Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function.

Spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating intracellular immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as antineoplastic and anti-inflammatory therapeutics and have also gained interest as antiplatelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659; four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib); and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Similarly, these TKIs reduced the percentage of activated integrin αIIbß3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to phosphoinositide 3-kinase (PI3K) and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

Laparoscopic supracervical hysterectomy versus endometrial ablation for women with heavy menstrual bleeding (HEALTH): a parallel-group, open-label, randomised controlled trial.

BACKGROUND: Heavy menstrual bleeding affects 25% of women in the UK, many of whom require surgery to treat it. Hysterectomy is effective but has more complications than endometrial ablation, which is less invasive but ultimately leads to hysterectomy in 20% of women. We compared laparoscopic supracervical hysterectomy with endometrial ablation in women seeking surgical treatment for heavy menstrual bleeding. METHODS: In this parallel-group, multicentre, open-label, randomised controlled trial in 31 hospitals in the UK, women younger than 50 years who were referred to a gynaecologist for surgical treatment of heavy menstrual bleeding and who were eligible for endometrial ablation were randomly allocated (1:1) to either laparoscopic supracervical hysterectomy or second generation endometrial ablation. Women were randomly assigned by either an interactive voice response telephone system or an internet-based application with a minimisation algorithm based on centre and age group (<40 years vs ≥40 years). Laparoscopic supracervical hysterectomy involves laparoscopic (keyhole) surgery to remove the upper part of the uterus (the body) containing the endometrium. Endometrial ablation aims to treat heavy menstrual bleeding by destroying the endometrium, which is responsible for heavy periods. The co-primary clinical outcomes were patient satisfaction and condition-specific quality of life, measured with the menorrhagia multi-attribute quality of life scale (MMAS), assessed at 15 months after randomisation. Our analysis was based on the intention-to-treat principle. The trial was registered with the ISRCTN registry, number ISRCTN49013893. FINDINGS: Between May 21, 2014, and March 28, 2017, we enrolled and randomly assigned 660 women (330 in each group). 616 (93%) of 660 women were operated on within the study period, 588 (95%) of whom received the allocated procedure and 28 (5%) of whom had an alternative surgery. At 15 months after randomisation, more women allocated to laparoscopic supracervical hysterectomy were satisfied with their operation compared with those in the endometrial ablation group (270 [97%] of 278 women vs 244 [87%] of 280 women; adjusted percentage difference 9·8, 95% CI 5·1-14·5; adjusted odds ratio [OR] 2·53, 95% CI 1·83-3·48; p<0·0001). Women randomly assigned to laparoscopic supracervical hysterectomy were also more likely to have the best possible MMAS score of 100 than women assigned to endometrial ablation (180 [69%] of 262 women vs 146 [54%] of 268 women; adjusted percentage difference 13·3, 95% CI 3·8-22·8; adjusted OR 1·87, 95% CI 1·31-2·67; p=0·00058). 14 (5%) of 309 women in the laparoscopic supracervical hysterectomy group and 11 (4%) of 307 women in the endometrial ablation group had at least one serious adverse event (adjusted OR 1·30, 95% CI 0·56-3·02; p=0·54). INTERPRETATION: Laparoscopic supracervical hysterectomy is superior to endometrial ablation in terms of clinical effectiveness and has a similar proportion of complications, but takes longer to perform and is associated with a longer recovery. FUNDING: UK National Institute for Health Research Health Technology Assessment Programme.

Laparoscopic entry techniques.

