Plasmid Midiprep: A Method to Purify Plasmids for Recombinant DNA Studies

A fundamental aspect of molecular biology involves exploring the properties and functions of specific genes. The rise of recombinant DNA technology has vastly improved and simplified functional studies by allowing scientists to isolate specific genes using restriction enzymes and plasmids providing greater precision. Plasmids take great significance in downstream studies, which is why quantity and quality of the plasmids purified is important. In this study, we isolated and purified recombinant plasmids in microgram quantities to confirm the yield, quantity, and quality using spectral and size fractionation methods. We also assessed the plasmids for application in downstream studies. We found the plasmids that were isolated with a good yield ranging from 1-1.5 mg/ml. The high yield and purity suggest the Promega Midiprep kit is effective in producing high quality plasmids. The size of the plasmids was assessed using gel electrophoresis, and a transient transfection into mammalian cells confirmed their expression through fluorescence

Mucin MUC13 and YAP1 correlate with poor survival in colorectal cancer

Background: Metastatic disease contributes to over 90% of cancer-associated deaths. Colorectal cancer (CRC), the second lethal malignancy, has the greatest incidence and mortality rates in the Southern United States. Over 40-50% of CRC patients acquire metastasis at some point throughout their disease\u27s progression. CRC survival rate drops from 90%-14% when the disease is confined within the colon and therefore “early diagnosis” becomes imperative to determine timely and quality treatments. We have identified that MUC13 protein translocate to nucleus along with transcription factor Yes-Associated Protein 1 (YAP1) during anchorage independent conditions (metastatic phenotype). YAP1 is known to be overexpressed in CRC which promotes proliferation and survival of CRC cells. This study will provide information regarding MUC13 and YAP1 correlation and their role in CRC patient outcomes. Methods: The comparative analysis of MUC13 and YAP1 expression in CRC samples (Tissue Microarrays (TMA) of CRC patients (39 cases and 95 cores)) with Pathology grade, TNM Classification, Clinical stage, and Survival information were investigated using Immunohistochemistry (IHC) staining, followed by digital scanning by 3D-Histech scanner, and analysis using QuantCenter image analysis software. Results: IHC analysis revealed increased MUC13 expression in colon adenocarcinoma and metastatic adenocarcinoma compared to normal colon tissues. MUC13 expression was observed in nucleus, cytoplasm and membrane associated with mostly with poorly differentiated adenocarcinomas, while YAP1 was localized in the nucleus. The correlation of MUC13/YAP1 expression with patient outcome is in progress. Conclusion: This study will potentially establish a correlation between MUC13 and YAP1 with CRC patient outcome

Role of POTE-2 in hepatocellular carcinoma progression.

Background: Hepatocellular carcinoma (HCC) accounts for 85-90% of primary liver cancers. The Hispanic population had an incidence of 21.2 per 100,000 in Texas. Particularly, the Rio Grande Valley (RGV) is an underserved area facing disparities that increase risk factors of HCC and thus, yielding higher incidence and mortality. Therefore, early, faster, and inexpensive diagnostic biomarkers and methods are crucial to under-resourced areas such as the RGV. Recently, we have identified an extracellular cancer antigen, POTE-2. Preliminary data indicates high POTE-2 expression in HCC tumors. In this study, we will discuss the role of POTE-2 in HCC progression and its associated regulatory pathways. Methods: The Cancer Genome Atlas (TCGA) database of HCC patients (n=371 tumor; n=50 normal) was analyzed. Liver cancer cells were procured from ATCC. POTE-2 mRNA and protein expression analyzed via RT-PCR and western blot. Absolute copy number was determined using Digital Droplet PCR. Lentiviral-based plasmids were used for overexpression and knockdown studies. Signaling pathways were analyzed using Proteome Profiler array. Results: Comprehensive analysis of TCGA database revealed high POTE-2 expression tumors with upregulation in all stages of HCC. POTE-2 expression increases with nodal metastatic status leading to poor survival. The protein expression for POTE-2 was significantly higher in SK-HEP1 compared to C3A cells. Lentiviral transduction showed significant overexpression and knockdown of the POTE-2 protein. Modulation of POTE-2 expression led to changes in lncRNA and kinase pathways. Conclusion: These studies will help discover novel mechanisms of POTE-2 protein function, signaling pathways and roles in liver cancer progression

