Search

Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such asEscherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs forHalomonas alkaliphila, Halomonas neptunia,andShewanella electrodiphila. Furthermore, we expand the list of functional ORIs forDuganella zoogloeoides,Pseudomonas alcaliphila,Shewanella oneidensisandShewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.

The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. In the present study, we used structure-based molecular docking of DNA polymerase β (Pol-β) and identified a potent small molecular weight inhibitor, NSC-666715. We determined the specificity of this small molecular weight inhibitor for Pol-β by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β–directed single-nucleotide and long-patch BER. The binding specificity of NSC-666715 with Pol-β was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug temozolomide (TMZ) to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-β activity. It blocked Pol-β–directed single-nucleotide and long-patch BER without affecting the activity of APE1, Fen1, and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-β and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the “proof-of-concept” for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer. (Mol Cancer Res 2009;7(12):1973–83)

Supplementary Data from A Novel Inhibitor of DNA Polymerase β Enhances the Ability of Temozolomide to Impair the Growth of Colon Cancer Cells

Related Article from Molecular Imaging of Inflammation and Carcinogenesis

Supplementary Figure legends for figures S1 and S2. Figure S1 Legend provides experimental information for the effect of YM155 on Panc-1 cells. Figure S2 Legend provides experimental information for the effect of YM155 on PC-3 cells.

Figure S1 shows the effect of YM155 on Panc-1 cells as evidenced by hoechst staining, DNA ladder assay and western blotting analysis of caspases 3, 8 as well as BID and PARP. Figure S2 shows the effect of YM155 on another pancreatic cell line PC-3 as evidenced by hoechst staining and western blotting analysis of survivin and DR5 expression.

Development of imaging agents that can be used broadly for early detection of neoplasia at various tissue sites and at various stages of disease and that also can assess states of minimal residual disease would have tremendous utility in the diagnosis and management of cancer. In a series of articles culminating with a report in this issue of the journal (beginning on page 1536), Uddin and colleagues show their ability to systemically target the enzyme COX-2 with imaging probes that will serve as agents for early detection, risk assessment, prognosis, and intervention outcome measures. These probes will enable the detection and localization of regions of inflammation and a wide variety of premalignant lesions and cancers, with utility in monitoring the effects of cancer prevention and therapy. Cancer Prev Res; 4(10); 1523–6. ©2011 AACR.

There is an urgent need to understand the functional effects of mutations in emerging variants of SARS-CoV-2. Variants of concern (alpha, beta, gamma and delta) acquired four patterns of spike glycoprotein mutations that enhance transmissibility and immune evasion: 1) mutations in the N-terminal domain (NTD), 2) mutations in the Receptor Binding Domain (RBD), 3) mutations at interchain contacts of the spike trimer, and 4) furin cleavage site mutations. Most distinguishing mutations among variants of concern are exhibited in the NTD, localized to sites of high structural flexibility. Emerging variants of interest such as mu, lambda and C.1.2 exhibit the same patterns of mutations as variants of concern. There is a strong likelihood that SARS-CoV-2 variants will continue to emerge with mutations in these defined patterns, thus providing a basis for the development of next line antiviral drugs and vaccine candidates.

Supplementary Fig. from Z3, a novel Jak2 tyrosine kinase small-molecule inhibitor that suppresses Jak2-mediated pathologic cell growth

Jak2 tyrosine kinase is essential for animal development and hyperkinetic Jak2 function has been linked to a host of human diseases. Control of this pathway using Jak2-specific inhibitors would therefore potentially serve as a useful research tool and/or therapeutic agent. Here, we used a high-throughput program called DOCK to predict the ability of 20,000 small molecules to interact with a structural pocket adjacent to the ATP-binding site of murine Jak2. One small molecule, 2-methyl-1-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-1-one (herein designated as Z3), bound to Jak2 with a favorable energy score. Z3 inhibited Jak2-V617F and Jak2-WT autophosphorylation in a dose-dependent manner but was not cytotoxic to cells at concentrations that inhibited kinase activity. Z3 selectively inhibited Jak2 kinase function with no effect on Tyk2 or c-Src kinase function. Z3 significantly inhibited proliferation of the Jak2-V617F-expressing, human erythroleukemia cell line, HEL 92.1.7. The Z3-mediated reduction in cell proliferation correlated with reduced Jak2 and STAT3 tyrosine phosphorylation levels as well as marked cell cycle arrest. Finally, Z3 inhibited the growth of hematopoietic progenitor cells isolated from the bone marrow of an essential thrombocythemia patient harboring the Jak2-V617F mutation and a polycythemia vera patient carrying a Jak2-F537I mutation. Collectively, the data suggest that Z3 is a novel specific inhibitor of Jak2 tyrosine kinase. [Mol Cancer Ther 2008;7(8):2308–18]

