Enrolling High-Acuity Emergency General Surgery Patients in a Prospective Longitudinal Cohort Study.

Over three million patients are admitted to hospitals annually with high-acuity conditions mandating emergency abdominal or skin/soft-tissue operation. Patients with these high-acuity emergency general surgery (HA-EGS) diseases experience significant morbidity/mortality, yet the quality of life (QOL) impact on survivors is not well studied. Acuity, transfer patterns, and adverse social determinants of health (SDOH) documented in epidemiologic studies are cited reasons for inability to measure patient-reported outcomes (PROs) among HA-EGS survivors. We conducted a feasibility study to understand facilitators and barriers of conducting a prospective study of changes in QOL after surviving HA-EGS. From September 2019 to April 2021, we collected baseline (pre-admission) and 30/60-days post-surgery data on activities of daily living, depression, self-efficacy, resilience, pain, work limitations, social support, and substance use from patients who enrolled during index hospitalization. 100 patients were consented to participate in the study (71.9% enrollment rate). The retention rate was 65.9% for 30-day calls and 53.8% for 60-day calls. Median time to complete each time point remained under 25 minutes. Patients with a longer length of stay and nicotine users didn't complete their 30-day interview while those with systemic complications didn't complete their 60-day interview. These results set the foundation for future PRO studies.

Obesity Is Associated with Improved Early Survival but Increased Late Mortality in Surgical Patients with Sepsis: A Propensity Matched Analysis.

INTRODUCTION: While obesity is a risk factor for post-operative complications, its impact following sepsis is unclear. The primary objective of this study was to evaluate the association between obesity and mortality following admission to the surgical ICU (SICU) with sepsis. METHODS: We conducted a single center retrospective review of SICU patients grouped into obese (n = 766, BMI ≥30 kg/m2) and non-obese (n = 574, BMI 18-29.9 kg/m2) cohorts. Applying 1:1 propensity matching for age, sex, comorbidities, SOFA, and transfer status, demographic data, comorbidities, and sepsis presentation were compared between groups. Primary outcomes included in-hospital and 90-day mortality, ICU length of stay (LOS), need for mechanical ventilation (IMV) and renal replacement therapy (RRT). P < 0.05 was considered significant. RESULTS: Obesity associates with higher median ICU LOS (8.2 vs 5.6, p < 0.001), need for IMV (76% vs 67%, p = 0.001), ventilator days (5 vs 4, p < 0.004), and RRT (23% vs 12%, p < 0.001). In-hospital (29% vs 18%, p < 0.0001) and 90-day mortality (34% vs 24%, p = 0.0006) was higher for obese compared to non-obese groups. Obesity independently predicted need for IMV (OR 1.6, 95th CI: 1.2-2.1), RRT (OR 2.2, 95th CI: 1.5-3.1), in-hospital (OR 2.1, 95th CI: 1.5-2.8) and 90-day mortality (HR: 1.4, 95TH CI: 1.1-1.8), after adjusting for SOFA, age, sex, and comorbidities. Comparative survival analyses demonstrate a paradoxical early survival benefit for obese patients followed by a rapid decline after 7 days (logrank p = 0.0009). CONCLUSIONS: Obesity is an independent risk factor for 90-day mortality for surgical patients with sepsis, but its impact appeared later in hospitalization. Understanding differences in systemic responses between these cohorts may be important for optimizing critical care management. LEVEL OF EVIDENCE: III.

Area Deprivation Index Predicts Mortality for Critically Ill Surgical Patients With Sepsis.