Redox-responsive nano-self assemblies for targeted cancer therapeutics

Background: Despite significant advances in cancer therapeutics, it remains one of the leading causes of deaths due to poor response to available treatment modalities and drug resistance. Combination therapy has shown the potential to provide a synergistic therapeutic effect and to overcome drug resistance. However, smart delivery systems that can improve the bioavailability and the delivery of multiple hydrophobic anti-cancer drugs simultaneously at the tumor site without normal organ toxicity could be an effective strategy for cancer treatment. Methods: Here, a PEGylated drug-drug conjugate (CUR-PEG-S-S-CPT) have been successfully synthesized by conjugating two hydrophobic anti-cancer molecules, curcumin and camptothecin through an ester and a redox-sensitive disulfide linkage (-S-S-), respectively, with the PEG chain, via in situ two-step reaction. This amphiphilic polymeric-dual drug conjugate was characterized in the presence and absence of the tannic acid (TA, a physical crosslinker) using various in vitro biophysical, analytical, and functional bioassays. Results: The newly synthesized amphiphilic CUR-PEG-S-S-CPT polymer was found to spontaneously self-assembled in presence of tannic acid into anionic comparatively smaller sized stable nano-assemblies in water in comparison to parent conjugate, where the drug forms hydrophobic core of the particle with negative chirality and left-handed helical arrangement. TA, in addition to help forming stable nano-assemblies in water, it was able produce FRET pair in water between these two anticancer drugs. These nano-assemblies exhibited enhanced cellular uptake and antiproliferative effect in cancer cells (AsPC1 and SW480) in comparison to the individual drugs. Interestingly, our nanoassemblies showed preferential cleavage, breakdown and release of drugs in tumor-relevant redox environment leading to disappearance of the FRET signal, thus can be highly effective for targeted cancer treatment. Conclusions: Our promising in vitro results with novel redox stimuli-responsive (CUR-PEG-S-S-CPT) conjugate system in presence of TA can be a highly useful advanced theranostic platform for effective cancer treatment/management

Exploration of potential natural inhibitors against KRAS-G12D in PanCan: Protein centered pharmacophore HTVS approach.

Background: As per key statistics of American Cancer Society 2021, Pancreatic Cancer (PanCan) affects around 60,430 persons a year in the U.S. and is tricky to diagnose & treat. Studies revealed that African Americans have a 50–90% higher incidence of PanCan compared to other ethnic groups. Oncogenic KRAS mutation is the signature genetic incident in the progression and development of PDAC. KRAS is the most common protein which is 95% times mutated in PDAC condition. By considering this alarming situation our group is now focused on to develop therapeutic portfolio against KRAS-G12D mutation associated PanCan by using high through-put virtual screening (HTVS) approach. Methodology: In this study, prompt HTVS for vetting the best possible drug candidates from natural compound (NCs) databases has been implemented. Herein, time tested rigorous multi-layered drug screening process to narrow down 66,969 NCs for the identification of potential lead(s) is implemented. Druggability parameters, protein centered pharmacophore-based drug selections & different docking approaches (Rigid & Flexible) were employed in this study. Result: By using different NCs databases around 66,969 NCs were screened based on protein-centered pharmacophore fit score & binding energies. Less than 0.001% of potential NCs were selected against the known & reference KRAS-G12D inhibitor (BI2852). Conclusion: By using HTVS approach we have identified a pool of natural inhibitors against KRAS G12D

