Methylerythritol Phosphate Pathway: Enzymatic Evidence for a Rotation in the LytB/IspH-Catalyzed Reaction.

IspH/LytB, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of the methylerythritol phosphate (MEP) pathway, a target for the development of new antimicrobial agents. This metalloenzyme converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, the synthesis of (S)-[4-2 H1 ]HMBPP and (R)-[4-2 H1 ]HMBPP is reported together with a detailed NMR analysis of the products formed after their respective incubation with E. coli IspH/LytB in the presence of the biological reduction system used by E. coli to reduce the [4Fe-4S] center. (S)-[4-2 H1 ]HMBPP was converted into [4-2 H1 ]DMAPP and (E)-[4-2 H1 ]IPP, whereas (R)-[4-2 H1 ]HMBPP yielded [4-2 H1 ]DMAPP and (Z)-[4-2 H1 ]IPP, hence providing the direct enzymatic evidence that the mechanism catalyzed by IspH/LytB involves a rotation of the CH2 OH group of the substrate to display it away from the [4Fe-4S].

Population of Patients With Traumatic Brain Injury in Skilled Nursing Facilities: A Decade of Change.

OBJECTIVE: To describe the natural history of patients with traumatic brain injury (TBI) admitted to skilled nursing facilities (SNFs) following hospitalizations. SETTING: Between 2005 and 2014. PARTICIPANTS: Adults who had incident admissions to skilled nursing facilities (SNFs) with a diagnosis of TBI. DESIGN: Retrospective review of the Minimum Data Set. MAIN MEASURES: Main variables were cognitive and physical function, length of stay, presence of feeding tube, terminal condition, and dementia. RESULTS: Incident admissions to SNFs increased annually from 17 247 patients to 20 787 from 2005 to 2014. The percentage of patients with activities of daily living score 23 or more decreased from 25% to 14% (P < .05). The overall percentage of patients with severe cognitive impairment decreased from 18% to 10% (P < .05). More patients had a diagnosis of dementia in 2014 compared with previous years (P < .05), and the presence of a terminal condition increased from 1% to 1.5% over the 10-year period (P < .05). The percentage of patients who stayed fewer than 30 days was noted to increase steadily over the 10 years, starting with 48% in 2005 and ending with 53% in 2013 (P < .05). CONCLUSION: Understanding past trends in TBI admissions to SNFs is necessary to guide appropriate discharge and predict future demand, as well as inform SNF policy and practice necessary to care for this subgroup of patients.

Competition between phytoplankton and bacteria: exclusion and coexistence.

Resource-based competition between microorganisms species in continuous culture has been studied extensively both experimentally and theoretically, mostly for bacteria through Monod and Contois "constant yield" models, or for phytoplankton through the Droop "variable yield" models. For homogeneous populations of N bacterial species (Monod) or N phytoplanktonic species (Droop), with one limiting substrate and under constant controls, the theoretical studies indicated that competitive exclusion occurs: only one species wins the competition and displaces all the others (Armstrong and McGehee in Am Nat 115:151, 1980; Hsu and Hsu in SIAM J Appl Math 68:1600-1617, 2008). The winning species expected from theory is the one with the lowest "substrate subsistence concentration" s([star]), such that its corresponding equilibrium growth rate is equal to the dilution rate D. This theoretical result was validated experimentally with phytoplankton (Tilman and Sterner in Oecologia 61(2):197-200, 1984) and bacteria (Hansen and Hubell in Science 207(4438):1491-1493, 1980), and observed in a lake with microalgae (Tilman in Ecology 58(22):338-348, 1977). On the contrary for aggregating bacterial species described by a Contois model, theory predicts coexistence between several species (Grognard et al. in Discrete Contin Dyn Syst Ser B 8(1):73-93, 2007). In this paper we present a generalization of these results by studying a competition between three different types of microorganisms: planktonic (or free) bacteria (represented by a generalized Monod model), aggregating bacteria (represented by a Contois model) and free phytoplankton (represented by a Droop model). We prove that the outcome of the competition is a coexistence between several aggregating bacterial species with a free species of bacteria or phytoplankton, all the other free species being washed out. This demonstration is based mainly on the study of the substrate concentration's evolution caused by competition; it converges towards the lowest subsistence concentration s([star]), leading to three different types of competition outcomes: (1) the best free bacteria or phytoplankton competitor excludes all other species; (2) only some aggregating bacterial species coexist in the chemostat; (3) A coexistence between the single best free species, with one or several aggregating species.

