Interaction between microRNA-195 and HuR regulates Paneth cell function in the intestinal epithelium by altering SOX9 translation.

Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium.

Nuclear magnetic resonance reveals a two hairpin equilibrium near the 3'-splice site of influenza A segment 7 mRNA that can be shifted by oligonucleotides.

Influenza A kills hundreds of thousands of people globally every year and has the potential to generate more severe pandemics. Influenza A's RNA genome and transcriptome provide many potential therapeutic targets. Here, nuclear magnetic resonance (NMR) experiments suggest that one such target could be a hairpin loop of 8 nucleotides in a pseudoknot that sequesters a 3' splice site in canonical pairs until a conformational change releases it into a dynamic 2 × 2-nt internal loop. NMR experiments reveal that the hairpin loop is dynamic and able to bind oligonucleotides as short as pentamers. A 3D NMR structure of the complex contains 4 and likely 5 bp between pentamer and loop. Moreover, a hairpin sequence was discovered that mimics the equilibrium of the influenza hairpin between its structure in the pseudoknot and upon release of the splice site. Oligonucleotide binding shifts the equilibrium completely to the hairpin secondary structure required for pseudoknot folding. The results suggest this hairpin can be used to screen for compounds that stabilize the pseudoknot and potentially reduce splicing.

Laparoscopic surgery for adrenocortical carcinoma: Estimating the risk of margin-positive resection.

BACKGROUND: Over recent years, there has been increasing adoption of minimally invasive surgery (MIS) in the treatment of adrenocortical carcinoma (ACC). However, MIS has been associated with noncurative resection and locoregional recurrence. We aimed to identify risk factors for margin-positivity among patients who undergo MIS resection for ACC. We hypothesized that a simple nomogram can accurately identify patients most suitable for curative MIS resection. METHODS: Curative-intent resections for ACC were identified through the National Cancer Database spanning 2010-2018. Trends in MIS utilization were reported using Pearson correlation coefficients. Factors associated with margin-positive resection were identified among preoperatively available variables using multivariable logistic regression, then incorporated into a predictive model. Model quality was cross validated using an 80% training data set and 20% test data set. RESULTS: Among 1260 ACC cases, 38.6% (486) underwent MIS resection. MIS utilization increased over time at nonacademic centers (R = 0.818, p = 0.007), but not at academic centers (R = 0.009, p = 0.982). Factors associated with margin-positive MIS resection were increasing age, nonacademic center (odds ratio [OR]: 1.8, p = 0.006), cT3 (OR: 4.7, p < 0.001) or cT4 tumors (OR: 14.6, p < 0.001), and right-sided tumors (OR: 2.0, p = 0.006). A predictive model incorporating these four factors produced favorable c-statistics of 0.75 in the training data set and 0.72 in the test data set. A pragmatic nomogram was created to enable bedside risk stratification. CONCLUSIONS: An increasing proportion of ACC are resected via minimally invasive operations, particularly at nonacademic centers. Patient selection based on a few key factors can minimize the risk of noncurative surgery.

Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5'GGUC3' paired to 3'CUGG5' separately from other 5'GU3'/3'UG5' stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

miR-542-5p targets c-myc and negates the cell proliferation effect of SphK1 in intestinal epithelial cells.

Intestinal epithelial barrier defects occur commonly during a variety of pathological conditions, though their underlying mechanisms are not completely understood. Sphingosine-1-phosphate (S1P) has been shown to be a critical regulator of proliferation and of maintenance of an intact intestinal epithelial barrier, as is also sphingosine kinase 1 (SphK1), the rate-limiting enzyme for S1P synthesis. SphK1 has been shown to modulate its effect on intestinal epithelial proliferation through increased levels of c-myc. We conducted genome-wide profile analysis to search for differential microRNA expression related to overexpressed SphK1 demonstrating adjusted expression of microRNA 542-5p (miR-542-5p). Here, we show that miR-542-5p is regulated by SphK1 activity and is an effector of c-myc translation that ultimately serves as a critical regulator of the intestinal epithelial barrier. miR-542-5p directly regulates c-myc translation through direct binding to the c-myc mRNA. Exogenous S1P analogs administered in vivo protect murine intestinal barrier from damage due to mesenteric ischemia reperfusion, and damaged intestinal tissue had increased levels of miR-542-5p. These results indicate that miR-542-5p plays a critical role in the regulation of S1P-mediated intestinal barrier function, and may highlight a novel role in potential therapies.

