and 8 as well as PARP cleavage. QA also upregulated mRNA and protein levels of death receptor 5 (DR5). Further investigation revealed that QA did not alter DR5 gene promoter activity, but enhanced DR5 mRNA and protein stabilities. DR5 is one of the major components of the extrinsic pathway of apoptosis. Accordingly, Apo2L/TRAIL, the DR5 ligand, potentiated the anticancer effects of QA. Our results indicate that QA mediates its anticancer effects, at least in part, by engaging DR5-depentent pathway to induce apoptosis. Based on our results, we propose that QA in combination with Apo2L/TRAIL can be further investigated as a novel therapeutic approach for breast and lung cancers.

Artemisinin and its derivatives: a promising cancer therapy.

The world is experiencing a cancer epidemic and an increase in the prevalence of the disease. Cancer remains a major killer, accounting for more than half a million deaths annually. There is a wide range of natural products that have the potential to treat this disease. One of these products is artemisinin; a natural product from Artemisia plant. The Nobel Prize for Medicine was awarded in 2015 for the discovery of artemisinin in recognition of the drug's efficacy. Artemisinin produces highly reactive free radicals by the breakdown of two oxygen atoms that kill cancerous cells. These cells sequester iron and accumulate as much as 1000 times in comparison with normal cells. Generally, chemotherapy is toxic to both cancerous cells and normal cells, while no significant cytotoxicity from artemisinin to normal cells has been found in more than 4000 case studies, which makes it far different than conventional chemotherapy. The pleiotropic response of artemisinin in cancer cells is responsible for growth inhibition by multiple ways including inhibition of angiogenesis, apoptosis, cell cycle arrest, disruption of cell migration, and modulation of nuclear receptor responsiveness. It is very encouraging that artemisinin and its derivatives are anticipated to be a novel class of broad-spectrum antitumor agents based on efficacy and safety. This review aims to highlight these achievements and propose potential strategies to develop artemisinin and its derivatives as a new class of cancer therapeutic agents.

Pharmacological evaluation of Acacia modesta bark for antipyretic, anti-inflammatory, analgesic, antidepressant and anticoagulant activities in Sprague Dawley rats.

In this study the bark of Acacia modesta was evaluated for anti-inflammatory, antipyretic, analgesic, antidepressant and anticoagulant activity by carrageenan, hot plat, forced swim and capillary tube method respectively in rats. Highest anti-inflammatory activity was exhibited by chloroform (AMC) extract (74.96% inhibition) while other two active fractions being n-hexane (AMH) and ethyl acetate (AME) exhibited 71.26% and 52.87% inhibition of edema respectively. On the other hand, the aqueous (AMA) fraction showed most effective response with 67.06% analgesic activity. Additionally, the significant (p<0.05) post-treatment antipyretic effect was found by all fractions in time dependent manner. The current findings showed that AMC, AME and AMA had significant reduction in immobility time in the antidepressant test, while AMH showed mild antidepressant activity. In anticoagulant assay, the coagulation time of crude extract A. modesta and its all fractions were comparable to that of positive control aspirin (208s). Moreover, neither mortality nor lethality was observed in the tested animals. Overall, the plant extracts showed potent anti-inflammatory, antipyretic, analgesic, antidepressant and anticoagulant activities which concludes that the bark of A. modesta have significant therapeutic potential.

Silver nanoparticles conjugated with Neurotrophin 3 upregulate myelin gene transcription pathway.

Mathematical modeling is the art of converting problems from the biological area into handy mathematical formulations whose theoretical and numerical analysis provides understandings about the directions and solutions to the particular problem. Recently, the combination therapy treatments have been revealed exceptionally fruitful by using mathematical modeling technique. The human nervous system is composed of axons, covered by the myelin sheath. Axons carry signals and promote myelin development. The abnormalities in myelination formation due to mutations in myelin gene result in memory disorders and impaired cognitive activities. The ERBb gene family is responsible for causing abnormalities in myelin gene. Using this knowledge, the pathway of mutated myelin gene was retrieved and its model was developed. Modeling and simulation analysis was performed to determine the level of expression of several genes. The Neurotrophin 3 ligand-coated with silver nanoparticle was induced in the model to normalize the transcription of myelin gene. It was observed that the myelin gene expression level increases from 0 after two days of NT3 induction and reaches to the maximum level on the 10th day of drug induction along with an increase in ERBb expression. This research work can be used in the future as a part of drug discovery and formulation.