This study tested biological sensitivity to context theory in the peer context. Respiratory sinus arrythmia (RSA-R) and skin conductance level (SCL-R) reactivity to a peer stressor were collected for participants (N = 86; M age = 45.99 months old; 70.2% White) in the summer (Time 1). Children’s peer risk (i.e., physical and relational victimization) and protective (i.e., received prosocial behavior) factors were examined in the fall (T2) and relational and physical aggression were measured at T2 and in the spring (T3). Interactions were tested in regression analyses. Interactions emerged between relational victimization, RSA-R, and SCL-R in the prediction of T3 relational aggression and between received prosocial behavior, RSA-R, and SCL-R in the prediction of T3 relational and physical aggression, respectively. There was a positive relation between T2 relational victimization and T3 relational aggression for children with a coactivation pattern (i.e., increased RSA and SCL activity to a bullying stressor) but no relation for any other physiological pattern. Conversely, there was a negative relation between T2 received prosocial behavior and both forms of aggression at T3 for children with a reciprocal pattern (i.e., increased RSA and decreased SCL or decreased RSA and increased SCL activity) but no protective benefit of received prosocial behavior on subsequent aggression for children with a coactivation pattern. For children with a coinhibition pattern (i.e., decreased RSA and SCL activity), received prosocial behavior was negatively related to subsequent physical but not relational aggression. In sum, a coactivation pattern in response to stress may represent a vulnerability factor.

Introduction A multicenter adult inflammatory bowel disease learning health system (IBD Qorus) implemented clinical care process changes for reducing unplanned emergency department visits and hospitalizations using a Breakthrough Series Collaborative approach. Methods Using Markov decision models, we determined the health economic impact of participating in the Collaborative from the third-party payer perspective. Results Across all 23 sites, participation in the Collaborative was associated with lower annual costs by an average of $2,528 ± $233 per patient when compared with the baseline period. Discussion Implementing clinical care process changes using a Collaborative approach was associated with overall cost savings. Future work should examine which specific interventions are most effective and whether such cost savings are sustainable.

We apply dynamic programming to solve a long-horizon fund choice problem, given that the underlying market can switch between different regimes. The objective function is based on reaching a target level of wealth, following the paradigm of goal-based investing. In a world with a good regime (e.g., a bull market) and a bad regime (e.g., a bear market), we find that an investor who is cognizant of regime switching can potentially do much better over time than an investor who assumes there is only one regime. However, there is a caveat---an investor must be able to predict the regime they are in with reasonable levels of confidence, and if not, they are in fact worse off than an investor who assumes just one regime. Using data from recent history, we find that investors may be better off not switching from existing single-regime models to more complex multiple-regime models.

INTRODUCTION: There is significant variation in processes and outcomes of care for patients with inflammatory bowel disease (IBD), suggesting opportunities to improve quality of care. We aimed to determine whether a structured quality of care program can improve IBD outcomes, including the need for unplanned health care utilization. METHODS: We used a structured approach to improve adult IBD care in 27 community-based gastroenterology practices and academic medical centers. Patient-reported outcomes (PRO) and health care utilization were collected at clinical visits. Outcomes were monitored monthly using statistical process control charts; improvement was defined by special cause (nonrandom) variation over time. Multivariable logistic regression was applied to patient-level data. Nineteen process changes were offered to improve unplanned health care utilization. Ten outcomes were assessed, including disease activity, remission status, urgent care need, recent emergency department use, hospitalizations, computed tomography scans, health confidence, corticosteroid or opioid use, and clinic phone calls. RESULTS: We collected data prospectively from 20,382 discrete IBD visits. During the 15-month project period, improvement was noted across multiple measures, including need for urgent care, hospitalization, steroid use, and opioid utilization. Adjusted multivariable modeling showed significant improvements over time across multiple outcomes including urgent care need, health confidence, emergency department utilization, hospitalization, corticosteroid use, and opioid use. Attendance at monthly coached webinars was associated with improvement. DISCUSSION: Outcomes of IBD care were improved using a structured quality improvement program that facilitates small process changes, sharing of best practices, and ongoing feedback. Spread of these interventions may facilitate broad improvement in IBD care when applied to a large population.