BACKGROUND: Laparoscopy is a common procedure in many surgical specialties. Complications arising from laparoscopy are often related to initial entry into the abdomen. Life-threatening complications include injury to viscera (e.g. bowel, bladder) or to vasculature (e.g. major abdominal and anterior abdominal wall vessels). No clear consensus has been reached as to the optimal method of laparoscopic entry into the peritoneal cavity. OBJECTIVES: To evaluate the benefits and risks of different laparoscopic entry techniques in gynaecological and non-gynaecological surgery. SEARCH METHODS: We searched the Cochrane Gynaecology and Fertility (CGF) Group trials register, CENTRAL, MEDLINE, Embase, PsycINFO, and trials registers in January 2018. We also checked the references of articles retrieved. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared one laparoscopic entry technique versus another. Primary outcomes were major complications including mortality, vascular injury of major vessels and abdominal wall vessels, visceral injury of bladder or bowel, gas embolism, solid organ injury, and failed entry (inability to access the peritoneal cavity). Secondary outcomes were extraperitoneal insufflation, trocar site bleeding, trocar site infection, incisional hernia, omentum injury, and uterine bleeding. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies, assessed risk of bias, and extracted data. We expressed findings as Peto odds ratios (Peto ORs) with 95% confidence intervals (CIs). We assessed statistical heterogeneity using the I² statistic. We assessed the overall quality of evidence for the main comparisons using GRADE methods. MAIN RESULTS: The review included 57 RCTs including four multi-arm trials, with a total of 9865 participants, and evaluated 25 different laparoscopic entry techniques. Most studies selected low-risk patients, and many studies excluded patients with high body mass index (BMI) and previous abdominal surgery. Researchers did not find evidence of differences in major vascular or visceral complications, as would be anticipated given that event rates were very low and sample sizes were far too small to identify plausible differences in rare but serious adverse events.Open-entry versus closed-entryTen RCTs investigating Veress needle entry reported vascular injury as an outcome. There was a total of 1086 participants and 10 events of vascular injury were reported. Four RCTs looking at open entry technique reported vascular injury as an outcome. There was a total of 376 participants and 0 events of vascular injury were reported. This was not a direct comparison. In the direct comparison of Veress needle and Open-entry technique, there was insufficient evidence to determine whether there was a difference in rates of vascular injury (Peto OR 0.14, 95% CI 0.00 to 6.82; 4 RCTs; n = 915; I² = N/A, very low-quality evidence). Evidence was insufficient to show whether there were differences between groups for visceral injury (Peto OR 0.61, 95% CI 0.06 to 6.08; 4 RCTs; n = 915: I² = 0%; very low-quality evidence), or failed entry (Peto OR 0.45, 95% CI 0.14 to 1.42; 3 RCTs; n = 865; I² = 63%; very low-quality evidence). Two studies reported mortality with no events in either group. No studies reported gas embolism or solid organ injury.Direct trocar versus Veress needle entryTrial results show a reduction in failed entry into the abdomen with the use of a direct trocar in comparison with Veress needle entry (OR 0.24, 95% CI 0.17 to 0.34; 8 RCTs; N = 3185; I² = 45%; moderate-quality evidence). Evidence was insufficient to show whether there were differences between groups in rates of vascular injury (Peto OR 0.59, 95% CI 0.18 to 1.96; 6 RCTs; n = 1603; I² = 75%; very low-quality evidence), visceral injury (Peto OR 2.02, 95% CI 0.21 to 19.42; 5 RCTs; n = 1519; I² = 25%; very low-quality evidence), or solid organ injury (Peto OR 0.58, 95% Cl 0.06 to 5.65; 3 RCTs; n = 1079; I² = 61%; very low-quality evidence). Four studies reported mortality with no events in either group. Two studies reported gas embolism, with no events in either group.Direct vision entry versus Veress needle entryEvidence was insufficient to show whether there were differences between groups in rates of vascular injury (Peto OR 0.39, 95% CI 0.05 to 2.85; 1 RCT; n = 186; very low-quality evidence) or visceral injury (Peto OR 0.15, 95% CI 0.01 to 2.34; 2 RCTs; n = 380; I² = N/A; very low-quality evidence). Trials did not report our other primary outcomes.Direct vision entry versus open entryEvidence was insufficient to show whether there were differences between groups in rates of visceral injury (Peto OR 0.13, 95% CI 0.00 to 6.50; 2 RCTs; n = 392; I² = N/A; very low-quality evidence), solid organ injury (Peto OR 6.16, 95% CI 0.12 to 316.67; 1 RCT; n = 60; very low-quality evidence), or failed entry (Peto OR 0.40, 95% CI 0.04 to 4.09; 1 RCT; n = 60; very low-quality evidence). Two studies reported vascular injury with no events in either arm. Trials did not report our other primary outcomes.Radially expanding (STEP) trocars versus non-expanding trocarsEvidence was insufficient to show whether there were differences between groups in rates of vascular injury (Peto OR 0.24, 95% Cl 0.05 to 1.21; 2 RCTs; n = 331; I² = 0%; very low-quality evidence), visceral injury (Peto OR 0.13, 95% CI 0.00 to 6.37; 2 RCTs; n = 331; very low-quality evidence), or solid organ injury (Peto OR 1.05, 95% CI 0.07 to 16.91; 1 RCT; n = 244; very low-quality evidence). Trials did not report our other primary outcomes.Other studies compared a wide variety of other laparoscopic entry techniques, but all evidence was of very low quality and evidence was insufficient to support the use of one technique over another. AUTHORS' CONCLUSIONS: Overall, evidence was insufficient to support the use of one laparoscopic entry technique over another. Researchers noted an advantage of direct trocar entry over Veress needle entry for failed entry. Most evidence was of very low quality; the main limitations were imprecision (due to small sample sizes and very low event rates) and risk of bias associated with poor reporting of study methods.