Background: The impact of socioeconomic status on outcomes after sepsis has been challenging to define, and no polysocial metric has been shown to predict mortality in sepsis. The primary objective of this study was to evaluate the association between the Area Deprivation Index (ADI) and mortality in patients admitted to the surgical intensive care unit (SICU) with sepsis. Patients and Methods: All patients admitted to the SICU with sepsis (Sequential Organ Failure Assessment [SOFA] score ≥2) were retrospectively reviewed. The ADI scores were obtained and classified as "high ADI" (≥85th percentile, n = 400, representative of high socioeconomic deprivation) and "control ADI" (ADI <85th percentile, n = 976). Baseline demographic and clinical characteristics were compared between groups. The primary outcome was 90-day mortality. Results: High ADI patients were younger (mean age 58.5 vs. 60.8; p = 0.01) and more likely to be non-white (23.7% vs. 10.0%; p < 0.0005) and to present with chronic obstructive pulmonary disease (26.5% vs. 19.0%; p = 0.002). High ADI patients had increased in-hospital (27.3% vs. 21.6%; p = 0.025) and 90-day mortality (35.0% vs. 28.9%; p = 0.03). High ADI patients also had increased rates of renal failure (20.3% vs. 15.3%; p = 0.02). Both cohorts had similar intensive care unit (ICU) lengths of stay and median hospital stay, Charlson comorbidity index, and rate of discharge to home. High ADI is an independent risk factor for 90-day mortality after admission for surgical sepsis (odds ratio [OR], 1.39 ± 0.24; p = 0.014). Conclusions: High ADI is an independent predictor of 90-day mortality in patients with surgical sepsis. Targeted community interventions are needed to reduce sepsis mortality for these at-risk patients.

Distressed Communities Index Is Not Associated with Mortality for Critically Ill Surgical Patients with Sepsis.

Background: The impact of socioeconomic metrics on outcomes after sepsis is unclear. The Distressed Communities Index (DCI) is a composite score quantifying socioeconomic well-being by zip code. The primary objective of this study was to evaluate the association between DCI and mortality in patients with sepsis admitted to the surgical intensive care unit (SICU). Patients and Methods: All patients with sepsis admitted to the SICU (Sequential Organ Failure Assessment [SOFA] score ≥2) were reviewed retrospectively. Composite DCI scores were obtained for each patient and classified into high-distress (DCI ≥75th percentile; n = 331) and control distress (DCI <50th percentile; n = 666) groups. Baseline demographic and clinical characteristics were compared between groups. The primary outcomes were in-hospital and 90-day mortality. Results: The high-distress cohort was younger and more likely to be African American (19.6% vs. 6.2%), transferred from an outside facility (52% vs. 42%), have chronic obstructive pulmonary disease (25.1% vs. 18.8%), and baseline liver disease (8.2% vs. 4.2%). Sepsis presentation was comparable between groups. Compared with the control cohort, high-distress patients had similar in-house (23% vs. 24%) and 90-day mortality (30% vs. 28%) but were associated with longer hospital stay (23 vs. 19 days). High DCI failed to predict in-hospital or 90-day mortality but was an independent risk factor for longer hospital length of stay (odds ratio [OR], 2.83 ± 1.42; p = 0.047). Conclusions: High DCI was not associated with mortality but did independently predict longer length of stay. This may reflect limitations of DCI score in evaluating mortality for patients with sepsis. Future studies should elucidate its association with length of stay, re-admissions, and follow-up.

Structural basis for a six nucleotide genetic alphabet.

Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard "edge on" Watson-Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology.

A mechanochemical switch to control radical intermediates.

B12-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side reactions. Using hybrid quantum mechanical/molecular mechanical simulations, we follow the full catalytic cycle of an AdoB12-dependent enzyme and present the details of a mechanism that utilizes a highly effective mechanochemical switch. When the switch is "off", the 5'-deoxyadenosyl radical moiety is stabilized by releasing the internal strain of an enzyme-imposed conformation. Turning the switch "on," the enzyme environment becomes the driving force to impose a distinct conformation of the 5'-deoxyadenosyl radical to avoid deleterious radical transfer. This mechanochemical switch illustrates the elaborate way in which enzymes attain selectivity of extremely chemically challenging reactions.

Assigning the EPR fine structure parameters of the Mn(II) centers in Bacillus subtilis oxalate decarboxylase by site-directed mutagenesis and DFT/MM calculations.

Oxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants.

Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase.

The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 °C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels.

BACKGROUND AND PURPOSE: Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca(2+) -activated K(+) (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. EXPERIMENTAL APPROACH: We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. KEY RESULTS: We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 µM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. CONCLUSIONS AND IMPLICATIONS: Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity.

KV2.1 and electrically silent KV channel subunits control excitability and contractility of guinea pig detrusor smooth muscle.