Development of a Web-based surgical booking and informed consent system to reduce the potential for error and improve communication.

BACKGROUND: The decision to perform an elective procedure often originates during an office visit between surgeon and patient. Several administrative tasks follow, including scheduling or "booking" of the case and obtaining informed consent. These processes require communicating accurate information regarding diagnosis, procedure, and other patient-specific details necessary for the safe and effective performance of an operation. Nonstandardized and paper-based consents pose difficulty with legibility, portability, and consistency, thereby representing a source of potential error and inefficiency. There are numerous barriers to efficiently booking elective surgical procedures and obtaining a legible, complete, and easily retrievable informed consent. An integrated Web-based booking and consent system was developed at a multisite university-affiliated community hospital system to improve the speed and quality of work flow, as well as communication with both the patients and staff. METHODS: A booking and consent system was developed and made available over the intranet. This customized system was created by leveraging existing information systems. RESULTS: The electronic consent system uses surgeon-specific templates and allows for a consistent approach to each procedure. A printed consent form can be generated at any time from any of the health care system's three campuses and is commonly stored in the electronic medical record. Integration into our perioperative system allows for coordination with the operating room staff, administrative personal, financial coordinators, and central supply. Total systems expenditure for development was estimated at $40,000 (US). CONCLUSIONS: Organizations considering standardizing their own consent and operating room booking processes can review this experience in making their own "make or buy" decision for their own settings.

Stabilizing effect of cannibalism in a two stages population model.

In this paper we build a prey-predator model with discrete weight structure for the predator. This model will conserve the number of individuals and the biomass and both growth and reproduction of the predator will depend on the food ingested. Moreover the model allows cannibalism which means that the predator can eat the prey but also other predators. We will focus on a simple version with two weight classes or stage (larvae and adults) and present some general mathematical results. In the last part, we will assume that the dynamics of the prey is fast compared to the predator's one to go further in the results and eventually conclude that under some conditions, cannibalism can stabilize the system: more precisely, an unstable equilibrium without cannibalism will become almost globally stable with some cannibalism. Some numerical simulations are done to illustrate this result.

Needs Assessment for a Resuscitative Thoracotomy Curriculum for General Surgery Residents in the Northeast Region.

INTRODUCTION: Resuscitative thoracotomy (RT) is a high-acuity low occurrence (HALO) procedure with which general surgical resident (GSR) experience and confidence are unknown. We sought to identify and describe this educational gap by conducting a targeted needs assessment for an RT curriculum for GSRs. METHODS: An online regional needs assessment survey was conducted for an RT curriculum for GSRs. The survey was developed by a group of trauma stakeholders and revised after being piloted on a small, representative group of GSRs. We surveyed GSRs in the Northeast region regarding their experience and confidence with RT; interest in an RT curriculum; and content, format, and scope for an RT curriculum. RESULTS: The survey response rate was 43%, reflecting the viewpoints of GSRs at 8 major training centers across the Northeast. Only 13% of respondents were interested in pursuing a career in Trauma and Critical Care despite 97% of them training at a Level I Trauma Center. Twenty-nine percent and 33% of GSRs had ever assisted with or performed RT, respectively. Twenty-one percent of GSRs reported feeling confident performing RT. Most respondents (98%) agreed or strongly agreed that an RT curriculum would add value to their general surgery education. The most positively rated content topics were resuscitative maneuvers (100% positive responses [PR]), when to cease resuscitative efforts (100% PR), and morbidity and mortality associated with RT (98% PR). The most highly rated learning methods were individual RT simulation time (97% PR) and a tour of the trauma bay equipment (97% PR). CONCLUSIONS: This needs assessment demonstrates a lack of experience and confidence with RT, a strong learner interest in an RT curriculum, and a desire for experiential learning methods. Learning objectives are defined herein, and the next steps involve developing educational materials for an RT curriculum for GSRs.

Improved amputation-free survival in unreconstructable critical limb ischemia and its implications for clinical trial design and quality measurement.

OBJECTIVE: Amputation-free survival (AFS), a composite endpoint of mortality and amputation, is the preferred outcome measure in critical limb ischemia (CLI). Given the improvements in systemic management of atherosclerosis and interventional management of limb ischemia over the past 2 decades, we examined whether these outcomes have changed in patients with CLI without revascularization options (no option-critical limb ischemia [NO-CLI]). METHODS: We reviewed the literature for published 1-year AFS, mortality, and amputation rates from control groups in NO-CLI trials. Summary proportions of events were estimated by conducting a random effects meta-analysis of proportions. To determine whether there had been any change in event rates over time, we performed a random effects meta-regression and a mixed effects logistic regression, both regressed against the variable "final year of recruitment." RESULTS: Eleven trials consisting of 886 patients satisfied search criteria, 7 of which presented AFS data. Summary proportion of events (95% confidence interval) were 0.551 (0.399 to 0.693) for AFS; 0.198 (0.116 to 0.317) for death; and 0.341 (0.209 to 0.487) for amputation. Regression analyses demonstrated that AFS has risen over time as mortality rates have fallen, and these improvements are statistically significant. The decrease in amputation rates failed to reach statistical significance. The lack of published data precluded a quantitative evaluation of any change in the clinical severity or comorbidities in the NO-CLI population. CONCLUSIONS: AFS and mortality rates in NO-CLI have improved over the past 2 decades. Due to declining event rates, clinical trials may underestimate treatment effects and thus fail to reach statistical significance unless sample sizes are increased or unless a subgroup with a higher event rate can be identified. Alternatively, comparing outcomes to historical values for quality measurement may overestimate treatment effects. Benchmark values of AFS and morality require periodic review and updating.

The role of amputation as an outcome measure in cellular therapy for critical limb ischemia: implications for clinical trial design.

BACKGROUND: Autologous bone marrow-derived stem cells have been ascribed an important therapeutic role in No-Option Critical limb Ischemia (NO-CLI). One primary endpoint for evaluating NO-CLI therapy is major amputation (AMP), which is usually combined with mortality for AMP-free survival (AFS). Only a trial which is double blinded can eliminate physician and patient bias as to the timing and reason for AMP. We examined factors influencing AMP in a prospective double-blinded pilot RCT (2:1 therapy to control) of 48 patients treated with site of service obtained bone marrow cells (BMAC) as well as a systematic review of the literature. METHODS: Cells were injected intramuscularly in the CLI limbs as either BMAC or placebo (peripheral blood). Six month AMP rates were compared between the two arms. Both patient and treating team were blinded of the assignment in follow-up examinations. A search of the literature identified 9 NO-CLI trials, the control arms of which were used to determine 6 month AMP rates and the influence of tissue loss. RESULTS: Fifteen amputations occurred during the 6 month period, 86.7% of these during the first 4 months. One amputation occurred in a Rutherford 4 patient. The difference in amputation rate between patients with rest pain (5.6%) and those with tissue loss (46.7%), irrespective of treatment group, was significant (p = 0.0029). In patients with tissue loss, treatment with BMAC demonstrated a lower amputation rate than placebo (39.1% vs. 71.4%, p = 0.1337). The Kaplan-Meier time to amputation was longer in the BMAC group than in the placebo group (p = 0.067). Projecting these results to a pivotal trial, a bootstrap simulation model showed significant difference in AFS between BMAC and placebo with a power of 95% for a sample size of 210 patients. Meta-analysis of the literature confirmed a difference in amputation rate between patients with tissue loss and rest pain. CONCLUSIONS: BMAC shows promise in improving AMP-free survival if the trends in this pilot study are validated in a larger pivotal trial. The difference in amp rate between Rutherford 4 & 5 patients suggests that these patients should be stratified in future RCTs.

Optimizing surgical stabilization of rib fractures using intraoperative ultrasound localization.

BACKGROUND: Surgical stabilization of rib fractures (SSRF) significantly improve the outcomes of patients with rib fractures. Ultrasound is a specific modality for localizing rib fractures. We hypothesized that use of perioperative ultrasound localization of fracture sites optimizes surgical approach and clinical outcomes. METHODS: We performed a retrospective cohort study of adult patients undergoing SSRF and compared those with and without adjunctive perioperative ultrasound fracture localization. Our primary outcome was improved surgical efficiency as measured by incision length and total operative time. Secondary clinical outcomes included numeric pain score on follow-up visit and daily morphine milligram equivalent prescribed within 30 days from discharge. RESULTS: We performed 49 surgical rib fixations between 2015 and 2020; of which, 13 (26.5%) additionally underwent ultrasound localization (26.5%). There were no significant differences between groups in age, sex, number of ribs repaired, or days till surgery. More patients in the ultrasound group had nonflail chest wall injury (76.9% vs. 27.8%, p = 0.003). Use of perioperative ultrasound was associated with shorter incision length (median, 9 vs. 15.5 cm; p = 0.0001), shorter operative time (median, 120 vs. 174 minutes; p = 0.003), less daily morphine milligram equivalent (25 vs. 68 mg, p = 0.009), and reduced numeric pain score on follow up (median, 4 vs. 7, p = 0.05). CONCLUSION: Use of perioperative ultrasound localization of rib fractures to optimize surgical approach for SSRF was associated with reduced incision length, operative time, and opioid requirements on patient discharge. We recommend considering routine perioperative localization to improve surgical approach and efficiency during SSRF. LEVEL OF EVIDENCE: Therapeutic, level IV.

How does abundance scale with body size in coupled size-structured food webs?

1. Widely observed macro-ecological patterns in log abundance vs. log body mass of organisms can be explained by simple scaling theory based on food (energy) availability across a spectrum of body sizes. The theory predicts that when food availability falls with body size (as in most aquatic food webs where larger predators eat smaller prey), the scaling between log N vs. log m is steeper than when organisms of different sizes compete for a shared unstructured resource (e.g. autotrophs, herbivores and detritivores; hereafter dubbed 'detritivores'). 2. In real communities, the mix of feeding characteristics gives rise to complex food webs. Such complexities make empirical tests of scaling predictions prone to error if: (i) the data are not disaggregated in accordance with the assumptions of the theory being tested, or (ii) the theory does not account for all of the trophic interactions within and across the communities sampled. 3. We disaggregated whole community data collected in the North Sea into predator and detritivore components and report slopes of log abundance vs. log body mass relationships. Observed slopes for fish and epifaunal predator communities (-1.2 to -2.25) were significantly steeper than those for infaunal detritivore communities (-0.56 to -0.87). 4. We present a model describing the dynamics of coupled size spectra, to explain how coupling of predator and detritivore communities affects the scaling of log N vs. log m. The model captures the trophic interactions and recycling of material that occur in many aquatic ecosystems. 5. Our simulations demonstrate that the biological processes underlying growth and mortality in the two distinct size spectra lead to patterns consistent with data. Slopes of log N vs. log m were steeper and growth rates faster for predators compared to detritivores. Size spectra were truncated when primary production was too low for predators and when detritivores experienced predation pressure. 6. The approach also allows us to assess the effects of external sources of mortality (e.g. harvesting). Removal of large predators resulted in steeper predator spectra and increases in their prey (small fish and detritivores). The model predictions are remarkably consistent with observed patterns of exploited ecosystems.

About the Crystallization of Abiotic Coded Matter.

The crystallization of digitally encoded polyurethanes was studied by electron diffraction. A series of oligomers with different primary structures was analyzed in this work. They all form hydrogen-bonding-directed lamellar single crystals with a base-centered orthorhombic unit cell. Although crystal morphology was the same in all cases, the digital coding of the oligomers has a small influence on the intersheet distance in the crystals. The crystal lattices allow calculation of the volume occupied by one basic information unit, which is in the range 148-188 Å3. Interestingly, this volume is about 3× smaller than that occupied by a coded nucleotide in a DNA double helix. Furthermore, crystallization of blends of oligourethanes with different coded primary structures was investigated. Oligomers with drastically different monomer compositions form structures that are not cocrystals but more probably segregated crystals containing distinct domains of different composition.

Efficient Protocol for the Synthesis of "N-Coded" Oligo- and Poly(N-Substituted Urethanes).

Sequence-defined poly(N-substituted urethanes) were synthesized via a solid-phase iterative protocol including two successive orthogonal coupling steps: the formation of an activated carbonate and its chemoselective reaction with the secondary amine group of amino alcohol building blocks. This simple method was used to write binary information on the formed polymers using four-coded molecules, 2-(methylamino)ethanol, 2-(ethylamino)ethanol, 2-(propylamino)ethanol, and 2-(butylamino)ethanol, symbolizing binary dyads 00, 01, 10, and 11, respectively. The method is fast and allows synthesis of uniform oligomers and polymers with controlled lengths (4-mer to 28-mer) and digital information sequences. Furthermore, the coded poly(N-substituted urethanes) were easily characterized by electrospray mass spectrometry and decoded by tandem mass spectrometry. Overall, these digital macromolecules offer interesting advantages over conventional sequence-coded polyurethanes, i.e., synthesis of longer chains, reduced synthesis times, and better solubility and processing in common organic solvents.

RNA interference demonstrates a novel role for integrin-linked kinase as a determinant of pancreatic adenocarcinoma cell gemcitabine chemoresistance.

Integrin-linked kinase (ILK) facilitates signal transduction between extracellular events and important intracellular survival pathways involving protein kinase B/Akt. We examined the role of ILK in determining pancreatic adenocarcinoma cellular chemoresistance to the nucleoside analogue gemcitabine. Cellular ILK expression was quantified by Western blot analysis. We examined the effects of overexpression of active ILK and of ILK knockdown induced by RNA interference on gemcitabine chemoresistance. We also examined the effects of modulating ILK expression on gemcitabine-induced caspase 3-mediated apoptosis, phosphorylation status of Akt (Ser473) and glycogen synthase kinase. Overexpression of ILK increased cellular gemcitabine chemoresistance, whereas ILK knockdown induced chemosensitization via increased caspase 3-mediated apoptosis. ILK knockdown attenuated Akt Ser473 and glycogen synthase kinase phosphorylation, whereas overexpression of constitutively active myristoylated Akt was sufficient to induce significant recovery in gemcitabine chemoresistance in the presence of ILK knockdown. Levels of ILK expression affect gemcitabine chemoresistance in pancreatic adenocarcinoma cells. This novel finding suggests that therapies directed against ILK and its downstream signaling targets may have the potential to enhance the efficacy of gemcitabine-based chemotherapy.

A novel role for carcinoembryonic antigen-related cell adhesion molecule 6 as a determinant of gemcitabine chemoresistance in pancreatic adenocarcinoma cells.

Most patients with pancreatic adenocarcinoma present with surgically incurable disease. Gemcitabine, the principal agent used to treat such patients, has little impact on outcome. Overexpression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6, a feature of this malignancy, is associated with resistance to anoikis and increased metastasis. The purpose of this study was to determine the role of CEACAM6 in cellular chemoresistance to gemcitabine. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express very low levels of the protein. Suppression of CEACAM6 expression was achieved in BxPC3 cells, which inherently overexpress CEACAM6, by stable transfection of a CEACAM6 small interfering RNA-generating vector. The effects of modulating CEACAM6 expression on gemcitabine-induced cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay, flow cytometric apoptosis quantification, caspase profiling, and Western analysis of cytoplasmic cytochrome c release. The roles of Akt and c-Src kinases as downstream targets of CEACAM6 signaling were examined. Stable overexpression of CEACAM6 in Capan2 increased gemcitabine chemoresistance, whereas CEACAM6 gene silencing in BxPC3 markedly increased the sensitivity of these cells to gemcitabine. Differential expression of CEACAM6 modulates Akt activity in a c-Src-dependent manner, and CEACAM6 overexpression appears to protect cells from cytochrome c-induced caspase 3 activation and apoptosis.

Prostaglandin E2 enhances pancreatic cancer invasiveness through an Ets-1-dependent induction of matrix metalloproteinase-2.

Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2) in the pathogenesis of a wide range of malignancies. Here we tested the hypothesis that the COX-2 product prostaglandin E(2) (PGE(2)) increases cellular invasive potential by inducing matrix metalloproteinase-2 (MMP-2) expression and activity through an extracellular signal-regulated kinase (ERK)/Ets-1-dependent mechanism in pancreatic cancer. PANC-1 and MIAPaCa-2 pancreatic cancer cells were treated with PGE(2) or rofecoxib, a selective COX-2 inhibitor. MMP-2 expression and activity were assayed using Western blot analysis and zymography, respectively. MMP-2 promoter activity was analyzed with a luciferase-based assay. Ets-1 activity was analyzed using gel shift assay. Ets-1 expression was specifically silenced using RNA interference. Cellular invasive and migratory potentials were determined using a Boyden chamber assay with or without Matrigel, respectively. Exogenous PGE(2) induced MMP-2 expression and activity and increased ERK1/2 phosphorylation, Ets-1 binding activity, and MMP-2 promoter activity. PGE(2) also increased cellular migratory and invasive potentials. The mitogen-activated protein kinase kinase inhibitor PD98059 and Ets-1 silencing each abolished PGE(2)-induced increases in MMP-2 expression. PD98059 and Ets-1 silencing each abrogated the effect of PGE(2) on cellular invasive potential but not on cellular migratory potential. Rofecoxib suppressed MMP-2 expression and activity, Ets-1 binding activity, MMP-2 promoter activity, and cellular migratory and invasive potentials. These results suggest that PGE(2) mediates pancreatic cancer cellular invasiveness through an ERK/Ets-1-dependent induction of MMP-2 expression and activity. They also suggest that COX-2 inhibition may represent a strategy to inhibit invasive potential in pancreatic cancer.

Overexpression of CEACAM6 promotes insulin-like growth factor I-induced pancreatic adenocarcinoma cellular invasiveness.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycosylphosphatidylinositol-linked immunoglobulin superfamily member that is overexpressed in a variety of human cancers. We have recently reported that suppression of CEACAM6 expression impairs pancreatic adenocarcinoma progression in vivo. In order to characterize the mechanisms through which CEACAM6 influences the malignant phenotype, CEACAM6-overexpressing Capan2 pancreatic adenocarcinoma cells were established by stable transfection. We determined the effect of CEACAM6 overexpression on cellular invasiveness towards insulin-like growth factor I (IGF-I), a peptide of critical importance in pancreatic cancer malignant cellular behavior and tumor progression. IGF-I-induced cellular invasiveness and IGF-IR expression were significantly increased in clones overexpressing CEACAM6. Using inhibitory anti-IGF-IR antibody, a requirement for IGF-IR signaling in the enhanced invasiveness towards IGF-I induced by CEACAM6 overexpression was confirmed. CEACAM6-overexpressing clones exhibited increased Akt and c-Src kinase activities, as well as higher levels of matrix metalloproteinase-2 (MMP-2) expression and activity in the presence of IGF-I. While Akt kinase is both necessary and sufficient to induce IGF-IR upregulation, c-Src kinase activity is necessary, but alone is insufficient to upregulate IGF-IR expression. CEACAM6 is an important determinant of pancreatic adenocarcinoma malignant cellular behavior and, together with its downstream targets, warrants further investigation as a therapeutic target in this disease.

Retrovirally mediated RNA interference targeting the M2 subunit of ribonucleotide reductase: A novel therapeutic strategy in pancreatic cancer.

BACKGROUND: Ribonucleotide reductase M2 subunit (RRM2) overexpression enhances tumor chemoresistance and cellular invasiveness. We hypothesized that the RNA interference (RNAi) induced by retrovirally delivered small interfering RNA (siRNA) would sensitize pancreatic adenocarcinoma cells to gemcitabine and attenuate their invasive potential. METHODS: Stable suppression of RRM2 expression in PANC1, MIAPaCa2, BxPC3, and Capan2 cells was induced by exposure to a novel replication-deficient retrovirus, engineered to express RRM2-specific siRNA (psiRRM2), and confirmed by Western blot analysis. Single-base mismatch vector (psiControl) served as control. Ribonucleotide reductase activity was quantified, and gemcitabine 50% inhibitory concentrations were calculated. TUNEL staining and caspase profiling were performed after gemcitabine exposure. Cellular invasiveness was quantified in a Matrigel Boyden chamber. NF-kappaB activity and matrix metalloproteinase-9 (MMP-9) expression and activity were measured. RESULTS: RRM2 expression was stably and specifically suppressed in psiRRM2, but not psiControl transfectants. psiRRM2 transfectants exhibited lower 50% inhibitory concentrations, increased gemcitabine-induced apoptosis, and greater caspase-3 activation, relative to psiControl transfectants. Invasiveness was attenuated in psiRRM2 transfectants, as was NF-kappaB activity, MMP-9 expression, and MMP-9 activity, relative to psiControl transfectants. CONCLUSIONS: RRM2 gene silencing attenuates pancreatic adenocarcinoma cellular invasiveness and gemcitabine chemoresistance. Retroviral siRNA delivery can efficiently induce stable RNAi, allowing dissection of gene function and potentially representing a new therapeutic modality.