Biotechnological approaches for artemisinin production in Artemisia.

Artemisinin and its analogues are naturally occurring most effective antimalarial secondary metabolites. These compounds also possess activity against various types of cancer cells, schistosomiasis, and some viral diseases. Artemisinin and its derivatives (A&D) are found in very low amounts in the only natural source i.e. Artemisia plant. To meet the global needs, plant sources have been exploited for the enhanced production of these natural products because their chemical synthesis is not profitable. The generally adopted approaches include non-transgenic (tissue and cell cultures) and transgenic together with the cell, tissue, and whole transgenic plant cultures. The genes targeted for the overproduction of A&D include the biosynthetic pathway genes, trichome development genes and rol genes, etc. Artemisinin is naturally produced in trichomes of leaves. At the same time, transgenic hairy roots are considered a good source to harvest artemisinin. However, the absence of trichomes in hairy roots suggests that artemisinin biosynthesis is not limited to trichomes. Moreover, the expression of the gene involved in trichome development and sesquiterpenoid biosynthesis (TFAR1) in transgenic and non-transgenic roots provokes researchers to look for new insight of artemisinin biosynthesis. Here we discuss and review precisely the various biotechnological approaches for the enhanced biosynthesis of A&D.

Rol genes enhance the biosynthesis of antioxidants in Artemisia carvifolia Buch.

BACKGROUND: The secondary metabolites of the Artemisia genus are well known for their important therapeutic properties. This genus is one of the valuable sources of flavonoids and other polyphenols, but due to the low contents of these important metabolites, there is a need to either enhance their concentration in the original plant or seek alternative sources for them. The aim of the current study was to detect and enhance the yield of antioxidant compounds of Artemisia carvifolia Buch. HPLC analysis was performed to detect the antioxidants. With the aim of increasing flavonoid content, Rol gene transgenics of A. carvifolia were established. Two genes of the flavonoid biosynthetic pathway, phenylalanine ammonia-lyase and chalcone synthase, were studied by real time qPCR. Antioxidant potential was determined by performing different antioxidant assays. RESULTS: HPLC analysis of wild-type A. carvifolia revealed the presence of flavonoids such as caffeic acid (30 µg/g DW), quercetin (10 µg/g DW), isoquercetin (400 µg/g DW) and rutin (300 µg/g DW). Compared to the untransformed plants, flavonoid levels increased 1.9-6-fold and 1.6-4-fold in rol B and rol C transgenics, respectively. RT qPCR analysis showed a variable expression of the flavonoid biosynthetic genes, including those encoding phenylalanine ammonia-lyase and chalcone synthase, which were found to be relatively more expressed in transformed than wild-type plants, thus correlating with the metabolite concentration. Methanolic extracts of transgenics showed higher antioxidant capacity, reducing power, and protection against free radical-induced DNA damage. Among the transgenic plants, those harboring rol B were slightly more active than the rol C-transformants. CONCLUSION: As well as demonstrating the effectiveness of rol genes in inducing plant secondary metabolism, this study provides insight into the molecular dynamics of the flavonoid accumulation pattern, which correlated with the expression of biosynthetic genes.

Evaluation of Ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities.

