A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions.

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively.

Riluzole Enhancing Anti-PD-1 Efficacy by Activating cGAS/STING Signaling in Colorectal Cancer.

Colorectal cancer is the second leading cause of cancer mortality in the United States. Although immune checkpoint blockade therapies including anti-PD-1/PD-L1 have been successful in treating a subset of patients with colorectal cancer, the response rates remain low. We have found that riluzole, a well-tolerated FDA-approved oral medicine for treating amyotrophic lateral sclerosis, increased intratumoral CD8+ T cells and suppressed tumor growth of colon cancer cells in syngeneic immune-competent mice.

DNA strand breaks and gaps target retroviral intasome binding and integration.

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration.

Paroxysmal atrial fibrillation ablation with a novel temperature-controlled CF-sensing catheter: Q-FFICIENCY clinical and healthcare utilization benefits.

Introduction: The prospective, nonrandomized, multicenter Q-FFICIENCY study demonstrated the safety and 12-month efficacy of paroxysmal atrial fibrillation (AF) ablation with the novel QDOT MICRO temperature-controlled, contact force-sensing, radiofrequency (RF) catheter. Participants underwent pulmonary vein isolation with very high-power short-duration (vHPSD) mode (90 W, ≤4 s) alone or combined with conventional-power temperature-controlled (CPTC) mode (25-50 W). This study aimed to assess quality-of-life (QOL) and healthcare utilization (HCU) benefits experienced by Q-FFICIENCY study participants.

Very High-Power Short-Duration, Temperature-Controlled Radiofrequency Ablation in Paroxysmal Atrial Fibrillation: The Prospective Multicenter Q-FFICIENCY Trial.

Background: QDOT MICRO (QDM) is a novel contact force-sensing catheter optimized for temperature-controlled radiofrequency (RF) ablation. The very high-power short-duration (vHPSD) algorithm modulates power, maintaining target temperature during 90 W ablations for ≤4 seconds.

Objectives: This study aims to evaluate safety and 12-month effectiveness of the QDM catheter in paroxysmal atrial fibrillation (AF) ablation using the vHPSD mode combined with conventional-power temperature-controlled (CPTC) mode.

MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair.

Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD).

Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA.

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins.

Mass COVID-19 patient screening using UvsX and UvsY mediated DNA recombination and high throughput parallel sequencing.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions.

Evolutionary advantage of a dissociative search mechanism in DNA mismatch repair.

Protein complexes involved in DNA mismatch repair diffuse along dsDNA as sliding clamps in order to locate a hemimethylated incision site. They have been observed to use a dissociative mechanism, in which two proteins, while continuously remaining attached to the DNA, sometimes associate into a single complex sliding on the DNA and sometimes dissociate into two independently sliding proteins. Here, we study the probability that these complexes locate a given target site via a semi-analytic, Monte Carlo calculation that tracks the association and dissociation of the sliding complexes.

Strategies for Targeting Retroviral Integration for Safer Gene Therapy: Advances and Challenges.

Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration.

Mismatch repair: Choreographing accurate strand excision.

State-of-the-art genetic and cellular studies uniquely implicate the S. cerevisiae Pms1 endonuclease (human PMS2) and ExoI as the major components that produce and/or maintain the strand-specific nicks that precisely direct mismatch repair.

Retroviral prototype foamy virus intasome binding to a nucleosome target does not determine integration efficiency.

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD).

Linker domain function predicts pathogenic MLH1 missense variants.

The pathogenic consequences of 369 unique human HsMLH1 missense variants has been hampered by the lack of a detailed function in mismatch repair (MMR). Here single-molecule images show that HsMSH2-HsMSH6 provides a platform for HsMLH1-HsPMS2 to form a stable sliding clamp on mismatched DNA. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of clinically relevant HsMLH1 missense mutations.

Free Nipple Grafting and Nipple Sharing in Autologous Breast Reconstruction after Mastectomy.

Nipple sparing mastectomy is gaining popularity in recent years, as it provides superior aesthetic results and has a positive impact on the psychological well-being of patients. However, patients with macromastia and high grade ptosis are not good candidates for nipple sparing mastectomy due to a high risk for nipple necrosis; for these patients, the free nipple grafting (FNG) is an excellent option following autologous reconstruction. We herein present our experience with FNG for women with large and ptotic breasts undergoing mastectomy and autologous reconstruction.

Examination of the Therapeutic Potential of Mouse Oral Mucosa Stem Cells in a Wound-Healing Diabetic Mice Model.

Diabetic wounds' delayed healing response is still considered a major therapeutic challenge. Stem cells and derived cellular products have been an active field of research for novel therapies referred to as regenerative medicine. It has recently been shown that human oral mucosa stem cells (hOMSCs) are a readily accessible source for obtaining large quantities of stem cells.

Long-term safety and effectiveness of paroxysmal atrial fibrillation ablation using a porous tip contact force-sensing catheter from the SMART SF trial.

Purpose: The prospective, multicenter SMART SF trial demonstrated the acute safety and effectiveness of the 56-hole porous tip irrigated contact force (CF) catheter for drug-refractory paroxysmal atrial fibrillation (PAF) ablation with a low primary adverse event rate (2.5%), leading to FDA approval of the catheter. Here, we are reporting the long-term effectiveness and safety results that have not yet been reported.

A novel tissue culture model for evaluating the effect of aging on stem cell fate in adult microvascular networks.

In vitro models of angiogenesis are valuable tools for understanding the underlying mechanisms of pathological conditions and for the preclinical evaluation of therapies. Our laboratory developed the rat mesentery culture model as a new tool for investigating mechanistic cell-cell interactions at specific locations across intact blood and lymphatic microvascular networks ex vivo. The objective of this study was to report a method for evaluating the effect of aging on human stem cell differentiation into pericytes during angiogenesis in cultured microvascular networks.

MutL sliding clamps coordinate exonuclease-independent Escherichia coli mismatch repair.

A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites.

Nucleosome DNA unwrapping does not affect prototype foamy virus integration efficiency or site selection.

Eukaryotic DNA binding proteins must access genomic DNA that is packaged into chromatin in vivo. During a productive infection, retroviral integrases (IN) must similarly interact with chromatin to integrate the viral cDNA genome. Here we examine the role of nucleosome DNA unwrapping in the retroviral integrase search for a target site.

MutS homolog sliding clamps shield the DNA from binding proteins.

Sliding clamps on DNA consist of evolutionarily conserved enzymes that coordinate DNA replication, repair, and the cellular DNA damage response. MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recognizing mispaired nucleotides and in the presence of ATP form stable sliding clamps that randomly diffuse along the DNA. The MSH sliding clamps subsequently load MutL homolog (MLH/PMS) proteins that form a second extremely stable sliding clamp, which together coordinate downstream MMR components with the excision-initiation site that may be hundreds to thousands of nucleotides distant from the mismatch.

Expression and purification of nuclease-free protocatechuate 3,4-dioxygenase for prolonged single-molecule fluorescence imaging.

Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments.

Stochastic Processes and Component Plasticity Governing DNA Mismatch Repair.

DNA mismatch repair (MMR) is a DNA excision-resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch.

Coordinating Multi-Protein Mismatch Repair by Managing Diffusion Mechanics on the DNA.

DNA mismatch repair (MMR) corrects DNA base-pairing errors that occur during DNA replication. MMR catalyzes strand-specific DNA degradation and resynthesis by dynamic molecular coordination of sequential downstream pathways. The temporal and mechanistic order of molecular events is essential to insure interactions in MMR that occur over long distances on the DNA.