Cloning, sequencing, and characterization of the hexahydro-1,3,5-trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xpLA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization

A Novel Mechanism for Calmodulin Dependent Inactivation of Transient Receptor Potential Vanilloid 6

The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the Transient Receptor Potential (TRP) superfamily of ion channels and both play an important role in overall Cahomeostasis. The functioning of the channels centres on a tightly controlled Ca-dependent feedback mechanism where the direct binding of the universal Ca-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular [CaCaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When cytosolic [Ca] increases charging the apo CaM N-lobe with Ca, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges between two TRPV6 monomers resulting in closure of the channel pore

The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(•-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes

A Matched-Pair Analysis after Robotic and Retropubic Radical Prostatectomy: A New Definition of Continence and the Impact of Different Surgical Techniques

Radical prostatectomy is considered the gold-standard treatment for patients with localized prostate cancer. The literature suggests there is no difference in oncological and functional outcomes between robotic-assisted radical prostatectomy (RARP) and open (RRP). (2) Methods: The aim of this study was to compare continence recovery rates after RARP and RRP measured with 24 h pad weights and the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF). After matching the population (1:1), 482 met the inclusion criteria, 241 patients per group. Continent patients with a 24 h pad test showing <20 g of urinary leakage were considered, despite severe incontinence, and categorized as having >200 g of urinary leakage. (3) Results: There was no difference between preoperative data. As for urinary continence (UC) and incontinence (UI) rates, RARP performed significantly better than RRP based on objective and subjective results at all evaluations. Univariable and multivariable Cox Regression Analysis pointed out that the only significant predictors of continence rates were the bilateral nerve sparing technique (1.25 (CI 1.02,1.54), p = 0.03) and the robotic surgical approach (1.42 (CI 1.18,1.69) p ≤ 0.001). (4) Conclusions: The literature reports different incidences of UC depending on assessment and definition of continence "without pads" or "social continence" based on number of used pads per day. In this, our first evaluation, the advantage of objective measurement through the weight of the 24 h and subjective measurement with the ICIQ-SF questionnaire best demonstrates the difference between the two surgical techniques by enhancing the use of robotic surgery over traditional surgery

Characteristics of Sepsis or Acute Pyelonephritis Combined with Ureteral Stone in the United States: A Retrospective Analysis of Large National Cohort

To identify the characteristics of patients with sepsis or acute pyelonephritis (APN) combined with ureteral calculi and to analyze the risk factors in its causation. Methods: We included patients with sepsis or APN caused by ureteral calculi who received treatment in the United States from January 2003 to December 2017 using the Optum® deidentified Clinformatics® Datamart. Demographic factors and risk factors for the receipt of sepsis or APN were subsequently analyzed for statistical significance. Results: Of 467,502 urinary stone patients, age-matched multivariate analysis revealed that a history of urinary tract infection (OR 11.31, 95% CI 10.68–11.99, p < 0.0001) and female gender (OR 2.73, 95% CI 2.62–2.84, p < 0.0001) were significantly related to an increased risk of sepsis or APN. Conversely, a previous past medical history of urolithiasis (OR 0.91, 95% CI 0.87–0.95, p < 0.0001) and cancer (OR 0.91, 95% CI 0.87–0.95, p < 0.0001) were associated with a decreased risk of sepsis or APN. With regards to comorbidities, when more than one comorbidity was present, there was an additive effect with higher OR point estimates, rising to 11.31 (10.68–11.99) when three or more comorbidities present. History of urinary tract infection and female gender are risk factors for sepsis or APN in patients with ureteral calculi. Conclusions: This large national cohort reveals the characteristics of sepsis or APN combined with ureteral stone and provides an important baseline for the treatment of urolithiasis in the future

Recent Trends in the Diagnostic and Surgical Management of Benign Prostatic Hyperplasia in the U.S. from 2004 to 2017: Annual Changes in the Selection of Treatment Options and Medical Costs

Background: Transurethral resection of the prostate (TURP) is the gold-standard treatment for benign prostatic hyperplasia (BPH). However, laser surgery techniques (e.g., photoselective vaporization of the prostate (PVP), holmium laser, thulium laser enucleation of the prostate (HoLEP or ThuLEP)), and minimally invasive treatment options (e.g., UroLift) are increasingly replacing TURP. This study seeks to report the annual incidence, management trends, and costs of BPH procedures in the U.S. Methods: Data analyses of U.S. health insurance claims from 2004 to 2017, collected from the de-identified Optum Clinformatics Claims Database, were performed to determine the number of BPH patients and the treatment selected. Results: A total of 51,448 patients underwent BPH procedures from 2004 to 2017. There was a significant increase in the annual rate from 770 in 2004 to 6571 in 2017. The mean patient age (±SD) increased from 67.6 years old (±8.4) in 2004 to 73.4 years old (±8.4) in 2017. More than 60% of patients underwent cystourethroscopy and a post-void residual urine check for workup prior to surgical management. TURP was the most-common, and PVP was the second-most-common BPH procedure. Medical and total treatment costs increased, while the detection rate of prostate cancer after BPH surgery gradually decreased from 19.87% in 2004 to 5.78% in 2017. Conclusions: Our study demonstrates a recent trend in BPH management that replaces the traditional TURP technique with alternative methods. Due to rising costs, future studies should assess whether these newer methods are cost effective over the long ter