BACKGROUND: Ajuga bracteosa has been extensively used traditionally for the treatment of a variety of diseases. The aim of the study was to scientifically validate the wide-scale exploitation of A. bracteosa in folk medicine various in vitro and in vivo assays. Moreover, these activities were related to the intrinsic biologically active phytoecdysteroids of A. bracteosa. METHODS: Aerial and root parts of A. bracteosa were first extracted separately with chloroform (AbCA and AbCR) and the residue was again extracted with methanol (AbMA and AbMR). Total flavonoid and phenolic contents were assayed as quercetin (QE) and gallic acid equivalents (GAE), respectively. These extracts were analyzed for in vitro antioxidant assessment including DPPH and H2O2 (% inhibition of free radicals), and reducing power and phosphomolybdenum methods (ascorbic acid equivalents AAE mg/g DW). Further, these extracts were assayed in vivo in separate groups of Sprague-Dawley rats for carrageenan induced rat paw edema inhibition, hotplate antinociception, forced swim antidepression and anticoagulation. Dose of each crude extract and standard drug given to rats was 200 mg/Kg- and 10 mg/10 mL/Kg body weight respectively. Plant extracts and standard drugs were administered orally, 60 min prior to the conduction of assays. Moreover, biologically active phytoecdysteroids were screened in A. bracteosa with the help of RP-HPLC. RESULTS: AbMA represented highest values of flavonoids (QE 1.98 % DW) and phenolic contents (GAE 5.94 % DW), significantly scavenged DPPH radicles (IC50 36.9) and reduced ferric ions with 718.4 mg ascorbic acid equivalent/g (AAE). Highest total antioxidant capacity was expressed by AbMR (927 mg AAE) with an IC50 value 19.1 µg/mL. The extracts which were found potent anti-oxidants, were also good at in vivo activities. AbMA significantly reduced edema in all the three hours of treatment (67.9, 70.3 and 74.3 %). AbMA also showed maximum nociceptor suppression in analgesic assay by delaying the time to start licking of paws in rats (57.7 ± 4.9 s). In addition, maximum anti-coagulation was also exhibited by AbMA (89.3 s), while all extracts were found strong antidepressants (≤15.66 s immobility time). Screening of biologically active phytoecdysteroids revealed the presence of 20-hydroxyecdysone (20-HE), makisterone (MKA), cyasterone (CYP) and ajujalactone (AJL). Total phytoecdysteroid content found in A. bracteosa was 1232.5 µg/g DW and 20-HE was most abundant (1232.5 µg/g DW) as compared to other phytoecdysteroids. CONCLUSION: Based on the tested in vitro and in vivo activities, AbMA was found to be a promising bioactive extract. These activities can be attributed to the intrinsic polyphenols and phytoecdysteroids contents of A. bracteosa.

Enhanced artemisinin yield by expression of rol genes in Artemisia annua.

BACKGROUND: Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug. METHODS: In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined. RESULTS: Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant. CONCLUSION: Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

Artemisia carvifolia Buch silver nanoparticles downregulate the Rap2A gene in liver cancer.

Liver cancer is the second main reason of death globally. In the current study, Rap2A protein a member of Ras Gtpase was selected as a drug target for liver cancer which has been identified as an oncogene in different types of tumors. The present study aimed to evaluate Artemisia carvifolia Buch extract and its silver nanoparticles against liver cancer targeting the Rap2A gene. The synthesized silver nanoparticles showed an absorbance peak at 450 nm by a UV-Vis spectrophotometer. SEM revealed that polyhedral silver nanoparticles had a size ranging from 80 ± 6 nm. Furthermore, amines, aldehydes, ketones and alcohols of Artemisia carvifolia were found involved in the reduction and stabilization of nanoparticles by FTIR. Moreover, XRD and EDX confirmed the cubic crystalline nature and particle elemental composition, respectively. Furthermore, the cytotoxicity against HePG2 cancer cell lines was also found significant with an IC50 value of 2.57 µM for silver nanoparticles and 11.57 µM for plant extract. The gene expression and protein level of Rap2A were also decreased in plant extract and nanoparticle-treated cells compared to control groups. The apoptotic potential of extract and nanoparticles was also determined by evaluating the apoptotic pathway genes and protein including BAX, caspase 3, 8 and 9. Significantly elevated levels of expression of these genes by real-time qPCR along with increased protein levels by ELISA were found. This is the first-ever report describing the synthesis and efficacy of silver nanoparticles of Artemisia carvifolia Buch against liver cancer.

Enhanced Antioxidant and Anticancer Potential of Artemisia carvifolia Buch Transformed with rol A Gene.