CEACAM7 expression contributes to early events of pancreatic cancer

Highlights The trends of pancreatic cancer (PanCa) incidence and mortality are on rising pattern, and it will a second leading cause of cancer related deaths by 2030. Approximately 85–90% PanCa are pancreatic ductal adenocarcinoma (PDAC), which is one of the most challenging and aggressive malignancy. PDAC exhibits with grim prognosis as mortality rate is very close to the incidence due lack of early detection methods and effective therapeutic regimen. Our team has identified a novel oncogenic protein, carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7), that can be useful for early PDAC diagnosis and predictor of patient survival. We also observed an increase of CEACAM7 expression in PDAC cell line panel model. However, poorly differentiated, and normal cell lines did not show any expression. Commercially available human tissue analysis also strengthened our data by showing strong and positive IHC staining in early-stage tumors. Abstract Background The trends of pancreatic cancer (PanCa) incidence and mortality are on rising pattern, and it will be a second leading cause of cancer related deaths by 2030. Pancreatic ductal adenocarcinoma (PDAC), major form of PanCa, exhibits a grim prognosis as mortality rate is very close to the incidence rate, due to lack of early detection methods and effective therapeutic regimen. Considering this alarming unmet clinic need, our team has identified a novel oncogenic protein, carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7), that can be useful for spotting early events of PDAC. Methodology This study includes bioinformatics pre-screening using publicly available cancer databases followed by molecular biology techniques in PDAC progressive cell line panel and human tissues to evaluate CEACAM7 expression in early events of pancreatic cancer. Results PanCa gene and protein expression analysis demonstrated the significantly higher expression of CEACAM7 in PDAC, compared to other cancers and normal pancreas. Overall survival analysis demonstrated an association between the higher expression of CEACAM7 and poor patients’ prognosis with high hazard ratio. Additionally, in a performance comparison analysis CEACAM7 outperformed S100A4 in relation to PDAC. We also observed an increase of CEACAM7 in PDAC cell line panel model. However, poorly differentiated, and normal cell lines did not show any expression. Human tissue analysis also strengthened our data by showing strong and positive IHC staining in early-stage tumors. Conclusion Our observations clearly cite that CEACAM7 can serve as a potential early diagnostic and/or prognostic marker of PDAC and may also potentiate the sensitivity of the existing biomarker panel of PDAC. However, further studies are warranted to determine its clinical significance

Modulation of POTE-2 Expression by ncRNAs in Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) has one of the highest incidents and mortality rates within the Hispanic population of South Texas. The Surveillance, Epidemiology, and End Results (SEER) cancer registry reports a 20.3% 5-year relative survival rate upon HCC diagnoses, which decreases in advanced stage cancers. The disproportionate impact on the Hispanic community and poor prognostics makes the search for better diagnostic measures imperative. A major step in bridging the disparity in HCC occurrence is the identification of potential biomarkers aiding in HCC diagnosis, surveillance, and treatment. POTE ankyrin domain members have been recognized as key promotors of tumorigenesis. POTE-2, a novel protein, has shown to be differentially regulated in liver cancer. Micro-RNAs (miRNAs) regulate protein expression through translational inhibition or mRNA degradation. This study aims to investigate possible role of miRNA-3662 in POTE-2 expression regulation in HCC cell lines. Methods: POTE-2 mRNA and protein were analyzed using RT-PCR and western blot respectively in liver cancer cell lines, SK-HEP1, C3A, HEPG2, and HEP-3B. POTE-2 mRNA was analyzed for potential miRNA binding sites using miRNAdb.org. Identified miRNAs were verified using miRNA specific RT-PCR. Results: SK-HEP1 yielded relatively low mRNA with high protein, and the opposite was observed in C3A cells. SK-HEP1 cells showed higher proliferation, migration, and invasion. Analysis of POTE-2 mRNA using miRNA database identified potential miRNAs binding sites. MiRNA-3662 being the top candidate is being analyzed for its role in POTE-2 regulation. Conclusions: Regulation of POTE-2 mRNA by miRNA-3662 makes it a potential candidate for miRNA-based therapeutics in HCC

In silico CD4 + T-cell multiepitope prediction and HLA distribution analysis for Marburg Virus—A strategy for vaccine designing

Marburg, a RNA virus (MRV), is responsible for causing hemorrhagic fever that affects humans and non-human primates. World Health Organization (WHO), National Institutes of Health (NIH) and Centre of Disease Control and Prevention (CDC) considered this as an extremely dangerous virus, thus categorised as risk group 4, category A priority pathogen and category “A” bioterrorism agent, respectively. Despite of all these alarming concerns, no prophylaxis arrangements are available against this virus till date. In fact, the construction of immunogenic vaccine candidates by traditional molecular immunology methods is time consuming and very expensive. Considering these concerns, herein, we have designed CD4 + T Cell multiepitopes against MRV using in silico approach. The pin-point criteria of the screening and selection of potential epitopes are, non-mutagenic, antigenic, large HLAs coverage, non-toxic and high world population coverage. This kind of methodology and investigations can precisely reduce the expenditure and valuable time for experimental planning in development of vaccines in laboratories. In current scenario, researchers are frequently using in silico approaches to speed up their vaccine-based lab studies. The computational studies are highly valuable for the screening of large epitope dataset into smaller one prior to in vitro and in vivo confirmatory analyses

YB-1 transcription factor promotes Sorafenib resistance in Liver Cancer

Background: Hepatocellular carcinoma (HCC) is a primary malignant liver tumor that commonly occurs as a progression of chronic liver inflammation. Sorafenib is the standard first-line systemic drug for advanced HCC, but the acquired resistance to sorafenib results in limited benefits. The mechanism underlying sorafenib resistance in HCC remains unclear. Recently, we have identified a multifunctional oncoprotein Y-box binding protein-1 (YB-1) that dysregulates a wide range of genes involved in drug resistance in other cancers and is responsible for increasing the IC-50 of sorafenib in HCC cell lines. In this study we will analyze the signaling pathways and genes regulated by YB-1, that is responsible for increasing sorafenib resistant in liver cancer cells. Methods: HCC cell lines SK-Hep-1, C3A, HepG2 and Hep-3B were treated with Sorafenib and the IC-50 was calculated using MTT assay. RNA and protein of YB-1 was analyzed using RT-PCR and western blot respectively. Lentiviral based overexpression and knockdown of YB1 was performed in these cell lines and sorafenib IC50 were calculated to verify its role in Sorafenib resistance. Development of sorafenib resistant cell line is in progress. Results: IC-50 values calculated from MTT assays of the HCC cell lines were compared with the YB-1 protein expression in four liver cancer cell lines. Knockdown of YB-1 re-sensitized cell lines to Sorafenib. We have developed Sorafenib resistant cell lines to further study the mechanism of YB-1 mediated drug resistance. Conclusion: This study will establish oncogenic YB-1 protein as an effective therapeutic target to overcome sorafenib resistance in liver cancer

Non-methane volatile organic compounds emitted from domestic fuels in Delhi: Emission factors and total city-wide emissions

In controlled laboratory conditions, 62 samples of domestic fuels collected from 56 grids of Delhi were burnt to quantify the emissions of 23 non-methane volatile organic compounds (NMVOCs), i.e., alkanes (11), alkenes (6), alkynes (1) and aromatic compounds (5). The domestic fuels used for residential activities were comprised of 20 unique types of fuel woods, 3 species of crop residue, dung cakes and coal. These fuels are primarily used for cooking and water/space heating during winters. The current study reports the total emission budget of NMVOCs from domestic burning over Delhi. Furthermore, this study also compares the differences in EFs of NMVOCs which are calculated for different burning cycles and sample collection methods. The EFs of NMVOCs calculated from the samples collected during the flaming stage using canisters were analysed for 23 NMVOCs and then compared with same species emitted from complete burning cycle. In addition to this, 10 consumption and emission hotspot grids were also identified in Delhi; based on the ground survey and laboratory simulated results. The total annual usage of domestic fuels for the year 2019 was found to be 0.415 Mt/yr (million tonnes) in Delhi. 12.01 Gg/yr of annual NMVOC emissions was calculated from domestic fuel burning in which the emissions from dung cake and fuel wood dominated with 6.6 Gg/yr and 5.4 Gg/yr, respectively. The EFs of NMVOCs calculated using canister and online collection method differ significantly from each other. The flaming stage presented enhanced emissions compared to the complete burning cycle by ~7 times which suggests that the method of data analysis and the period of sample collection play a pivotal role in the preparation of an emission inventory and estimating the budget