A Rapid, Simple DNA Mismatch Repair Substrate Construction Method

A more flexible and higher-yielding in vitro DNA mismatch repair (MMR) substrate construction method, which was developed initially by Wang and Hays, is described for the construction of a nucleotide-based chemical mismatch (G/IU) and a G/T mismatch. Our modifications use the combination of two endonuclease enzymes (NheI and BciVI) and two new redesigned plasmids (pWDAH1A and pWDSH1B). In our modified methodology, plasmids are initially digested with the nicking endonucleases, followed by the streptavidin treatment. The mismatch-containing oligo is then annealed to the gap DNA and finally ligated to produce a mismatch-containing DNA substrate. We report a high efficiency (up to 90%) of these mismatch substrates and confirm recognition using a functional assay. These modifications, coupled with the use of the redesigned plasmids, can be applied for the construction of other types of chemically induced mismatches as well as insertion-deletion loops for future in vitro studies of MMR processing by our group and others

Impact of Autophagy on Chemotherapy and Radiotherapy Mediated Tumor Cytotoxicity: “To Live or not to Live”

Autophagy, a highly regulated cell “self-eating” pathway, is controlled by the action of over 34 autophagy-related proteins (collectively termed Atgs). Although they are fundamentally different processes, autophagy and apoptosis (type I programmed cell death), under certain circumstances, can be regulated by common signaling mediators. Current cancer therapies including chemotherapy and ionizing radiation are known to induce autophagy within tumor cells. However, autophagy plays a dual role of either pro-cell survival or pro-cell death in response to these cancer treatments, depending on the cellular context and the nature of the treatment. We review the current basic and translational cancer research literature on how autophagy impacts tumor cell survival (“to live”) and death (“not to live”) following treatment as well as the role of chemical mediators of autophagy

In silico and in vitro screening for potential anticancer candidates targeting GPR120

The G-protein coupled receptor - GPR120 has recently been implicated as a novel target for colorectal cancer (CRC) and other cancer managements. In this study, a homology model of GPR120S (short isoform) was generated to identify potential anti-cancer compounds targeting the GPR120 receptor using a combined in silico docking-based virtual screening (DBVS), structure-activity relationships (SAR) and in vitro screening approach. SPECS database of synthetic chemical compounds (~350,000) was screened using the developed GPR120S model to identify molecules binding to the orthosteric binding pocket followed by an AutoDock SMINA rigid-flexible docking protocol. The best 13 hit molecules were then tested in vitro to evaluate their cytotoxic activity against SW480 - human CRC cell line expressing GPR120. The test compound 1 (3-(4-methylphenyl)-2-[(2-oxo-2-phenylethyl)sulfanyl]-5,6-dihydrospiro(benzo[h]quinazoline-5,1\u27-cyclopentane)-4(3H)-one) showed ~ 90% inhibitory effects on cell growth with micromolar affinities (IC50 = 23.21-26.69 µM). Finally, SAR analysis of compound 1 led to the identification of a more active compound from the SPECS database showing better efficacy during cell-based cytotoxicity assay -5 (IC50 = 5.89-6.715 µM), while a significant reduction in cytotoxic effects of 5 was observed in GPR120-siRNA pre-treated SW480 cells. The GPR120S homology model generated, and SAR analysis conducted by this work discovered a potential chemical scaffold, dihydrospiro(benzo[h]quinazoline-5,1\u27-cyclopentane)-4(3H)-one, which will aid future research on anti-cancer drug development for CRC management

DETECTING ARCHAEOLOGICAL SIGNATURES IN SHALLOW WATER: A STUDY OF THE CHICAMACOMICO RACES BATTLESCAPE (1-5 OCTOBER 1861)