Secondary metabolites have been shown to possess a range of biological functions. Flavonoids, due to their ability to scavenge ROS, are famous antioxidants. The plants of Artemisia species are rich sources of flavonoids; however, the amount of these metabolites is less. In the current study, the flavonoid content was detected and then enhanced by genetically modifying the Artemisia carvifolia Buch with Agrobacterium tumefaciens strain GV3101 carrying rol A gene. The transformation of rol A gene was confirmed with PCR and the gene copy number was confirmed by Southern blot analysis. The HPLC analysis revealed the presence of catechin (3.19 ug/mg DW) and geutisic acid (2.22 ug/mg DW) in transformed plants, unlike wild-type plants. In transformed plants, all detected flavonoids (vanillic acid, rutin, catechine, gallic acid, syringic acid, caffeic acid, coumaric acid, geutisic acid, ferulic acid, and cinnamic acid) were increased up to several folds. Real-time qPCR revealed the higher expression levels of the genes for flavonoid biosynthesis enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in plants transformed with rol A genes, as the expression levels were increased up to 9-20-fold and 2-6-fold, respectively. The rol A transgenic lines T3 and T5 carrying two copies of rol A gene, particularly showed higher expression of both PAL and CHS gene, with the highest expression in T3 line. The transgenic lines demonstrated an average increase of 1.4-fold in the total phenolic content and 1-2-fold in the total flavonoid content as compared to wild-type plants. Total antioxidant capacity and total reducing power were increased up to an average of 1-2-fold and 1.5-2-fold respectively, along with increased free radical scavenging ability. Furthermore, the rol A gene transgenics were found to have much greater cytotoxic capacity than the A. carvifolia wild-type plant against the MCF7, HeLA, and HePG2 cancer cell lines. Current findings show that the rol A gene effectively increases the flavonoid content of A. carvifolia Buch, boosting the plant's capacity as an antioxidant and an anticancer. This is the first-ever report, demonstrating the genetic transformation of Artemisia carvifolia Buch with rol A gene.

Molecular Screening of Bioactive Compounds of Garlic for Therapeutic Effects against COVID-19.

An outbreak of pneumonia occurred on December 2019 in Wuhan, China, which caused a serious public health emergency by spreading around the globe. Globally, natural products are being focused on more than synthetic ones. So, keeping that in view, the current study was conducted to discover potential antiviral compounds from Allium sativum. Twenty-five phytocompounds of this plant were selected from the literature and databases including 3-(Allylsulphinyl)-L-alanine, Allicin, Diallyl sulfide, Diallyl disulfide, Diallyl trisulfide, Glutathione, L-Cysteine, S-allyl-mercapto-glutathione, Quercetin, Myricetin, Thiocysteine, Gamma-glutamyl-Lcysteine, Gamma-glutamylallyl-cysteine, Fructan, Lauricacid, Linoleicacid, Allixin, Ajoene, Diazinon Kaempferol, Levamisole, Caffeicacid, Ethyl linoleate, Scutellarein, and S-allylcysteine methyl-ester. Virtual screening of these selected ligands was carried out against drug target 3CL protease by CB-dock. Pharmacokinetic and pharmacodynamic properties defined the final destiny of compounds as drug or non-drug molecules. The best five compounds screened were Allicin, Diallyl Sulfide, Diallyl Disulfide, Diallyl Trisulfide, Ajoene, and Levamisole, which showed themselves as hit compounds. Further refining by screening filters represented Levamisole as a lead compound. All the interaction visualization analysis studies were performed using the PyMol molecular visualization tool and LigPlot+. Conclusively, Levamisole was screened as a likely antiviral compound which might be a drug candidate to treat SARS-CoV-2 in the future. Nevertheless, further research needs to be carried out to study their potential medicinal use.

Bioassays guided isolation of berberine from Berberis lycium and its neuroprotective role in aluminium chloride induced rat model of Alzheimer's disease combined with insilico molecular docking.

