Real-World Use of Oral and Subcutaneous Semaglutide in Routine Clinical Practice in the UK: A Single-Centre, Retrospective Observational Study.

INTRODUCTION: Semaglutide, the only glucagon-like peptide-1 receptor agonist (GLP-1 RA) available in subcutaneous and oral formulation for treatment of type 2 diabetes (T2D), has demonstrated clinically significant improvements in glycaemic control and weight in clinical trials. This study aimed to gain insights into the use of both formulations and evaluate their clinical effectiveness in a secondary care clinic in Wales. METHODS: This was a retrospective observational analysis of adults with T2D initiated on oral or subcutaneous semaglutide. Changes from baseline in glycated haemoglobin (HbA1c), weight and other metabolic parameters were evaluated. RESULTS: At baseline, participants (n = 103) had a mean age of 57.3 years, mean HbA1c of 79.1 mmol/mol (9.38%), mean weight of 111.8 kg and body mass index (BMI) of 39.6 kg/m2 (no statistically significant differences between oral and subcutaneous groups). At 6-month follow-up, statistically significant improvements in HbA1c (- 19.3 mmol/mol [- 1.77%] and - 20.8 mmol/mol [- 1.90%]), body weight (- 9.0 kg and - 7.2 kg), and BMI (- 3.3 kg/m2 and - 2.5 kg/m2) were observed for oral and subcutaneous semaglutide, respectively. No statistically significant differences between the formulations were observed, and safety profiles were comparable. CONCLUSIONS: Both formulations of semaglutide provided clinically and statistically significant reductions in HbA1c and weight in real-world practice. Oral GLP-1 RA may offer a practical and effective option for the management of T2D.

Development and Multicenter Case-Control Validation of Urinary Comprehensive Genomic Profiling for Urothelial Carcinoma Diagnosis, Surveillance, and Risk-Prediction.

PURPOSE: Urinary comprehensive genomic profiling (uCGP) uses next-generation sequencing to identify mutations associated with urothelial carcinoma and has the potential to improve patient outcomes by noninvasively diagnosing disease, predicting grade and stage, and estimating recurrence risk. EXPERIMENTAL DESIGN: This is a multicenter case-control study using banked urine specimens collected from patients undergoing initial diagnosis/hematuria workup or urothelial carcinoma surveillance. A total of 581 samples were analyzed by uCGP: 333 for disease classification and grading algorithm development, and 248 for blinded validation. uCGP testing was done using the UroAmp platform, which identifies five classes of mutation: single-nucleotide variants, copy-number variants, small insertion-deletions, copy-neutral loss of heterozygosity, and aneuploidy. UroAmp algorithms predicting urothelial carcinoma tumor presence, grade, and recurrence risk were compared with cytology, cystoscopy, and pathology. RESULTS: uCGP algorithms had a validation sensitivity/specificity of 95%/90% for initial cancer diagnosis in patients with hematuria and demonstrated a negative predictive value (NPV) of 99%. A positive diagnostic likelihood ratio (DLR) of 9.2 and a negative DLR of 0.05 demonstrate the ability to risk-stratify patients presenting with hematuria. In surveillance patients, binary urothelial carcinoma classification demonstrated an NPV of 91%. uCGP recurrence-risk prediction significantly prognosticated future recurrence (hazard ratio, 6.2), whereas clinical risk factors did not. uCGP demonstrated positive predictive value (PPV) comparable with cytology (45% vs. 42%) with much higher sensitivity (79% vs. 25%). Finally, molecular grade predictions had a PPV of 88% and a specificity of 95%. CONCLUSIONS: uCGP enables noninvasive, accurate urothelial carcinoma diagnosis and risk stratification in both hematuria and urothelial carcinoma surveillance patients.

Efficacy of a Dual-Epitope Dendritic Cell Vaccine as Part of Combined Immunotherapy for HER2-Expressing Breast Tumors.

Previous work from our group and others has shown that patients with breast cancer can generate a T cell response against specific human epidermal growth factor 2 (HER2) epitopes. In addition, preclinical work has shown that this T cell response can be augmented by Ag-directed mAb therapy. This study evaluated the activity and safety of a combination of dendritic cell (DC) vaccination given with mAb and cytotoxic therapy. We performed a phase I/II study using autologous DCs pulsed with two different HER2 peptides given with trastuzumab and vinorelbine to a study cohort of patients with HER2-overexpressing and a second with HER2 nonoverexpressing metastatic breast cancer. Seventeen patients with HER2-overexpressing and seven with nonoverexpressing disease were treated. Treatment was well tolerated, with one patient removed from therapy because of toxicity and no deaths. Forty-six percent of patients had stable disease after therapy, with 4% achieving a partial response and no complete responses. Immune responses were generated in the majority of patients but did not correlate with clinical response. However, in one patient, who has survived >14 y since treatment in the trial, a robust immune response was demonstrated, with 25% of her T cells specific to one of the peptides in the vaccine at the peak of her response. These data suggest that autologous DC vaccination when given with anti-HER2-directed mAb therapy and vinorelbine is safe and can induce immune responses, including significant T cell clonal expansion, in a subset of patients.

Tackling antimicrobial resistance across sub-Saharan Africa: current challenges and implications for the future.

INTRODUCTION: Antimicrobial resistance (AMR) is a concern as this increases morbidity, mortality, and costs, with sub-Saharan Africa having the highest rates globally. Concerns with rising AMR have resulted in international, Pan-African, and country activities including the development of national action plans (NAPs). However, there is variable implementation across Africa with key challenges persisting. AREAS COVERED: Consequently, there is an urgent need to document current NAP activities and challenges across sub-Saharan Africa to provide future guidance. This builds on a narrative review of the literature. EXPERT OPINION: All surveyed sub-Saharan African countries have developed their NAPs; however, there is variable implementation. Countries including Botswana and Namibia are yet to officially launch their NAPs with Eswatini only recently launching its NAP. Cameroon is further ahead with its NAP than these countries; though there are concerns with implementation. South Africa appears to have made the greatest strides with implementing its NAP including regular monitoring of activities and instigation of antimicrobial stewardship programs. Key challenges remain across Africa. These include available personnel, expertise, capacity, and resources to undertake agreed NAP activities including active surveillance, lack of focal points to drive NAPs, and competing demands and priorities including among donors. These challenges are being addressed, with further co-ordinated efforts needed to reduce AMR.

Tomatoes: An Extensive Review of the Associated Health Impacts of Tomatoes and Factors That Can Affect Their Cultivation.

This review outlines the health benefits associated with the regular consumption of tomatoes and tomato products. The first section provides a detailed account of the horticultural techniques that can impact the quality of the fruit and its nutritional properties, including water availability, light intensity, temperature, and growing media. The next section provides information on the components of tomato that are likely to contribute to its health effects. The review then details some of the health benefits associated with tomato consumption, including anticancer properties, cardiovascular and neurodegenerative diseases and skin health. This review also discusses the impact tomatoes can have on the gut microbiome and associated health benefits, including reducing the risk of inflammatory bowel diseases. Other health benefits of eating tomatoes are also discussed in relation to effects on diabetes, the immune response, exercise recovery, and fertility. Finally, this review also addresses the negative effects that can occur as a result of overconsumption of tomato products and lycopene supplements.

Role of Position K+4 in the Phosphorylation and Dephosphorylation Reaction Kinetics of the CheY Response Regulator.

Two-component signaling is a primary method by which microorganisms interact with their environments. A kinase detects stimuli and modulates autophosphorylation activity. The signal propagates by phosphotransfer from the kinase to a response regulator, eliciting a response. Response regulators operate over a range of time scales, corresponding to their related biological processes. Response regulator active site chemistry is highly conserved, but certain variable residues can influence phosphorylation kinetics. An Ala-to-Pro substitution (K+4, residue 113) in the Escherichia coli response regulator CheY triggers a constitutively active phenotype; however, the A113P substitution is too far from the active site to directly affect phosphochemistry. To better understand the activating mechanism(s) of the substitution, we analyzed receiver domain sequences to characterize the evolutionary role of the K+4 position. Although most featured Pro, Leu, Ile, and Val residues, chemotaxis-related proteins exhibited atypical Ala, Gly, Asp, and Glu residues at K+4. Structural and in silico analyses revealed that CheY A113P adopted a partially active configuration. Biochemical data showed that A113P shifted CheY toward a more activated state, enhancing autophosphorylation. By characterizing CheY variants, we determined that this functionality was transmitted through a hydrophobic network bounded by the ß5α5 loop and the α1 helix of CheY. This region also interacts with the phosphodonor CheAP1, suggesting that binding generates an activating perturbation similar to the A113P substitution. Atypical residues like Ala at the K+4 position likely serve two purposes. First, restricting autophosphorylation may minimize background noise generated by intracellular phosphodonors such as acetyl phosphate. Second, optimizing interactions with upstream partners may help prime the receiver domain for phosphorylation.

Computational modeling and confirmation of leukemia-associated minor histocompatibility antigens.

T-cell responses to minor histocompatibility antigens (mHAs) mediate both antitumor immunity (graft-versus-leukemia [GVL]) and graft-versus-host disease (GVHD) in allogeneic stem cell transplant. Identifying mHAs with high allele frequency, tight binding affinity to common HLA molecules, and narrow tissue restriction could enhance immunotherapy against leukemia. Genotyping and HLA allele data from 101 HLA-matched donor-recipient pairs (DRPs) were computationally analyzed to predict both class I and class II mHAs likely to induce either GVL or GVHD. Roughly twice as many mHAs were predicted in HLA-matched unrelated donor (MUD) stem cell transplantation (SCT) compared with HLA-matched related transplants, an expected result given greater genetic disparity in MUD SCT. Computational analysis predicted 14 of 18 previously identified mHAs, with 2 minor antigen mismatches not being contained in the patient cohort, 1 missed mHA resulting from a noncanonical translation of the peptide antigen, and 1 case of poor binding prediction. A predicted peptide epitope derived from GRK4, a protein expressed in acute myeloid leukemia and testis, was confirmed by targeted differential ion mobility spectrometry-tandem mass spectrometry. T cells specific to UNC-GRK4-V were identified by tetramer analysis both in DRPs where a minor antigen mismatch was predicted and in DRPs where the donor contained the allele encoding UNC-GRK4-V, suggesting that this antigen could be both an mHA and a cancer-testis antigen. Computational analysis of genomic and transcriptomic data can reliably predict leukemia-associated mHA and can be used to guide targeted mHA discovery.

The canine MHC class Ia allele DLA-88*508:01 presents diverse self- and canine distemper virus-origin peptides of varying length that have a conserved binding motif.

Ideally, CD8+ T-cell responses against virally infected or malignant cells are defined at the level of the specific peptide and restricting MHC class I element, a determination not yet made in the dog. To advance the discovery of canine CTL epitopes, we sought to determine whether a putative classical MHC class Ia gene, Dog Leukocyte Antigen (DLA)-88, presents peptides from a viral pathogen, canine distemper virus (CDV). To investigate this possibility, DLA-88*508:01, an allele prevalent in Golden Retrievers, was expressed as a FLAG-tagged construct in canine histiocytic cells to allow affinity purification of peptide-DLA-88 complexes and subsequent elution of bound peptides. Pattern analysis of self peptide sequences, which were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), permitted binding preferences to be inferred. DLA-88*508:01 binds peptides that are 9-to-12 amino acids in length, with a modest preference for 9- and 11-mers. Hydrophobic residues are favored at positions 2 and 3, as are K, R or F residues at the C-terminus. Testing motif-matched and -unmatched synthetic peptides via peptide-MHC surface stabilization assay using a DLA-88*508:01-transfected, TAP-deficient RMA-S line supported these conclusions. With CDV infection, 22 viral peptides ranging from 9-to-12 residues in length were identified in DLA-88*508:01 eluates by LC-MS/MS. Combined motif analysis and surface stabilization assay data suggested that 11 of these 22 peptides, derived from CDV hemagglutinin, large polymerase, matrix, nucleocapsid, and V proteins, were processed and presented, and thus, potential targets of anti-viral CTL in DLA-88*508:01-bearing dogs. The presentation of diverse self and viral peptides indicates that DLA-88 is a classical MHC class Ia gene.

Imidazole as a Small Molecule Analogue in Two-Component Signal Transduction.

In two-component signal transduction systems (TCSs), responses to stimuli are mediated through phosphotransfer between protein components. Canonical TCSs use His → Asp phosphotransfer in which phosphoryl groups are transferred from a conserved His on a sensory histidine kinase (HK) to a conserved Asp on a response regulator (RR). RRs contain the catalytic core of His → Asp phosphotransfer, evidenced by the ability of RRs to autophosphorylate with small molecule analogues of phospho-His proteins. Phosphorelays are a more complex variation of TCSs that additionally utilize Asp → His phosphotransfer through the use of an additional component, the histidine-containing phosphotransfer domain (Hpt), which reacts with RRs both as phosphodonors and phosphoacceptors. Here we show that imidazole has features of a rudimentary Hpt. Imidazole acted as a nucleophile and attacked phosphorylated RRs (RR-P) to produce monophosphoimidazole (MPI) and unphosphorylated RR. Phosphotransfer from RR-P to imidazole required the intact RR active site, indicating that the RR provided the core catalytic machinery for Asp → His phosphotransfer. Imidazole functioned in an artificial phosphorelay to transfer phosphoryl groups between unrelated RRs. The X-ray crystal structure of an activated RR·imidazole complex showed imidazole oriented in the RR active site similarly to the His of an Hpt. Imidazole interacted with RR nonconserved active site residues, which influenced the relative reactivity of RR-P with imidazole versus water. Rate constants for reaction of imidazole or MPI with chimeric RRs suggested that the RR active site contributes to the kinetic preferences exhibited by the YPD1 Hpt.

The Code of Professional Conduct for the Neurocritical Care Society.

Part of the responsibility of a professional society is to establish the expectations for appropriate behavior for its members. Some codes are so essential to a society that the code itself becomes the central document defining the organization and its tenets, as we see with the Hippocratic Oath. In that tradition, we have revised the code of professional conduct for the Neurocritical Care Society into its current version, which emphasizes guidelines for personal behavior, relationships with fellow members, relationships with patients, and our interactions with society as a whole. This will be a living document and updated as the needs of our society change in time.Available online: http://www.neurocriticalcare.org/about-us/bylaws-procedures-and-code-professional-conduct (1) Code of professional conduct (this document) (2) Leadership code of conduct (3) Disciplinary policy.

Salivary Biomarkers Associated With Bone Deterioration in Patients With Medication-Related Osteonecrosis of the Jaws.

PURPOSE: The purpose of this study was to investigate the use of saliva as a medium for the identification of biomarkers associated with bone resorption and formation. The authors hypothesized that biomarkers, such as N-telopeptide of type I collagen (NTX) and bone-specific alkaline phosphatase (B-AP), could be identified in saliva. They further hypothesized that there would be a difference between these biomarkers in the saliva of patients with medication-relation osteonecrosis of the jaws (MRONJ) and those who have no risk factors for the development of MRONJ. PATIENTS AND METHODS: This case-and-control study compared 2 salivary biomarkers, NTX and B-AP, in a group of patients with MRONJ and a control group. The predictor variable was the presence or absence of the disease (MRONJ or control group); the outcome variables were the levels of the 2 salivary biomarkers, NTX and B-AP. Saliva samples from 20 patients with a diagnosis of MRONJ and 14 control participants who were comparable to the study group with no history of antiresorptive medication use were collected. The saliva samples were analyzed using 2 commercially available assays for NTX and B-AP to evaluate for levels of each marker. A 2-tailed t test for 2 groups of unequal distribution was used for statistical analysis, with P values less than .05 considered statistically. RESULTS: The 2 biomarkers, NTX and B-AP, were detected in saliva samples from the MRONJ and control groups. A statistically significant difference was found in the levels of NTX in saliva of patients with MRONJ compared with the control participants (P = .0067). CONCLUSIONS: In this exploratory study, the 2 bone deterioration biomarkers (NTX and B-AP) were detected in saliva. There was a statistical difference in the levels of salivary NTX between patients with MRONJ and controls. Saliva evaluation could provide a novel method to detect, diagnose, stage, and potentially guide treatment decisions and monitor outcomes for patients with MRONJ in the future.

Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA.

BACKGROUND: Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis. RESULTS: LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein. CONCLUSION: These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance.

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

Accurate structure prediction of peptide-MHC complexes for identifying highly immunogenic antigens.

Designing an optimal HIV-1 vaccine faces the challenge of identifying antigens that induce a broad immune capacity. One factor to control the breadth of T cell responses is the surface morphology of a peptide-MHC complex. Here, we present an in silico protocol for predicting peptide-MHC structure. A robust signature of a conformational transition was identified during all-atom molecular dynamics, which results in a model with high accuracy. A large test set was used in constructing our protocol and we went another step further using a blind test with a wild-type peptide and two highly immunogenic mutants, which predicted substantial conformational changes in both mutants. The center residues at position five of the analogs were configured to be accessible to solvent, forming a prominent surface, while the residue of the wild-type peptide was to point laterally toward the side of the binding cleft. We then experimentally determined the structures of the blind test set, using high resolution of X-ray crystallography, which verified predicted conformational changes. Our observation strongly supports a positive association of the surface morphology of a peptide-MHC complex to its immunogenicity. Our study offers the prospect of enhancing immunogenicity of vaccines by identifying MHC binding immunogens.

T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64)Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

Francisella tularensis RipA protein topology and identification of functional domains.

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1ß (IL-1ß) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.

Evidence for dynamics in proteins as a mechanism for ligand dissociation.

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.

180-W XPS GreenLight laser therapy for benign prostate hyperplasia: early safety, efficacy, and perioperative outcome after 201 procedures.

BACKGROUND: Photoselective vaporisation of the prostate has evolved from the GreenLight 80-W KTP powered laser to the latest 180-W XPS laser involving a MoXy fibre. OBJECTIVE: Evaluate the prevalence of perioperative complications and short-term outcome for the first time with the XPS laser in men with lower urinary tract symptoms (LUTS) due to benign prostatic enlargement (BPE). DESIGN, SETTING, AND PARTICIPANTS: Prospective data were collected from consecutive patients at seven centres worldwide during June 2010 and March 2011. Indication for surgery was based on the European Association of Urology and the American Urological Association guidelines. Patients receiving anticoagulants or those with retention were included and analysed separately. INTERVENTION: 180-W XPS GreenLight laser prostatectomy using the MoXy fibre. MEASUREMENTS: Standard parameters associated with transurethral prostate surgery and perioperative prevalence of surgery-associated problems or complications were documented. RESULTS AND LIMITATIONS: A total of 201 patients were included in the study. Mean follow-up was 5.8 mo (standard deviation [SD]: 2.8; range: 1-12 mo). A quarter of the patients had a prostate volume≥80 ml. For prostates between 51 and 60 ml, a mean of 300 kJ (SD: 112) of energy was applied (lasing time: 35.0 min; SD: 15). Statistically significant improvements were noted in all key parameters postoperatively. The prevalence of perioperative complications was low. Limitations of the study are short duration of follow-up and limited number of available patients for the functional follow-up. CONCLUSIONS: The 180-W GreenLight XPS laser is a new effective treatment option with a low prevalence of perioperative complications for patients suffering from LUTS due to BPE.

Direct detection of structurally resolved dynamics in a multiconformation receptor-ligand complex.

Structure-based drug design relies on static protein structures despite significant evidence for the need to include protein dynamics as a serious consideration. In practice, dynamic motions are neglected because they are not understood well enough to model, a situation resulting from a lack of explicit experimental examples of dynamic receptor-ligand complexes. Here, we report high-resolution details of pronounced ~1 ms time scale motions of a receptor-small molecule complex using a combination of NMR and X-ray crystallography. Large conformational dynamics in Escherichia coli dihydrofolate reductase are driven by internal switching motions of the drug-like, nanomolar-affinity inhibitor. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments and NOEs revealed the crystal structure to contain critical elements of the high energy protein-ligand conformation. The availability of accurate, structurally resolved dynamics in a protein-ligand complex should serve as a valuable benchmark for modeling dynamics in other receptor-ligand complexes and prediction of binding affinities.

Matching biochemical reaction kinetics to the timescales of life: structural determinants that influence the autodephosphorylation rate of response regulator proteins.

In two-component regulatory systems, covalent phosphorylation typically activates the response regulator signaling protein, and hydrolysis of the phosphoryl group reestablishes the inactive state. Despite highly conserved three-dimensional structures and active-site features, the rates of catalytic autodephosphorylation for different response regulators vary by a factor of almost 10(6). Previous studies identified two variable active-site residues, corresponding to Escherichia coli CheY residues 59 and 89, that modulate response regulator autodephosphorylation rates about 100-fold. Here, a set of five CheY mutants, which match other "model" response regulators (ArcA, CusR, DctD, FixJ, PhoB, or Spo0F) at variable active-site positions corresponding to CheY residues 14, 59, and 89, were characterized functionally and structurally in an attempt to identify mechanisms that modulate autodephosphorylation rate. As expected, the autodephosphorylation rates of the CheY mutants were reduced 6- to 40-fold relative to wild-type CheY, but all still autodephosphorylated 12- to 80-fold faster than their respective model response regulators. Comparison of X-ray crystal structures of the five CheY mutants (complexed with the phosphoryl group analogue BeF(3)(-)) to wild-type CheY or corresponding model response regulator structures gave strong evidence for steric obstruction of the phosphoryl group from the attacking water molecule as one mechanism to enhance phosphoryl group stability. Structural data also suggested that impeding the change of a response regulator from the active to the inactive conformation might retard the autodephosphorylation reaction if the two processes are coupled, and that the residue at position '58' may contribute to rate modulation. A given combination of amino acids at positions '14', '59', and '89' adopted similar conformations regardless of protein context (CheY or model response regulator), suggesting that knowledge of residue identity may be sufficient to predict autodephosphorylation rate, and hence the kinetics of the signaling response, in the response regulator family of proteins.