Hit discovery of potential CDK8 inhibitors and analysis of amino acid mutations for cancer therapy through computer-aided drug discovery.

Cyclin-dependent kinase 8 (CDK8) has emerged as a promising target for inhibiting cancer cell function, intensifying efforts towards the development of CDK8 inhibitors as potential cancer therapeutics. Mutations in CDK8, a protein kinase, are also implicated as a primary factor associated with tumor formation. In this study, we identified potential inhibitors through virtual screening for CDK8 and single amino acid mutations in CDK8, namely D173A (Aspartate 173 mutate to Alanine), D189N (Aspartate 189 mutate to Asparagine), T196A (Threonine 196 mutate to Alanine) and T196D (Threonine 196 mutate to Aspartate). Four databases (CHEMBEL, ZINC, MCULE, and MolPort) containing 65,209,131 molecules have been searched to identify new inhibitors for CDK8 and its single mutations. In the first step, structure-based pharmacophore modeling in the Pharmit server was used to select the compounds to know the inhibitors. Then molecules with better predicted drug-like molecule properties were selected. The final filter used to select more effective inhibitors among the previously selected molecules was molecular docking. Finally, 13 hits for CDK8, 11 hits for D173A, 11 hits for D189N, 15 hits for T196A, and 12 hits for T196D were considered potential inhibitors. A majority of the virtual screening hits exhibited satisfactorily predict pharmacokinetic characteristics and toxicity properties.

Colorimetric bacteria sensing of Pseudomonas aeruginosa using gold nanoparticle probes.

BACKGROUND: Due to the advantages of molecular methods over biochemical methods, the use of molecular methods for diagnosing nosocomial infections such as Pseudomonas can be an appropriate and rapid way to choose the right diagnosis and treatment of infection and prevent further complications caused by the infection. The present article provides a description of the development of a nanoparticle-based detection technique for sensitive and specific deoxyribonucleic acid-based diagnostic of Pseudomonas aeruginosa. Specific thiolated oligonucleotide probes for one of the hypervariable regions of the 16S rDNA gene were designed and applied for colorimetric detection of the bacteria. RESULTS: The results of gold nanoprobe-nucleic sequence amplification indicated the probe attached to gold nanoparticles in the presence of the target deoxyribonucleic acid. It caused aggregation of gold nanoparticles in the form of connected networks resulting in color change and indicating the presence of the target molecule in the sample, which could be observed by the naked eye. In addition, the wavelength of gold nanoparticles changed from 524 to 558 nm. Multiplex polymerase chain reactions were performed using four specific genes of Pseudomonas aeruginosa (oprL, oprI, toxA, and 16S rDNA). The sensitivity and specificity of the two techniques were assessed. According to the observations, the specificity of both techniques was 100%, and the sensitivity was 0.5 ng/µL and 0.01 ng/µL of genomic deoxyribonucleic acid for multiplex polymerase chain reaction and colorimetric assay, respectively. CONCLUSIONS: The sensitivity of colorimetric detection was about 50 times higher than the polymerase chain reaction using the 16SrDNA gene. The results of our study proved to be highly specific with potential use for early detection of Pseudomonas aeruginosa.

Simple innovative adaptor to improve genome walking with convenient PCR.

BACKGROUND: Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. RESULTS: The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5' phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5' phosphate group. CONCLUSION: PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

Chromium Supplementation; Negotiation with Diabetes Mellitus, Hyperlipidemia and Depression.

Chromium (Cr) is an essential trace element which found naturally in a daily diet and available in the form of supplementary tablets to boost disorders like diabetes mellitus (DM) and functions like lipid metabolism and beneficial on depression too. Diabetes is one of the most prevalent endocrine diseases or in other words, the most severe metabolic syndrome (MS), which associated with high production of free-radicals which is out of bodies detoxifying machine capacity or high oxidative stress (HOS), vasculitis and elevated lipid profile. many research papers and clinical trials published about the significance of chromium on biological activities, pre and post clinical. For this review research articles, clinical trials, from 1st Jan'10 to 31st Dec'18 and refer literature for the biochemical, pharmacological and biological activity of Chromium. Primarily articles gathered from the above search engines. Then precisely according to our aim and goal and regarding designed objectives dismisses similar articles and finally came to 84 articles for the above said period. This review trying to cover the entire picture from what chromium is to the recent updates on their greater role in increasing insulin sensitivity of cells and enhancing lipid metabolism and even recent findings suggest its positive effects including prevention and ameliorating properties on depression. The biological activities, pharmacological features, clinical implications including efficacy and role of chromium compounds on the glycaemic index will be discussed. The outcome of this review is to bring the pros and cons of chromium supplementation along with is safety and toxicity concern beside molecular pathways, biochemistry and clinical trials, all in one comprehensive review.

Moiré Flat Bands in Twisted Double Bilayer Graphene.

We investigate twisted double bilayer graphene (TDBG), a four-layer system composed of two AB-stacked graphene bilayers rotated with respect to each other by a small angle. Our ab initio band structure calculations reveal a considerable energy gap at the charge-neutrality point that we assign to the intrinsic symmetric polarization (ISP). We then introduce the ISP effect into the tight-binding parametrization and perform calculations on TDBG models that include lattice relaxation effects down to very small twist angles. We identify a narrow region around the magic angle characterized by a manifold of remarkably flat bands gapped out from other states even without external electric fields. To understand the fundamental origin of the magic angle in TDBG, we construct a continuum model that points to a hidden mathematical link to the twisted bilayer graphene model, thus indicating that the band flattening is a fundamental feature of TDBG and is not a result of external fields.

Gold nanoparticle biosensors, a novel application in gene transformation and expression.