OBJECTIVE: Berberis lycium is an indigenous plant of Pakistan that is known for its medicinal properties. In the current study, we investigated the anti-Alzheimer's effect of berberine isolated from Berberis lycium. METHODS: Root extract of B. lycium was subjected to acetylcholinesterase inhibition assay and column chromatography for bioassays guided isolation of a compound. The neuroprotective and memory improving effects of isolated compound were evaluated by aluminium chloride induced Alzheimer's disease rat model, elevated plus maze (EPM) and Morris water maze (MWM) tests., Levels of dopamine and serotonin in rats brains were determined using HPLC. Moreover, western blot and docking were performed to determine interaction between berberine and ß-secretase. RESULTS: During fractionation, ethyl acetate and methanol (3:7) fraction was collected from solvent mixture of ethyl acetate and methanol. This fraction showed the highest anti-acetylcholinesterase activity and was alkaloid positive. The results of TLC and HPLC analysis indicated the presence of the isolated compound as berberine. Additionally, the confirmation of isolated compound as berberine was carried out using FTIR and NMR analysis. In vivo EPM and MWM tests showed improved memory patterns after berberine treatment in Alzheimer's disease model. The levels of dopamine, serotonin and activity of antioxidant enzymes were significantly (p<0.05) enhanced in brain tissue homogenates of berberine treated group. This was supported by decreased expression of ß-secretase in berberine treated rat brain homogenates and good binding affinity of berberine with ß-secretase in docking studies. Binding energies for interaction of ß-secretase with berberine and drug Rivastigmine is -7.0 kcal/mol and -5.8 kcal/mol respectively representing the strong interactions. The results of docked complex of secretase with berberine and Rivastigmine was carried out using Gromacs which showed significant stability of complex in terms of RMSD and radius of gyration. Overall, the study presents berberine as a potential drug against Alzheimer's disease by providing evidence of its effects in improving memory, neurotransmitter levels and reducing ß-secretase expression in the Alzheimer's disease model.

Effects of Phoenix dactylifera against Streptozotocin-Aluminium Chloride Induced Alzheimer's Rats and Their In Silico Study.

Phoenix dactylifera is known for medicinal importance due to its antioxidant, antidiabetic, antidepressant, and anti-inflammatory properties. This study is aimed at evaluating the effect of P. dactylifera seeds to cure Alzheimer's disease (AD). AD was induced in the rats with streptozotocin + aluminium chloride followed by treatment of methanolic extract of P. dactylifera seeds. The blood glucose levels were determined at regular intervals, which showed a prominent decrease in the extracts treated group. Behavior tests, including the Elevated Plus Maze (EPM) test and Morris Water Maze (MWM) test, were used to evaluate memory patterns in rats. The results indicated that extract-treated rats significantly improved memory behavior compared to the diseased group. After dissection, the serum electrolytes, antioxidant enzymes, and choline esterase enzymes were measured in different organs. The serum parameters creatinine, urea, and bilirubin increased after extract treatment. Similarly, the level of antioxidant enzymes like peroxidases (POD), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and thiobarbituric acid reactive substance (TBARS) in the extract-treated group showed improved results that were close to the normal control group. The enzyme (lipase, insulin, amylase, and acetylcholine) levels were found enhanced in extract groups as compared to diseased rats. High-performance liquid chromatography (HPLC) was used to determine the level of dopamine and serotonin neurotransmitters, which were increased significantly for P. dactylifera seeds with values of 0.18 µg/mg tissue and 0.56 µg/mg tissue, respectively. Overall, results showed that P. dactylifera seeds proved to be quite efficient in improving the memory and behavior of treated rats. The antioxidants and enzymes were also increased; therefore, it may be a potential candidate for treating AD.

Predicting 3D Structure, Cross Talks, and Prognostic Significance of KLF9 in Cervical Cancer.