After the Confederate surrender at Forts Hatteras and Clark in August of 1861, the Union took control of Pamlico Sound. Confederate soldiers, however, remained in control of an outpost on nearby Roanoke Island. This was their last line of defense against Union dominance of eastern North Carolina. In September 1861, Union forces set up an outpost at Chicamacomico (present-day Rodanthe on Hatteras Island). Once the Confederates discerned the Union's presence at Chicamacomico, they launched an attack to retake Pamlico Sound. Both Union and Confederate forces engaged each other on land and at sea. There were few casualties and the balance of power did not shift between the two sides. The "Chicamacomico Encounter" (also known as the "Chicamacomico Affair," and "Chicamacomico Races") has been documented in the historical record; however there has not been extensive archaeological study of the battlefield. This may be because it represents a challenge to battlefield archaeologists. The area of conflict is not only inundated with sand and vegetation, but it has also gone through drastic coastal change. Moreover, its marine battlescape is potentially expansive, and lies within exceptionally shallow water -- requiring the adaptation and combination of terrestrial and underwater archaeological surveying techniques to study it. The present study is an archaeological analysis of the battlescape which utilized the principles of KOCOA survey techniques from the American Battlefield Protection Program (ABPP) to reconstruct battlefield behavior, analyze tactics and strategy, and in doing so considered the pros and cons of various surveying and remote sensing methodologies

Rubric Design and Development for English Speaking Practice and Performance in the First-year University Classroom

As part of an ongoing action research project researchers collaborated on the creation,administration, and evaluation of a formative speaking assessment tool. A primary element of this tool was an analytic rubric to help first- and second year university students across departments practice and improve their speaking skills.The impetus of this research emerged from the results of previous speaking assessment studies involving second-year students. Though the previous learning outcomes appeared to have been achieved on average, the evaluation results of the second-year students tended to range around the high end of the grading rubric. This prompted researchers to have a closer look at the most recent rubric in use and improve its clarity, comprehensibility and transparency.The assessment tool and rubric integrated clear learning objectives, independent practice, peer evaluation and transparent instructor evaluation enabling researchers to focus on supporting student learning and confidence. The research questions were: 1) How did editing and revising the rubric affect student results? 2) How did editing and revising the rubric increase student confidence?This study took a mixed method approach where data from student grades and survey results were collected and analyzed. The data from the assessments showed that after introducing the newest version of the rubric student grades showed less extreme off shoots and were centered around the expected results. In addition, survey responses were positive regarding the rubric’s efficacy in supporting students’ English language speaking confidence

G-Protein-Coupled Receptors as Therapeutic Targets for Glioblastoma

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour in adults. Treatments include surgical resection, radiotherapy, and chemotherapy. Despite this, the prognosis remains poor, with an impacted quality of life during treatment coupled with brain tumour recurrence; thus, new treatments are desperately needed. In this review, we focus on recent advances in G-protein-coupled receptor (GPCR) targets. To date, the most promising targets are the chemokine, cannabinoid, and dopamine receptors, but future work should further examine the melanocortin receptor-4 (MC4R), adhesion, lysophosphatidic acid (LPA) and smoothened (Smo) receptors to initiate new drug-screening strategies and targeted delivery of safe and effective GBM therapies

Cerebrospinal fluid immunoglobulin light chain ratios predict disease progression in multiple sclerosis

Objective To determine whether the ratio of cerebrospinal fluid (CSF) immunoglobulin kappa to lambda light chains at time of multiple sclerosis (MS) diagnosis predicts disease progression and whether this was intrinsic to CSF plasmablasts. Methods CSF and peripheral blood were obtained from patients undergoing elective diagnostic lumbar puncture and included clinically isolated syndrome (CIS) (n=43), relapsing remitting MS (RRMS; n=50), primary progressive MS (PPMS; n=20) and other neurological disease controls, both inflammatory (ONID; n=23) and non-inflammatory (OND; n=114). CSF samples were assayed for free and immunoglobulin-associated light chains and on B cells and plasmablasts. Clinical follow-up data were collected during a 5-year follow-up period where available. Results There was an increased median CSF κ:λ free light chain (FLC) in all MS groups (CIS: 18.2, 95% CI 6.8 to 30.3; RRMS: 4.4, 95% CI 2.7 to 11.4; PPMS: 12.0, 95% CI 3.6 to 37.1) but not controls (OND: 1.61, 95% CI 1.4 to 1.9; ONID: 1.7, 95% CI 1.3 to 2.2; p10) CSF κ:λ FLC (0.0, 95% CI 0 to 2.5 vs 2.5, 95% CI 0 to 4, high vs low; p=0.049). CSF κ:λ FLC correlated with CSF IgG1 κ:λ (r=0.776; p<0.0001) and was intrinsic to CSF plasmablasts (r=0.65; p=0.026). Conclusions These data demonstrate that CSF immunoglobulin κ:λ ratios, determined at the time of diagnostic lumbar puncture, predict MS disease progression and may therefore be useful prognostic markers for early therapeutic stratification