The conventional techniques of PCR, Southern blot, northern blot, in situ hybridization, and RNase protection assay have long been used to investigate transformation and expression of genes, but most of them are time-consuming and have relatively low sensitivity. In recent years, applying biosensors for molecular identification of biomolecules has been expanding significantly. Hence in this study, Zabol melon was used as a model plant to introduce new DNA and RNA-based biosensors for confirming gene transformation and expression. First, the melon seeds were grown in vivo and Agrobacterium tumefaciens LBA4404 was used to introduce GUS reporter gene to the plant. In order to analyze GUS gene transformation and expression, probes were designed based on DNA, RNA, and cDNA of GUS gene sequence. Then, the analysis was performed using probes attached to gold nanoparticles to observe color change of the solution in presence of the target biomolecules. Hybridization of the probes with target molecules was evaluated at a wavelength of 400-700 nm and maximum change was observed in the wavelength range of 550-650 nm. In addition, lower detection limit of the assay was 0.25 ng/µL and linear regression showed the relationship between different concentrations of the genomic DNA and absorbance. Consequently, results showed that application of detectors attached to gold nanoparticles for investigation on gene transformation and expression is more rapid, specific and economic compared to the biochemical and molecular techniques. These tests can be carried out with initial optimization at research centers using the least facilities; hence there will be no need for special equipment.

Thiol-Capped Gold Nanoparticle Biosensors for Rapid and Sensitive Visual Colorimetric Detection of Klebsiella pneumoniae.

In the last few years, gold nanoparticle biosensors have been developed for rapid, precise, easy and inexpensive with high specificity and sensitivity detection of human, plant and animal pathogens. Klebsiella pneumoniae serotype K2 is one of the common gram-negative pathogens with high prevalence. Therefore, it is essential to provide the effective and exclusive method to detect the bacteria. Klebsiella pneumoniae serotype K2 strain ATCC9997 genomic DNA was applied to establish the detection protocol either with thiol-capped oligonucleotide probes and gold nanoparticles or polymerase chain reaction based on K2A gene sequence. In the presence of the genomic DNA and oligonucleotide probes, a change in the color of gold nanoparticles and maximum changes in wavelength at 550-650 nm was achieved. In addition, the result showed specificity of 15 × 105 CFU/mL and 9 pg/µL by gold nanoparticles probes. The lower limit of detection obtained by PCR method was 1 pg/µL. Moreover, results demonstrated a great specificity of the designed primers and probes for colorimetric detection assay and PCR. Colorimetric detection using gold nanoparticle probe with advantages such as the lower time required for detection and no need for expensive detection instrumentation compared to the biochemical and molecular methods could be introduced for rapid, accurate detection of the bacteria.

Simultaneous Expression of GUS and Actin Genes by Using the Multiplex RT-PCR and Multiplex Gold Nanoparticle Probes.

Gene expression analysis is considered to be extremely important in many different biological researches. DNA-based diagnostic test, which contributes to DNA identification, has higher specificity, cost, and speed than some biochemical and molecular methods. In this study, we try to use the novel nano technology approach with Multiplex RT-PCR and Gold nano particular probes (GNPs-probes) in order to get gene expression in Curcumas melons. We used Agrobacterium tumefactions for gene transfer and GUS reporter gene as a reporter. After cDNA synthesis, Multiplex PCR and Multiplex RT-PCR techniques were used. Finally, probes were designed for RNA of GUS and Actin genes, and then the analysis of the gene expression using the probes attached to GNPs was carried out and the color changes in the GNPs were applied. In the following, probes hybridization was checked with DNA between 400 to 700 nm wavelengths and the highest rate was observed in the 550 to 650 nm. The results show that the simultaneous use of GNP-attached detectors and Multiplex RT-PCRcan reduce time and costmore considerably than somelaboratory methods for gene expiration investigation. Additionally, it can be seen thatthere is an increase in sensitivity and specificity of our investigation. Based on our findings, this can bea novel study doneusingMultiplex RT-PCRand unmodified AuNPs for gene transfer and expression detection to plants. We can claim that this assay has a remarkable advantage including rapid, cost-effectiveness, specificity and accuracy to detect transfer and expression genes in plants. Also,we can use this technique from other gene expressionsin many different biology samples.

Thiolated AuNP probes and multiplex PCR for molecular detection of Staphylococcus epidermidis.

The emergence of nanotechnology in biology helps to apply the gold nanoparticle probes for fast and accurate identification of pathogens compared to the time-consuming and non-precise phenotypic methods. In this study, two molecular methods have been established for the accurate identification of staphylococcus epidermidis from other coagulase-negative staphylococci. Multiplex PCR was performed using designed primers for Gmk2 and pta housekeeping genes, and SESB specific gene of S. epidermidis. Colorimetric detection by gold nanoparticle probes was carried out using two 20-base thiolated probes designed based on the sequence of pta housekeeping gene of S. epidermidis. The specificity of multiplex PCR and colorimetric assays were determined using genomic DNA of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii as negative controls and no alteration was detected. To investigate the sensitivity of the primers and gold nanoparticle probes, different concentrations of the extracted DNA from S. epidermidis were used. Based on the results, the minimum required quantity of target DNA for multiplex PCR amplification was 1 ng/µL and for color and absorption alteration of solution in colorimetric assay was 20 ng/µL. Our results revealed that both methods were sufficiently specific and sensitive to detect S. epidermidis.

An efficient Agrobacterium-mediated transformation of strawberry cv. Camarosa by a dual plasmid system.

An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T(1) seedlings of tomato exposed to metal ions.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the ß-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T(0)). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T(1)) produced the highest GUS activity when treated with 150 µM Cu(2+) compared to the control (without Cu(2+)). However, Zn(2+) and Fe(2+) treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T(1) seedlings of tomato when subjected to Cu(2+) ions.