Our study aimed to identify the new blood-based biomarkers for the diagnosis and prognosis of cervical cancer. Moreover, the three-dimensional (3D) structure of Kruppel-like factor 9 (KLF9) was also determined in order to better understand its function, and a signaling pathway was constructed to identity its upstream and downstream targets. In the current study, the co-expressions of tumor protein D52 (TPD52), KLF9, microRNA 223 (miR-223), and protein kinase C epsilon (PKCϵ) were evaluated in cervical cancer patients and a possible relation with disease outcome was revealed. The expressions of TPD52, KLF9, miR-223, and PKCϵ were studied in the blood of 100 cervical cancer patients and 100 healthy controls using real-time PCR. The 3D structure of KLF9 was determined through homology modeling via the SWISS-MODEL and assessed using the Ramachandran plot. The predicted 3D structure of KLF9 had a similarity index of 62% with its template (KLF4) with no bad bonds in it. In order to construct a genetic pathway, depicting the crosstalk between understudied genes, STRING analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG), and DAVID software were used. The constructed genetic pathway showed that all the understudied genes are linked to each other and involved in the PI3K/Akt signaling pathway. There was a 23-fold increase in TPD52 expression, a 2-fold increase in miR-223 expression, a 0.14-fold decrease in KLF9 expression, and a 0.05-fold decrease of PKCϵ expression in cervical cancer. In the present study, we observed an association of the expressions of TPD52, KLF9, miR-223, and PKCϵ with tumor stage, metastasis, and treatment status of cervical cancer patients. Elevated expressions of TPD52 and miR-223 and reduced expressions of KLF9 and PKCϵ in peripheral blood of cervical cancer patients may serve as predictors of disease diagnosis and prognosis. Nevertheless, further in vitro and tissue-level studies are required to strengthen their role as potential diagnostic and prognostic biomarkers.

Unravelling Structure, Localization, and Genetic Crosstalk of KLF3 in Human Breast Cancer.

Breast cancer is the most prevailing disease among women. It actually develops from breast tissue and has heterogeneous and complex nature that constitutes multiple tumor quiddities. These features are associated with different histological forms, distinctive biological characteristics, and clinical patterns. The predisposition of breast cancer has been attributed to a number of genetic factors, associated with the worst outcomes. Unfortunately, their behavior with relevance to clinical significance remained poorly understood. So, there is a need to further explore the nature of the disease at the transcriptome level. The focus of this study was to explore the influence of Krüppel-like factor 3 (KLF3), tumor protein D52 (TPD52), microRNA 124 (miR-124), and protein kinase C epsilon (PKCε) expression on breast cancer. Moreover, this study was also aimed at predicting the tertiary structure of KLF3 protein. Expression of genes was analyzed through real-time PCR using the delta cycle threshold method, and statistical significance was calculated by two-way ANOVA in Graphpad Prism. For the construction of a 3D model, various bioinformatics software programs, Swiss Model and UCSF Chimera, were employed. The expression of KLF3, miR-124, and PKCε genes was decreased (fold change: 0.076443, 0.06969, and 0.011597, respectively). However, there was 2-fold increased expression of TPD52 with p value < 0.001 relative to control. Tertiary structure of KLF3 exhibited 80.72% structure conservation with its template KLF4 and was 95.06% structurally favored by a Ramachandran plot. These genes might be predictors of stage, metastasis, receptor, and treatment status and used as new biomarkers for breast cancer diagnosis. However, extensive investigations at the tissue level and in in vivo are required to further strengthen their role as a potential biomarker for prognosis of breast cancer.

Synthesis of Functional Silver Nanoparticles and Microparticles with Modifiers and Evaluation of Their Antimicrobial, Anticancer, and Antioxidant Activity.

An accumulating body of evidence reports the synthesis and biomedical applications of silver nanoparticles. However, the studies regarding the use of maleic acid and citric acid in the synthesis of nano-sized silver particles (AgNPs) and micro-sized silver particles (AgMPs) as well as their antibacterial, antifungal, and anticancer activities have not been reported. In the current study, we synthesized AgNPs and AgMPs using maleic acid and citric acid as capping agents and have characterized them by UV-Vis, energy-dispersive X-Ray spectroscopy (EDS), X-Ray diffraction (XRD), and scanning electron microscope (SEM) analysis. The capped silver particles were examined for their antimicrobial activity and cytotoxicity against bacteria, fungi, and brine shrimp. Additionally, the anticancer activity of these particles was tested against human breast and liver cancer cell lines. The free radical scavenging activity of capped silver particles was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. SEM analysis revealed a round plate-like morphology of maleic acid capped particles with an average size of 39 ± 4 nm, whereas citric acid capped particles display flower-shaped morphology with rough surfaces and an average size of 250 ± 5 nm. The uncapped AgMPs were hexagonal with 500 ± 4 nm size. EDS and XRD analysis confirmed the presence of Ag and face-centered cubic crystalline nature, respectively. Functionally, capped silver particles exhibited antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus) and Gram-negative bacteria (Salmonella setubal, Enterobacter aerogenes, and Agrobacterium tumefaciens). The bactericidal activity was more active against Gram-negative bacteria with minimum inhibitory concentration (MIC) as low as 5 ppm as compared to 25 ppm for Gram-positive. Similarly, the silver particles demonstrated antifungal activity by inhibiting the growth of five fungal strains (Mucor species, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, and Fusarium solani) up to 50% at the concentration of 500 ppm. Additionally, these particles showed substantial toxicity against brine shrimp and also significantly inhibited the proliferation of breast cancer (MCF7) and liver cancer (HePG2) cell lines (IC50 8.9-18.56 µM). Uncapped AgMPs were less effective, inhibiting only the proliferation of MCF7 cells with IC50 46.54 µM. Besides cytotoxicity, these particles acted as potential antioxidants, showing free radical scavenging up to 74.4% in a concentration-dependent manner. Taken together, our results showed that the modifiers affect the shape and size of silver particles and may, in part, contribute to the antimicrobial and antioxidant activity of silver particles. However, the contribution of maleic acid and citric acid in enhancing the antimicrobial, anticancer, and antioxidant potential independent of silver nano and microparticles needs to be studied further. In vivo experiments may determine the therapeutic effectiveness of silver particles capped with these modifiers.

Production of biomass and medicinal metabolites through adventitious roots in Ajuga bracteosa under different spectral lights.

Ajuga bracteosa an important medicinal herb, is getting endangered worldwide due to destructive harvesting by pharmaceutical industries in its different habitats. It is in dire need for protection and demands conservation and sustainable utilization. In the present study, effects of α-naphthalene acetic acid (NAA) under different spectral lights were estimated on the growth, secondary metabolism and biosynthesis of phenolic acids in adventitious roots (AR) cultures of A. bracteosa. Among the different spectral lights, highest AR induction frequency (88%) and formation of biomass (72 g/L FW and 22 g/L DW) were recorded in explants incubated in the presence of 1.5 mg/L NAA under yellow light. Maximum production of poly phenols (TPC;44.2 mg) and flavonoids (TFC;2.51 mg) were recorded in the AR cultures grown in the presence of blue light. Further, highest total protein content of (401.6 µg) was detected in the AR in response to normal white light. Blue spectral light induced maximum superoxide dismutase (SOD; 2.5 nM) and peroxidase activity (POD;0.85 nM) respectively, in AR cultures. Compared with other monochromatic lights, red light significantly enhanced the antioxidant potential of the AR cultures. Analysis through High performance liquid chromatography (HPLC-DAD) revealed significant variations in the levels of important phenolic acids such as gallic acid, catechin, rutin, caffeic acid, myricetin and apigenin in the AR samples treated with the lights of different spectra.

Drier Climatic Conditions Increase Withanolide Content of Withania coagulans Enhancing Its Inhibitory Potential Against Human Prostate Cancer Cells.

Prostate cancer is one of the major causes of cancer-related deaths in men and there is a growing interest in identifying natural compounds for its management. We analyzed bioactive withanolides in Withania coagulans from 11 different sites in Pakistan and evaluated the antiprostate cancer activities of leaf extracts from two sites with the greatest amounts. Total withanolide concentration differed by ~ 17-fold between sites, ranging from 1.01 ± 0.01 mg/g dry weight (mean ± SE) at Jand to 16.83 ± 0.02 mg/g at Mohmand Agency. Different tissues varied in their total withanolide content with roots having the least (0.42 ± 0.07 mg/g dry weight) and leaves the most (2.45 ± 0.45 mg/g). We found strong inverse correlations between site annual precipitation versus withanolide amounts in fruits (r = - 0.84, P = 0.001), leaves (r = - 0.88, P < 0.001), roots (r = - 0.91, P < 0.001), and total (r = - 0.89, P < 0.001), but not stems (r = - 0.20, P = 0.556). Extracts made from Mianwali and Mohmand Agency leaves possessed high anticancer activity in terms of increased induction of apoptosis and decreased cell viability, cell proliferation, invasion, and migration of different prostate cancer cell lines. These results are useful for the selection of withanolide-rich germplasm with potent anticancer properties.