Mid-term Surgery Outcomes in Patients With COVID-19: Results From a Nationwide Analysis.

OBJECTIVE: Determine mid-term postoperative outcomes among coronavirus disease 2019 (COVID-19)-positive (+) patients compared with those who never tested positive before surgery. BACKGROUND: COVID-19 is thought to be associated with prohibitively high rates of postoperative complications. However, prior studies have only evaluated 30-day outcomes, and most did not adjust for demographic, clinical, or procedural characteristics. METHODS: We analyzed data from surgeries performed at all Veterans Affairs hospitals between March 2020 and 2021. Kaplan-Meier curves compared trends in mortality and Cox proportional hazards models estimated rates of mortality and pulmonary, thrombotic, and septic postoperative complications between patients with a positive preoperative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test [COVID (+)] and propensity score-matched COVID-negative (-) patients. RESULTS: Of 153,741 surgical patients, 4778 COVID (+) were matched to 14,101 COVID (-). COVID (+) status was associated with higher postoperative mortality ( P <0.0001) with a 6-month survival of 94.2% (95% confidence interval: 93.2-95.2) versus 96.0% (95% confidence interval: 95.7.0-96.4) in COVID (-). The highest mortality was in the first 30 postoperative days. Hazards for mortality and postoperative complications in COVID (+) decreased with increasing time between testing COVID (+) and date of surgery. COVID (+) patients undergoing elective surgery had similar rates of mortality, thrombotic and septic complications, but higher rates of pulmonary complications than COVID (-) patients. CONCLUSIONS: This is the first report of mid-term outcomes among COVID-19 patients undergoing surgery. COVID-19 is associated with decreased overall and complication-free survival primarily in the early postoperative period, delaying surgery by 5 weeks or more reduces risk of complications. Case urgency has a multiplicative effect on short-term and long-term risk of postoperative mortality and complications.

Growing Deficit in New Cancer Diagnoses 2 Years Into the COVID-19 Pandemic: A National Multicenter Study.

BACKGROUND: Large decreases in cancer diagnoses were seen early in the COVID-19 pandemic. However, the evolution of these deficits since the end of 2020 and the advent of widespread vaccination is unknown. METHODS: This study examined data from the Veterans Health Administration (VA) from 1 January 2018 through 28 February 2022 and identified patients with screening or diagnostic procedures or new cancer diagnoses for the four most common cancers in the VA health system: prostate, lung, colorectal, and bladder cancers. Monthly procedures and new diagnoses were calculated, and the pre-COVID era (January 2018 to February 2020) was compared with the COVID era (March 2020 to February 2022). RESULTS: The study identified 2.5 million patients who underwent a diagnostic or screening procedure related to the four cancers. A new cancer was diagnosed for 317,833 patients. During the first 2 years of the pandemic, VA medical centers performed 13,022 fewer prostate biopsies, 32,348 fewer cystoscopies, and 200,710 fewer colonoscopies than in 2018-2019. These persistent deficits added a cumulative deficit of nearly 19,000 undiagnosed prostate cancers and 3300 to 3700 undiagnosed cancers each for lung, colon, and bladder. Decreased diagnostic and screening procedures correlated with decreased new diagnoses of cancer, particularly cancer of the prostate (R = 0.44) and bladder (R = 0.27). CONCLUSION: Disruptions in new diagnoses of four common cancers (prostate, lung, bladder, and colorectal) seen early in the COVID-19 pandemic have persisted for 2 years. Although reductions improved from the early pandemic, new reductions during the Delta and Omicron waves demonstrate the continued impact of the COVID-19 pandemic on cancer care.

miR-195 regulates intestinal epithelial restitution after wounding by altering actin-related protein-2 translation.