Voltage-gated K(+) (K(V)) channels are implicated in detrusor smooth muscle (DSM) function. However, little is known about the functional role of the heterotetrameric K(V) channels in DSM. In this report, we provide molecular, electrophysiological, and functional evidence for the presence of K(V)2.1 and electrically silent K(V) channel subunits in guinea pig DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of the homotetrameric K(V)2.1, K(V)2.2, and K(V)4.2 as well as the heterotetrameric K(V)2.1/6.3 and K(V)2.1/9.3 channels, was used to examine the role of these K(V) channels in DSM function. RT-PCR indicated mRNA expression of K(V)2.1, K(V)6.2-6.3, K(V)8.2, and K(V)9.1-9.3 subunits in isolated DSM cells. K(V)2.1 protein expression was confirmed by Western blot and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the K(V) current in freshly isolated DSM cells. ScTx1 (100 nM) did not significantly change the steady-state activation and inactivation curves for K(V) current. However, ScTx1 (100 nM) decreased the activation time-constant of the K(V) current at positive voltages. Although our patch-clamp data could not exclude the presence of the homotetrameric K(V)2.1 channels, the biophysical characteristics of the ScTx1-sensitive current were consistent with the presence of heterotetrameric K(V)2.1/silent K(V) channels. Current-clamp recordings showed that ScTx1 (100 nM) did not change the DSM cell resting membrane potential. ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and muscle tone as well as the amplitude of the electrical field stimulation-induced contractions of isolated DSM strips. Collectively, our data revealed that K(V)2.1-containing channels are important physiological regulators of guinea pig DSM excitability and contractility.

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function.

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, ß(1)-, and ß(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, ß(1), and ß(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.

Voltage-gated K(+) channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle.

Members of the voltage-gated K(+) (K(V)) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits K(V)2.1, K(V)2.2, K(V)4.2, and K(V)2.1/9.3 channels. The objective of this study was to investigate whether K(V) channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 microM). This indicates that, under conditions of sustained membrane depolarization, the K(V) channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3, but not K(V)4.2 channel subunits. Protein expression of K(V)2.1 and K(V)2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that K(V)2.1 and K(V)2.2, but not K(V)4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions.

Stimulation of beta3-adrenoceptors relaxes rat urinary bladder smooth muscle via activation of the large-conductance Ca2+-activated K+ channels.

We investigated the role of large-conductance Ca(2+)-activated K(+) (BK) channels in beta3-adrenoceptor (beta3-AR)-induced relaxation in rat urinary bladder smooth muscle (UBSM). BRL 37344, a specific beta3-AR agonist, inhibits spontaneous contractions of isolated UBSM strips. SR59230A, a specific beta3-AR antagonist, and H89, a PKA inhibitor, reduced the inhibitory effect of BRL 37344. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration response curves for contraction amplitude, net muscle force, and tone to the right. Freshly dispersed UBSM cells and the perforated mode of the patch-clamp technique were used to determine further the role of beta3-AR stimulation by BRL 37344 on BK channel activity. BRL 37344 increased spontaneous, transient, outward BK current (STOC) frequency by 46.0 +/- 20.1%. In whole cell mode at a holding potential of V(h) = 0 mV, the single BK channel amplitude was 5.17 +/- 0.28 pA, whereas in the presence of BRL 37344, it was 5.55 +/- 0.41 pA. The BK channel open probability was also unchanged. In the presence of ryanodine and nifedipine, the current-voltage relationship in response to depolarization steps in the presence and absence of BRL 37344 was identical. In current-clamp mode, BRL 37344 caused membrane potential hyperpolarization from -26.1 +/- 2.1 mV (control) to -29.0 +/- 2.2 mV. The BRL 37344-induced hyperpolarization was eliminated by application of iberiotoxin, tetraethylammonium or ryanodine. The data indicate that stimulation of beta3-AR relaxes rat UBSM by increasing the BK channel STOC frequency, which causes membrane hyperpolarization and thus relaxation.

Beta-adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca2+-activated K+ channel.

In urinary bladder smooth muscle (UBSM), stimulation of beta-adrenergic receptors (beta-ARs) leads to activation of the large-conductance Ca2+-activated K+ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255-C1263, 2005). In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to beta-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K+ or 1 microM carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with approximately 12 mV. Isoproterenol, a nonspecific beta-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentration-dependent manner. In the presence of IBTX, the concentration-response curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol- and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the beta-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in beta-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.