The Effect of Magnesium Carbonate (Chalk) on Geometric Entropy, Force, and Electromyography During Rock Climbing.

Rock climbers believe chalk dries the hands of sweat and improves the static coefficient of friction between the hands and the surface of the rock. The purpose of this study was to assess whether chalk affects geometric entropy or muscular activity during rock climbing. Nineteen experienced recreational rock climbers (13 males, 6 females; 173.5 ± 7.0 cm; 67.5 ± 3.4 kg) completed 2 climbing trails with and without chalk. The body position of the climber and muscular activity of the finger flexors was recorded throughout the trial. Following the movement sequence participants hung from a standard climbing hold until they slipped from the climbing structure, while the coefficient of friction and the ratio of the vertical forces on the hands and feet were determined. Although there were no differences in the coefficient of friction (P = .748), geometric entropy (P = .359), the ratio of the vertical forces between the hands and feet (P = .570), or muscular activity (P = .968), participants were able to hang longer after the use of chalk 62.9 ± 36.7 s and 49.3 ± 25.2 s (P = .046). This is advantageous because it may allow for prolonged rests, and more time to plan the next series of climbing moves.

The thrombotic potential of circulating tumor microemboli: computational modeling of circulating tumor cell-induced coagulation.

Thrombotic events can herald the diagnosis of cancer, preceding any cancer-related clinical symptoms. Patients with cancer are at a 4- to 7-fold increased risk of suffering from venous thromboembolism (VTE), with ∼7,000 patients with lung cancer presenting from VTEs. However, the physical biology underlying cancer-associated VTE remains poorly understood. Several lines of evidence suggest that the shedding of tissue factor (TF)-positive circulating tumor cells (CTCs) and microparticles from primary tumors may serve as a trigger for cancer-associated thrombosis. To investigate the potential direct and indirect roles of CTCs in VTE, we characterized thrombin generation by CTCs in an interactive numerical model coupling blood flow with advection-diffusion kinetics. Geometric measurements of CTCs isolated from the peripheral blood of a lung cancer patient prior to undergoing lobectomy formed the basis of the simulations. Singlet, doublet, and aggregate circulating tumor microemboli (CTM) were investigated in the model. Our numerical model demonstrated that CTM could potentiate occlusive events that drastically reduce blood flow and serve as a platform for the promotion of thrombin generation in flowing blood. These results provide a characterization of CTM dynamics in the vasculature and demonstrate an integrative framework combining clinical, biophysical, and mathematical approaches to enhance our understanding of CTCs and their potential direct and indirect roles in VTE formation.

A physical sciences network characterization of circulating tumor cell aggregate transport.

Circulating tumor cells (CTC) have been implicated in the hematogenous spread of cancer. To investigate the fluid phase of cancer from a physical sciences perspective, the multi-institutional Physical Sciences-Oncology Center (PS-OC) Network performed multidisciplinary biophysical studies of single CTC and CTC aggregates from a patient with breast cancer. CTCs, ranging from single cells to aggregates comprised of 2-5 cells, were isolated using the high-definition CTC assay and biophysically profiled using quantitative phase microscopy. Single CTCs and aggregates were then modeled in an in vitro system comprised of multiple breast cancer cell lines and microfluidic devices used to model E-selectin mediated rolling in the vasculature. Using a numerical model coupling elastic collisions between red blood cells and CTCs, the dependence of CTC vascular margination on single CTCs and CTC aggregate morphology and stiffness was interrogated. These results provide a multifaceted characterization of single CTC and CTC aggregate dynamics in the vasculature and illustrate a framework to integrate clinical, biophysical, and mathematical approaches to enhance our understanding of the fluid phase of cancer.

Laparoscopic entry techniques.

BACKGROUND: Laparoscopy is a common procedure in many surgical specialities. Complications arising from laparoscopy are often related to initial entry into the abdomen. Life-threatening complications include injury to viscera e.g. the bowel or bladder, or to vasculature e.g. major abdominal and anterior abdominal wall vessels. Minor complications can also occur, such as postoperative wound infection, subcutaneous emphysema, and extraperitoneal insufflation. There is no clear consensus as to the optimal method of laparoscopic entry into the peritoneal cavity. OBJECTIVES: To evaluate the benefits and risks of different laparoscopic entry techniques in gynaecological and non-gynaecological surgery. SEARCH METHODS: This updated review has drawn on the search strategy developed by the Cochrane Menstrual Disorders and Subfertility Group. In addition, MEDLINE, EMBASE, CENTRAL and PsycINFO were searched through to September 2014. SELECTION CRITERIA: We included randomised controlled trials (RCTs) in which one laparoscopic entry technique was compared with another. DATA COLLECTION AND ANALYSIS: Two authors independently selected studies, assessed risk of bias, and extracted data. We expressed findings as Peto odds ratios (Peto ORs) with 95% confidence intervals (CIs). We assessed statistical heterogeneity using the I² statistic. We assessed the overall quality of evidence for the main comparisons using GRADE methods. MAIN RESULTS: The review included 46 RCTs including three multi-arm trials (7389 participants) and evaluated 13 laparoscopic entry techniques. Overall there was no evidence of advantage using any single technique for preventing major vascular or visceral complications. The evidence was generally of very low quality; the main limitations were imprecision and poor reporting of study methods. Open-entry versus closed-entry There was no evidence of a difference between the groups for vascular (Peto OR 0.14, 95% CI 0.00 to 6.82, three RCTs, n = 795, I(2) = n/a; very low quality evidence) or visceral injury (Peto OR 0.61, 95% CI 0.06 to 6.08, three RCTs, n = 795, I(2) = 0%; very low quality evidence). There was a lower risk of failed entry in the open-entry group (Peto OR 0.16, 95% CI 0.04 to 0.63, n = 665, two RCTs, I(2) = 0%; very low quality evidence). This suggests that for every 1000 patients operated on, 31 patients in the closed-entry group will have failed entry compared to between 1 to 20 patients in the open-entry group. No events were reported in any of the studies for mortality, gas embolism or solid organ injury. Direct trocar versus Veress needle entry There was a lower risk of vascular injury in the direct trocar group (Peto OR 0.13, 95% CI 0.03 to 0.66, five RCTs, n = 1522, I(2) = 0%; low quality evidence) and failed entry (Peto OR 0.21, 95% CI 0.14 to 0.30, seven RCTs, n = 3104; I ²= 0%; moderate quality evidence). This suggests that for every 1000 patients operated on, 8 patients in the Veress needle group will experience vascular injury compared to between 0 to 5 patients in the direct trocar group; and that 64 patients in the Veress needle group will experience failed entry compared to between 10 to 20 patients in the direct trocar group. The vascular injury significance is sensitive to choice of statistical analysis and may be unreliable. There was no evidence of a difference between the groups for visceral (Peto OR 1.02, 95% CI 0.06 to 16.24, four RCTs, n = 1438, I(2) = 49%; very low quality evidence) or solid organ injury (Peto OR 0.16, 95% Cl 0.01 to 2.53, two RCTs, n = 998, I(2) = n/a; very low quality evidence). No events were recorded for mortality or gas embolism. Direct vision entry versus Veress needle entry There was no evidence of a difference between the groups in the rates of visceral injury (Peto OR 0.15, 95% CI 0.01 to 2.34, one RCT, n = 194; very low quality evidence). Other primary outcomes were not reported. Direct vision entry versus open-entry There was no evidence of a difference between the groups in the rates of visceral injury (Peto OR 0.13, 95% CI 0.00 to 6.50, two RCTs, n = 392; low quality evidence), solid organ injury (Peto OR 6.16, 95% CI 0.12 to 316.67, one RCT, n = 60, I(2) = n/a; very low quality evidence), or failed entry (Peto OR 0.40, 95% CI 0.04 to 4.09, one RCT, n = 60; low quality evidence). Vascular injury was reported, however no events occurred. Our other primary outcomes were not reported. Radially expanding (STEP) trocars versus non-expanding trocars There was no evidence of a difference between the groups for vascular injury (Peto OR 0.24, 95% Cl 0.05 to 1.21, two RCTs, n = 331, I(2) = 0%; low quality evidence), visceral injury (Peto OR 0.13, 95% CI 0.00 to 6.37, two RCTs, n = 331, I(2) = n/a; low quality evidence), or solid organ injury (Peto OR 1.05, 95% CI 0.07 to 16.91, one RCT, n = 244; very low quality evidence). Other primary outcomes were not reported. Comparisons of other laparoscopic entry techniquesThere was a higher risk of failed entry in the group in which the abdominal wall was lifted before Veress needle insertion than in the not-lifted group (Peto OR 4.44, 95% CI 2.16 to 9.13, one RCT, n = 150; very low quality evidence). There was no evidence of a difference between the groups in rates of visceral injury or extraperitoneal insufflation. The studies had small numbers and excluded many patients with previous abdominal surgery, and women with a raised body mass index. These patients may have unusually high complication rates. AUTHORS' CONCLUSIONS: Overall, there is insufficient evidence to recommend one laparoscopic entry technique over another.An open-entry technique is associated with a reduction in failed entry when compared to a closed-entry technique, with no evidence of a difference in the incidence of visceral or vascular injury.An advantage of direct trocar entry over Veress needle entry was noted for failed entry and vascular injury. The evidence was generally of very low quality with small numbers of participants in most studies; our findings should be interpreted with caution.

Thrombin mutant W215A/E217A treatment improves neurological outcome and attenuates central nervous system damage in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination and axonal damage of the central nervous system. The pathogenesis of MS has also been linked to vascular inflammation and local activation of the coagulation system, resulting in perivascular fibrin deposition. Treatment of experimental autoimmune encephalomyelitis (EAE), a model of human MS, with antithrombotic and antiinflammatory activated protein C (APC) reduces disease severity. Since recombinant APC (Drotecogin alfa), originally approved for the treatment of severe sepsis, is not available for human MS studies, we tested the hypothesis that pharmacologic activation of endogenous protein C could likewise improve the outcome of EAE. Mice were immunized with murine myelin oligodendrocyte glycoprotein (MOG) peptides and at the onset of EAE symptoms, were treated every other day with either WE thrombin (25 µg/kg; i.v.), a selective recombinant protein C activator thrombin analog, or saline control. Mice were monitored for changes in disease score until euthanized for ex vivo analysis of inflammation. Administration of WE thrombin significantly ameliorated clinical severity of EAE, reduced inflammatory cell infiltration and demyelination, suppressed the activation of macrophages comprising the CD11b + population and reduced accumulation of fibrin (ogen) in the spinal cord. These data suggest that symptomatic MS may respond to a treatment strategy that involves temporal pharmacological enhancement of endogenous APC generation.

Physical biology in cancer. 2. The physical biology of circulating tumor cells.

The identification, isolation, and characterization of circulating tumor cells (CTCs) promises to enhance our understanding of the evolution of cancer in humans. CTCs provide a window into the hematogenous, or "fluid phase," of cancer, underlying the metastatic transition in which a locally contained tumor spreads to other locations in the body through the bloodstream. With the development of sensitive and specific CTC identification and isolation methodologies, the role of CTCs in clinical diagnostics, disease surveillance, and the physical basis of metastasis continues to be established. This review focuses on the quantification of the basic biophysical properties of CTCs and the use of these metrics to understand the hematogenous dissemination of these enigmatic cells.

Urinary comprehensive genomic profiling predicts urothelial carcinoma recurrence and identifies responders to intravesical therapy.

Intravesical therapy (IVT) is the standard of care to decrease risk of recurrence and progression for high-grade nonmuscle-invasive bladder cancer. However, post-IVT recurrence remains common and the ability to risk-stratify patients before or after IVT is limited. In this prospectively designed and accrued cohort study, we examine the utility of urinary comprehensive genomic profiling (uCGP) for predicting recurrence risk following transurethral resection of bladder tumor (TURBT) and evaluating longitudinal IVT response. Urine was collected before and after IVT instillation and uCGP testing was done using the UroAmp™ platform. Baseline uCGP following TURBT identified patients with high (61%) and low (39%) recurrence risk. At 24 months, recurrence-free survival (RFS) was 100% for low-risk and 45% for high-risk patients with a hazard ratio (HR) of 9.3. Longitudinal uCGP classified patients as minimal residual disease (MRD) Negative, IVT Responder, or IVT Refractory with 24-month RFS of 100%, 50%, and 32%, respectively. Compared with MRD Negative patients, IVT Refractory patients had a HR of 10.5. Collectively, uCGP enables noninvasive risk assessment of patients following TURBT and induction IVT. uCGP could inform surveillance cystoscopy schedules and identify high-risk patients in need of additional therapy.

Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation.

The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.

GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the ß-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the ß-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

Thyroid hormone receptor-ß agonists prevent hepatic steatosis in fat-fed rats but impair insulin sensitivity via discrete pathways.

Liver-specific thyroid hormone receptor-ß (TRß)-specific agonists are potent lipid-lowering drugs that also hold promise for treating nonalcoholic fatty liver disease and hepatic insulin resistance. We investigated the effect of two TRß agonists (GC-1 and KB-2115) in high-fat-fed male Sprague-Dawley rats treated for 10 days. GC-1 treatment reduced hepatic triglyceride content by 75%, but the rats developed fasting hyperglycemia and hyperinsulinemia, attributable to increased endogenous glucose production (EGP) and diminished hepatic insulin sensitivity. GC-1 also increased white adipose tissue lipolysis; the resulting increase in glycerol flux may have contributed to the increase in EGP. KB-2115, a more TRß- and liver-specific thyromimetic, also prevented hepatic steatosis but did not induce fasting hyperglycemia, increase basal EGP rate, or diminish hepatic insulin sensitivity. Surprisingly, insulin-stimulated peripheral glucose disposal was diminished because of a decrease in insulin-stimulated skeletal muscle glucose uptake. Skeletal muscle insulin signaling was unaffected. Instead, KB-2115 treatment was associated with a decrease in GLUT4 protein content. Thus, although both GC-1 and KB-2115 potently treat hepatic steatosis in fat-fed rats, they each worsen insulin action via specific and discrete mechanisms. The development of future TRß agonists must consider the potential adverse effects on insulin sensitivity.

IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A.

IL-23 and Th17 cells producing IL-17A and IL-22 are found in excess in skin affected by psoriasis. Previous studies showed that IL-22, but not IL-17A, mediates psoriasis-like epidermal hyperplasia following recombinant murine (rm)IL-23 injections into skin. To further investigate the role of IL-17A, ears of mice were injected with rmIL-23. Investigators blinded to treatment conditions and mouse genotypes measured ear swelling, epidermal thickness, and cytokine expression. In wild-type (WT) mice, rmIL-23 induced ear swelling (p < 0.001, all p values versus saline), epidermal hyperplasia by histology (p < 0.001) and confocal microscopy (p < 0.004), and expression of both IL-17A and IL-22. As expected, rmIL-23 injections into IL-22(-/-) mice resulted in relatively little ear swelling (p < 0.09) and epidermal hyperplasia (p < 0.51 by histology and p < 0.75 by confocal microscopy). Notably, rmIL-23 injections into IL-17A(-/-) mice produced little ear swelling (p < 0.001, versus IL-23-injected WT mice) and epidermal hyperplasia (p < 0.001 by histology and p < 0.005 by confocal microscopy), even though IL-22 was readily induced in these mice. Furthermore, systemic delivery of blocking Abs directed against either IL-22 or IL-17A completely inhibited IL-23-induced epidermal hyperplasia in WT mice. These results demonstrate that IL-17A, like IL-22, is a downstream mediator for IL-23-induced changes in murine skin and that both of these Th17 cytokines are necessary to produce IL-23-mediated skin pathology. IL-17A may represent an attractive therapeutic target in individuals with psoriasis by blocking downstream effects of IL-23.

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.

Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-ß-lactamase active site.

Metallo-ß-lactamases catalyze the hydrolysis of a broad range of ß-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 ß-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of ß-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward ß-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 µM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable.

Measurement of single cell refractive index, dry mass, volume, and density using a transillumination microscope.

Phase contrast microscopy has become ubiquitous in the field of biology, particularly in qualitative investigations of cellular morphology. However, the use of quantitative phase retrieval methods and their connection to cellular refractive index and dry mass density remain under utilized. This is due in part to the restriction of phase and cellular mass determination to custom built instruments, involved mathematical analysis, and prohibitive sample perturbations. We introduce tomographic bright field imaging, an accessible optical imaging technique enabling the three dimensional measurement of cellular refractive index and dry mass density using a standard transillumination optical microscope. The validity of the technique is demonstrated on polystyrene spheres. The technique is then applied to the measurement of the refractive index, dry mass, volume, and density of red blood cells. This optical technique enables a simple and robust means to perform quantitative investigations of engineered and biological specimens in three dimensions using standard optical microscopes.

Laparoscopic entry techniques.

BACKGROUND: Laparoscopy is a common procedure in gynaecology. Complications associated with laparoscopy are often related to entry. Life-threatening complications include injury to the bowel, bladder, major abdominal vessels, and an anterior abdominal-wall vessel. Other less serious complications can also occur, such as post-operative infection, subcutaneous emphysema and extraperitoneal insufflation. There is no clear consensus as to the optimal method of entry into the peritoneal cavity. This is an update of a Cochrane review first published in 2008. OBJECTIVES: To evaluate the benefits and risks of different laparoscopic techniques in gynaecological and non-gynaecological surgery. SEARCH METHODS: This review has drawn on the search strategy developed by the Cochrane Menstrual Disorders and Subfertility Group. In addition, MEDLINE, EMBASE, CENTRAL and PsycINFO were searched through to February 2011. SELECTION CRITERIA: Randomised controlled trials were included when one laparoscopic entry technique was compared with another. DATA COLLECTION AND ANALYSIS: Data were extracted independently by the first three authors. Differences of opinion were registered and resolved by the fourth author. Results for each study were expressed as odds ratio (Peto OR) with 95% confidence interval (CI). MAIN RESULTS: The review included 28 randomised controlled trials with 4860 individuals undergoing laparoscopy and evaluated 14 comparisons. Overall there was no evidence of advantage using any single technique in terms of preventing major vascular or visceral complications. Using an open-entry technique compared to a Veress Needle demonstrated a reduction in the incidence of failed entry, Peto OR 0.12 (95% CI 0.02 to 0.92). There were three advantages with direct-trocar entry when compared with Veress Needle entry, in terms of lower rates of failed entry (Peto OR 0.21, 95% Cl 0.14 to 0.31), extraperitoneal insufflation (Peto OR 0.18, 95% Cl 0.13 to 0.26), and omental injury (Peto OR 0.28, 95% CI 0.14 to 0.55).There was also an advantage with radially expanding access system (STEP) trocar entry when compared with standard trocar entry, in terms of trocar site bleeding (Peto OR 0.31, 95% Cl 0.15 to 0.62). Finally, there was an advantage of not lifting the abdominal wall before Veress Needle insertion when compared to lifting in terms of failed entry, without an increase in the complication rate (Peto OR 4.44, 95% CI 2.16 to 9.13). However, studies were limited to small numbers, excluding many patients with previous abdominal surgery and women with a raised body mass index who may have unusually high complication rates. AUTHORS' CONCLUSIONS: An open-entry technique is associated with a significant reduction in failed entry when compared to a closed-entry technique, with no difference in the incidence of visceral or vascular injury.Significant benefits were noted with the use of a direct-entry technique when compared to the Veress Needle. The use of the Veress Needle was associated with an increased incidence of failed entry, extraperitoneal insufflation and omental injury; direct-trocar entry is therefore a safer closed-entry technique.The low rate of reported complications associated with laparoscopic entry and the small number of participants within the included studies may account for the lack of significant difference in terms of major vascular and visceral injury between entry techniques. Results should be interpreted with caution for outcomes where only single studies were included.

Gaining ligand selectivity in thyroid hormone receptors via entropy.

Nuclear receptors are important targets for pharmaceuticals, but similarities between family members cause difficulties in obtaining highly selective compounds. Synthetic ligands that are selective for thyroid hormone (TH) receptor beta (TRbeta) vs. TRalpha reduce cholesterol and fat without effects on heart rate; thus, it is important to understand TRbeta-selective binding. Binding of 3 selective ligands (GC-1, KB141, and GC-24) is characterized at the atomic level; preferential binding depends on a nonconserved residue (Asn-331beta) in the TRbeta ligand-binding cavity (LBC), and GC-24 gains extra selectivity from insertion of a bulky side group into an extension of the LBC that only opens up with this ligand. Here we report that the natural TH 3,5,3'-triodothyroacetic acid (Triac) exhibits a previously unrecognized mechanism of TRbeta selectivity. TR x-ray structures reveal better fit of ligand with the TRalpha LBC. The TRbeta LBC, however, expands relative to TRalpha in the presence of Triac (549 A(3) vs. 461 A(3)), and molecular dynamics simulations reveal that water occupies the extra space. Increased solvation compensates for weaker interactions of ligand with TRbeta and permits greater flexibility of the Triac carboxylate group in TRbeta than in TRalpha. We propose that this effect results in lower entropic restraint and decreases free energy of interactions between Triac and TRbeta, explaining subtype-selective binding. Similar effects could potentially be exploited in nuclear receptor drug design.