Early gut epithelial restitution reseals superficial wounds after acute injury, but the exact mechanism underlying this rapid mucosal repair remains largely unknown. MicroRNA-195 (miR-195) is highly expressed in the gut epithelium and involved in many aspects of mucosal pathobiology. Actin-related proteins (ARPs) are key components essential for stimulation of actin polymerization and regulate cell motility. Here, we reported that miR-195 modulates early intestinal epithelial restitution by altering ARP-2 expression at the translation level. miR-195 directly interacted with the ARP-2 mRNA, and ectopically expressed miR-195 decreased ARP-2 protein without effect on its mRNA content. In contrast, miR-195 silencing by transfection with anti-miR-195 oligo increased ARP-2 expression. Decreased ARP-2 levels by miR-195 overexpression were associated with an inhibition of early epithelial restitution, as indicated by a decrease in cell migration over the wounded area. Elevation of cellular ARP-2 levels by transfection with its transgene restored cell migration after wounding in cells overexpressing miR-195. Polyamines were found to decrease miR-195 abundance and enhanced ARP-2 translation, thus promoting epithelial restitution after wounding. Moreover, increasing the levels of miR-195 disrupted F-actin cytoskeleton organization, which was prevented by ARP2 overexpression. These results indicate that miR-195 inhibits early epithelial restitution by decreasing ARP-2 translation and that miR-195 expression is negatively regulated by cellular polyamines.

Impact of the COVID-19 pandemic on diagnosis of new cancers: A national multicenter study of the Veterans Affairs Healthcare System.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused disruptions in treatment for cancer. Less is known about its impact on new cancer diagnoses, where delays could cause worsening long-term outcomes. This study quantifies decreases in encounters related to prostate, lung, bladder and colorectal cancers, procedures that facilitate their diagnosis, and new diagnoses of those cancers in the COVID era compared to pre-COVID era. METHODS: All encounters at Veterans' Affairs facilities nationwide from 2016 through 2020 were reviewed. The authors quantified trends in new diagnoses of cancer and in procedures facilitating their diagnosis, from January 1, 2018 onward. Using 2018 to 2019 as baseline, reductions in procedures and new cancer diagnoses in 2020 were estimated. Calculated absolute and percentage differences in annual volume and observed-to-expected volume ratios were calculated. Heat maps and funnel plots of volume changes were generated. RESULTS: From 2018 through 2020, there were 4.1 million cancer-related encounters, 3.9 million relevant procedures, and 251,647 new cancers diagnosed. Compared to the annual averages in 2018 through 2019, colonoscopies in 2020 decreased by 45% whereas prostate biopsies, chest computed tomography scans, and cystoscopies decreased by 29%, 10%, and 21%, respectively. New cancer diagnoses decreased by 13% to 23%. These drops varied by state and continued to accumulate despite reductions in pandemic-related restrictions. CONCLUSION: The authors identified substantial reductions in procedures used to diagnose cancer and subsequent reductions in new diagnoses of cancer across the United States because of the COVID-19 pandemic. A nomogram is provided to identify and resolve these unmet health care needs and avoid worse long-term cancer outcomes. LAY SUMMARY: The disruptions due to the COVID-19 pandemic have led to substantial reductions in new cancers being diagnosed. This study quantifies those reductions in a national health care system and offers a method for understanding the backlog of cases and the resources needed to resolve them.

COVID-19 Vaccination Associated With Reduced Postoperative SARS-CoV-2 Infection and Morbidity.

