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

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

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

Stress induced lncRNA MALAT1 in colorectal cancer health disparity

Background: Health disparities in the lower Rio Grande Valley are well documented and can play a critical role in cancer prognosis. Chronic stress, arguably exacerbated by these disparities, can also lead to a poor outcome after diagnosis through dysregulation of molecular markers known to be involved in cancer progression, resistance, and recurrence. lncRNA have been a relatively recent point of interest in the field of cancer research and play a role in cancer initiation and progression across tissue types. We have found that lncRNA MALAT1 is stress induced through transcription factor NFATc1. Here, we propose to investigate the association of stress factors with NFATc1 and MALAT1 expression, and the role of these novel molecular drivers in CRC progression and metastasis. Methods: CRC tissues of different ethnicities were stained using Novel Z Probe based technology (lncRNA MALAT1) and immunohistochemistry (NFATc1). Stained tissues were scanned on digital scanner and scored. CRC cell lines were profiled for MALAT1 expression using RT-PCR. Lentiviral based overexpression (SW480) and knockdown (SW620) of MALAT1 in CRC cells was performed to study proliferation, invasion, migration, and colony forming capacity Results: MALAT1 and NFATc1 expression was scored to be high in underserved population. MALAT1 expression was lower in less aggressive SW480 cell line as compared to highly metastatic SW620 cell line. Overexpression of MALAT1 in SW480, and knockdown in SW620 resulted in changes in their oncogenic profiles. Conclusions: Understanding the mechanistic roles molecular drivers influenced by biochemical stressors can provide pivotal information pertinent to CRC progression and metastasis

Effect of sub-optimal moisture levels on the quality of groundnut (Arachis hypogaea L.) during storage in triple-layer hermetic storage bags

Storage is an important aspect of groundnut, as the in-shell and shelled kernels are prone to infestation by insects, pests, and fungi. Among several storage pests, the groundnut bruchid, Caryedon serratus, causes serious losses. Farmers often resort to different management practices, including hermetic storage, to control it. The moisture content of the commodity plays an important role in insect infestation during storage. Drying to safe moisture levels before storage is known to prevent the activity of various living organisms, such as storage pests. However, drying to low levels of moisture may not be economical for farmers, as they may not have access to devices to accurately check product moisture. In this regard, we wanted to demonstrate the efficacy of triple-layer hermetic storage bags in preventing the damage caused by C. serratus when the groundnuts are stored at intermediate (10%) and high (14%) levels of moisture compared to traditionally used bags such as polypropylene bags and jute bags. Groundnut pods at 10% moisture content and 14% moisture content were separately inoculated with adult bruchids and a toxigenic strain of Aspergillus flavus fungal inoculum before storing them for 6 months. Results from groundnut samples taken at two-month intervals indicated that groundnut pods stored in triple-layer hermetic bags were completely free from infestation by C. serratus by recording a zero number of eggs laid, number of pupae, adult emergence, percentage of loss, and percentage of damage up to 6 months of storage, by creating low oxygen (hypoxia) and high carbon dioxide (hypercarbia) conditions. Results also indicate no loss of pod weight stored in triple-layer bags, but a slight reduction in germination percentage was recorded due to a slight increase in fungal activity, but the reduction was significantly less in triple-layer plastic bags compared to other bag types. Similarly, biochemical constituents such as oil and protein content were slightly reduced in triple-layer plastic bags when pods were stored at a 10% moisture level, but a higher reduction was observed at a 14% moisture level. However, the reduction was very high and significant in other bag types at both 10 and 14% moisture levels

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

Long Non-Coding RNAs: New Insights in Neurodegenerative Diseases

Neurodegenerative diseases (NDDs), including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are gradually becoming a burden to society. The adverse effects and mortality/morbidity rates associated with these NDDs are a cause of many healthcare concerns. The pathologic alterations of NDDs are related to mitochondrial dysfunction, oxidative stress, and inflammation, which further stimulate the progression of NDDs. Recently, long non-coding RNAs (lncRNAs) have attracted ample attention as critical mediators in the pathology of NDDs. However, there is a significant gap in understanding the biological function, molecular mechanisms, and potential importance of lncRNAs in NDDs. This review documents the current research on lncRNAs and their implications in NDDs. We further summarize the potential implication of lncRNAs to serve as novel therapeutic targets and biomarkers for patients with NDDs