OBJECTIVE: The purpose of this study was to determine the effect of COVID-19 vaccination on postoperative mortality, pulmonary and thrombotic complications, readmissions and hospital lengths of stay among patients undergoing surgery in the United States. BACKGROUND: While vaccination prevents COVID-19, little is known about its impact on postoperative complications. METHODS: This is a nationwide observational cohort study of all 1,255 Veterans Affairs facilities nationwide. We compared patients undergoing surgery at least 2 weeks after their second dose of the Pfizer BioNTech or Moderna vaccines, to contemporary propensity score matched controls. Primary endpoints were 30-day mortality and postoperative COVID-19 infection. Secondary endpoints were pulmonary or thrombotic complications, readmissions, and hospital lengths of stay. RESULTS: 30,681 patients met inclusion criteria. After matching, there were 3,104 in the vaccination group (1,903 received the Pfizer BioNTech, and 1,201 received the Moderna vaccine) and 7,438 controls. Full COVID-19 vaccination was associated with lower rates of postoperative 30-day COVID-19 infection (Incidence Rate Ratio and 95% confidence intervals, 0.09 [0.01,0.44]), pulmonary complications (0.54 [0.39, 0.72]), thrombotic complications (0.68 [0.46, 0.99]) and decreased hospital lengths of stay (0.78 [0.69, 0.89]). Complications were also low in vaccinated patients who tested COVID-19 positive before surgery but events were too few to detect a significant difference compared to controls. CONCLUSION: COVID-19 vaccination is associated with lower rates of postoperative morbidity. The benefit is most pronounced among individuals who have never had a COVID-19 infection before surgery.

Low-Risk Papillary Thyroid Cancer: Treatment De-Escalation and Cost Implications.

INTRODUCTION: The American Thyroid Association (ATA) updated consensus guidelines in 2015 for radioactive iodine (RAI) and resection for low-risk papillary thyroid cancer. The objective of this study was to describe the evolution of institutional practice patterns and estimate the cost implications of these trends. MATERIALS AND METHODS: Patients with cT1-T2N0 papillary thyroid cancer were identified via an institutional tumor registry. Incidences of total thyroidectomy or RAI were tracked longitudinally using cumulative sum. Real-world costs for RAI and each surgical encounter were adjusted for inflation and standardized to national average costs from National Inpatient Sample cost data. RESULTS: Sixty-one patients met inclusion criteria between 2007 and 2018. Among these, 28 patients underwent total thyroidectomies and received RAI treatments based on criteria pre-dating the 2015 ATA guidelines. Cumulative sum revealed significant decreases in the rate of total thyroidectomy following May 2015 (15.8% versus 59.5%, P = 0.002) and RAI following March 2013 (3.0% versus 32.1%, P = 0.002). There were no locoregional recurrences in either period. The average cost savings attributable to these institutional practice changes was $1580 per patient. CONCLUSIONS: De-escalation in surgical and RAI utilization for low-risk papillary thyroid cancer according to 2015 ATA guidelines is associated with a substantial decrease in real-world costs.

A multicenter randomized controlled trial indicates that paclitaxel-coated balloons provide no benefit for arteriovenous fistulas.

The role of paclitaxel-coated balloons has been established in the coronary and peripheral arterial circulations with recent interest in the use of paclitaxel-coated balloons to improve patency rates following angioplasty of arteriovenous fistulas. To assess the efficacy of paclitaxel-coated angioplasty balloons to prolong the survival time of target lesion primary patency in arteriovenous fistulas, we designed an investigator-led multi-center randomized controlled trial with follow up time variable for a minimum of one year. Patients with an arteriovenous fistula who were undergoing an angioplasty for a clinical indication were included but patients with one or more lesions outside the treatment segment were excluded. Following successful treatment with a high-pressure balloon, 212 patients were randomized. In the intervention arm, the second component was insertion of a paclitaxel-coated balloon. In the control arm, an identical procedure was followed, but using a standard balloon. The primary endpoint was time to loss of clinically driven target lesion primary patency. Primary analysis showed no significant evidence for a difference in time to end of target lesion primary patency between groups: hazard ratio 1.18 with a 95% confidence interval of 0.78 to 1.79. There were no significant differences for any secondary outcomes, including patency outcomes and adverse events. Thus, our study demonstrated no evidence that paclitaxel-coated balloons provide benefit, following standard care high-pressure balloon angioplasty, in the treatment of arteriovenous fistulas. Hence, in view of the benefit suggested by other trials, the role of paclitaxel-coated angioplasty balloons remains uncertain.

A Nation-Wide Review of Elective Surgery and COVID-Surge Capacity.

BACKGROUND: The COVID-19 pandemic has resulted in over 225,000 excess deaths in the United States. A moratorium on elective surgery was placed early in the pandemic to reduce risk to patients and staff and preserve critical care resources. This report evaluates the impact of the elective surgical moratorium on case volumes and intensive care unit (ICU) bed utilization. METHODS: This retrospective review used a national convenience sample to correlate trends in the weekly rates of surgical cases at 170 Veterans Affairs Hospitals around the United States from January 1 to September 30, 2020 to national trends in the COVID-19 pandemic. We reviewed data on weekly number of procedures performed and ICU bed usage, stratified by level of urgency (elective, urgent, emergency), and whether an ICU bed was required within 24 hours of surgery. National data on the proportion of COVID-19 positive test results and mortality rates were obtained from the Center for Disease Control website. RESULTS: 198,911 unique surgical procedures performed during the study period. The total number of cases performed from January 1 to March 16 was 86,004 compared with 15,699 from March 17 to May 17. The reduction in volume occurred before an increase in the percentage of COVID-19 positive test results and deaths nationally. There was a 91% reduction from baseline in the number of elective surgeries performed allowing 78% of surgical ICU beds to be available for COVID-19 positive patients. CONCLUSION: The moratorium on elective surgical cases was timely and effective in creating bed capacity for critically ill COVID-19 patients. Further analyses will allow targeted resource allocation for future pandemic planning.

In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs.

The influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals.

Molecular dynamics correctly models the unusual major conformation of the GAGU RNA internal loop and with NMR reveals an unusual minor conformation.

The RNA "GAGU" duplex, (5'GACGAGUGUCA)2, contains the internal loop (5'-GAGU-3')2 , which has two conformations in solution as determined by NMR spectroscopy. The major conformation has a loop structure consisting of trans-Watson-Crick/Hoogsteen GG pairs, A residues stacked on each other, U residues bulged outside the helix, and all sugars with a C2'-endo conformation. This differs markedly from the internal loops, (5'-GAGC-3')2, (5'-AAGU-3')2, and (5'-UAGG-3')2, which all have cis-Watson-Crick/Watson-Crick AG "imino" pairs flanked by cis-Watson-Crick/Watson-Crick canonical pairs resulting in maximal hydrogen bonding. Here, molecular dynamics was used to test whether the Amber force field (ff99 + bsc0 + OL3) approximates molecular interactions well enough to keep stable the unexpected conformation of the GAGU major duplex structure and the NMR structures of the duplexes containing (5'-GAGC-3')2, (5'-AAGU-3')2, and (5'-UAGG-3')2 internal loops. One-microsecond simulations were repeated four times for each of the duplexes starting in their NMR conformations. With the exception of (5'-UAGG-3')2, equivalent simulations were also run starting with alternative conformations. Results indicate that the Amber force field keeps the NMR conformations of the duplexes stable for at least 1 µsec. They also demonstrate an unexpected minor conformation for the (5'-GAGU-3')2 loop that is consistent with newly measured NMR spectra of duplexes with natural and modified nucleotides. Thus, unrestrained simulations led to the determination of the previously unknown minor conformation. The stability of the native (5'-GAGU-3')2 internal loop as compared to other loops can be explained by changes in hydrogen bonding and stacking as the flanking bases are changed.

Improving RNA nearest neighbor parameters for helices by going beyond the two-state model.

RNA folding free energy change nearest neighbor parameters are widely used to predict folding stabilities of secondary structures. They were determined by linear regression to datasets of optical melting experiments on small model systems. Traditionally, the optical melting experiments are analyzed assuming a two-state model, i.e. a structure is either complete or denatured. Experimental evidence, however, shows that structures exist in an ensemble of conformations. Partition functions calculated with existing nearest neighbor parameters predict that secondary structures can be partially denatured, which also directly conflicts with the two-state model. Here, a new approach for determining RNA nearest neighbor parameters is presented. Available optical melting data for 34 Watson-Crick helices were fit directly to a partition function model that allows an ensemble of conformations. Fitting parameters were the enthalpy and entropy changes for helix initiation, terminal AU pairs, stacks of Watson-Crick pairs and disordered internal loops. The resulting set of nearest neighbor parameters shows a 38.5% improvement in the sum of residuals in fitting the experimental melting curves compared to the current literature set.

Using High-Fidelity Simulation to Evaluate Clinical Skills in Prelicensure Nursing Students.

A prelicensure associate degree nursing program identified the need to formally validate clinical skills and decision-making within nursing students across the program and implemented a process using high-fidelity simulation. The simulated experiences provided an opportunity for both formative and summative evaluation while also affording an opportunity to level student performance across the program. Faculty reported improved consistency in their expectations of student performance within each nursing course, and students reported improved confidence in their clinical practice skills.

Nuclear Magnetic Resonance Reveals That GU Base Pairs Flanking Internal Loops Can Adopt Diverse Structures.

RNA thermodynamics play an important role in determining the two- and three-dimensional structures of RNA. Internal loops of the sequence 5'-GMNU/3'-UNMG are relatively unstable thermodynamically. Here, five duplexes with GU-flanked 2 × 2 nucleotide internal loops were structurally investigated to reveal determinants of their instability. The following internal loops were investigated: 5'-GCAU/3'-UACG, 5'-UUCG/3'-GCUU, 5'-GCUU/3'-UUCG, 5'-GUCU/3'-UCUG, and 5'-GCCU/3'-UCCG. Two-dimensional nuclear magnetic resonance spectra indicate the absence of GU wobble base pairing in 5'-GCUU/3'-UUCG, 5'-GUCU/3'-UCUG, and 5'-GCCU/3'-UCCG. The 5'-GCUU/3'-UUCG loop has an unusual conformation of the GU base pairs, in which U's O2 carbonyl forms a bifurcated hydrogen bond with G's amino and imino protons. The internal loop of 5'-GUCU/3'-UCUG displays a shifted configuration in which GC pairs flank a U-U pair and several U's are in fast exchange between positions inside and outside the helix. In contrast, 5'-GCAU/3'-UACG and 5'-UUCG/3'-GCUU both have the expected GU wobble base pairs flanking the internal loop. Evidently, GU base pairs flanking internal loops are more likely to display atypical structures relative to Watson-Crick base pairs flanking internal loops. This appears to be more likely when the G of the GU pair is 5' to the loop. Such unusual structures could serve as recognition elements for biological function and as benchmarks for structure prediction methods.

Surprising Sequence Effects on GU Closure of Symmetric 2 × 2 Nucleotide RNA Internal Loops.

GU base pairs are important RNA structural motifs and often close loops. Accurate prediction of RNA structures relies upon understanding the interactions determining structure. The thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs are not well understood. Here, several self-complementary oligonucleotide sequences expected to form duplexes with 2 × 2 nucleotide internal loops closed by GU pairs were investigated. Surprisingly, nuclear magnetic resonance revealed that many of the sequences exist in equilibrium between hairpin and duplex conformations. This equilibrium is not observed with loops closed by Watson-Crick pairs. To measure the thermodynamics of some 2 × 2 nucleotide internal loops closed by GU pairs, non-self-complementary sequences that preclude formation of hairpins were designed. The measured thermodynamics indicate that some internal loops closed by GU pairs are unusually unstable. This instability accounts for the observed equilibria between duplex and hairpin conformations. Moreover, it suggests that future three-dimensional structures of loops closed by GU pairs may reveal interactions that unexpectedly destabilize folding.

ß-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding.

Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor (ß-PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of ß-PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of ß-PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous ß-PIX by its gene-silencing destabilized ß-PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of ß-PIX increased the levels of ß-PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated ß-PIX/GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of ß-PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of ß-PIX or GIT1. These results indicate that ß-PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of ß-PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines.