Search

The generation of distinct cell fates during development depends on asymmetric cell division of progenitor cells. In the central and peripheral nervous system of Drosophila, progenitor cells respectively called neuroblasts or sensory organ precursors use PAR polarity during mitosis to control cell fate determination in their daughter cells. How polarity and the cell cycle are coupled, and how the cell cycle machinery regulates PAR protein function and cell fate determination is poorly understood. Here, we generate an analog sensitive allele of CDK1 and reveal that its partial inhibition weakens but does not abolish apical polarity in embryonic and larval neuroblasts and leads to defects in polarisation of fate determinants. We describe a novel in vivo phosphorylation of Bazooka, the Drosophila homolog of PAR-3, on Serine180, a consensus CDK phosphorylation site. In some tissular contexts, phosphorylation of Serine180 occurs in asymmetrically dividing cells but not in their symmetrically dividing neighbours. In neuroblasts, Serine180 phosphomutants disrupt the timing of basal polarisation. Serine180 phosphomutants also affect the specification and binary cell fate determination of sensory organ precursors as well as Baz localisation during their asymmetric cell divisions. Finally, we show that CDK1 phosphorylates Serine-S180 and an equivalent Serine on human PAR-3 in vitro., Competing Interests: NL, EH, HS, AP, DM, GF, JJ No competing interests declared, (© 2024, Loyer et al.)

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner., (© 2024 Maib et al.)

Background: Lutetium-177-prostate-specific membrane antigen- 617 (Lu-PSMA) is an effective therapy for metastatic castration-resistant prostate cancer (mCRPC). However, treatment responses are heterogeneous despite stringent positron emission tomography (PET)-based imaging selection criteria. Molecularly based biomarkers have potential to refine patient selection and optimise outcomes., Objective: To identify circulating tumour DNA (ctDNA) features associated with treatment outcomes for men treated with Lu-PSMA., Design Setting and Participants: ctDNA from men treated with Lu-PSMA in combination with idronoxil for progressive mCRPC were analysed using an 85-gene customised sequencing assay. ctDNA fractions, molecular profiles, and the presence of alterations in aggressive-variant prostate cancer (AVPC) genes were analysed at baseline, cycle 3 and at disease progression., Intervention: Men received Lu-PSMA with idronoxil every 6 wk for up to six cycles., Outcome Measurements and Statistical Analysis: Baseline and exit PSMA and fluorodeoxyglucose PET/computed tomography (CT) imaging was conducted at baseline and study exit. Single-photon emission CT (SPECT) scans were performed 24 h after Lu-PSMA. Blood samples were collected at baseline,cycle 3 and at disease progression. Cox proportional-hazards models were used to assess associations and derive hazard ratios (HRs) and confidence intervals (CIs) for associations between molecular factors, imaging features, and clinical outcomes., Results and Limitations: Sixty samples from 32 men were sequenced (32 at baseline, 24 at cycle 3, four from patients with disease progression); two samples (baseline, on-treatment) from one individual were excluded from analysis owing to poor quality of the baseline sequencing data. Alterations in AVPC genes were associated with shorter prostate-specific antigen (PSA) progression-free survival (PFS) and overall survival (OS) in univariate (HR 3.4, 95% CI 1.5-7.7; p  = 0.0036; and HR 3.3, 95% CI 1.4-7.7; p  = 0.0063, respectively) and multivariate analyses (HR 4.8, 95% CI 1.8-13; p  = 0.0014; and HR 4.1, 95% CI 1.6-11; p  = 0.004)., Conclusions: ctDNA alterations in AVPC genes were associated with shorter PSA PFS and OS among men treated with Lu-PSMA and intermittent idronoxil. These candidate molecular biomarkers warrant further study to determine whether they have predictive value and potential to guide synergistic combination strategies to enhance outcomes for men treated with Lu-PSMA for mCRPC., Patient Summary: Certain DNA/gene changes detected in the blood of men with advanced prostate cancer were associated with shorter benefit from lutetium PSMA, a targeted radioactive therapy. This information may be useful in determining which men may benefit most from this treatment, but additional research is needed., (© 2023 The Author(s).)

Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2., (© 2023 Vines et al.)

Idiopathic sudden sensorineural hearing loss (ISSNHL) is a condition affecting 5-30 per 100,000 individuals with the potential to significantly reduce one's quality of life. The true incidence of this condition is not known because it often goes undiagnosed and/or recovers within a few days. ISSNHL is defined as a ≥30 dB loss of hearing over 3 consecutive audiometric octaves within 3 days with no known cause. The disorder is typically unilateral and most of the cases spontaneously recover to functional hearing within 30 days. High frequency losses, ageing, and vertigo are associated with a poorer prognosis. Multiple causes of ISSNHL have been postulated and the most common are vascular obstruction, viral infection, or labyrinthine membrane breaks. Corticosteroids are the standard treatment option but this practice is not without opposition. Post mortem analyses of temporal bones of ISSNHL cases have been inconclusive. This report analyzed ISSNHL studies administering corticosteroids that met strict inclusion criteria and identified a number of methodologic shortcomings that compromise the interpretation of results. We discuss the issues and conclude that the data do not support present treatment practices. The current status on ISSNHL calls for a multi-institutional, randomized, double-blind trial with validated outcome measures to provide science-based treatment guidance., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P 2 , driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.

Purpose: Methylation in IKZF1 and BCAT1 are common events in colorectal cancer (CRC). They are often detected in blood as circulating tumor DNA (ctDNA) at diagnosis and disappear after surgery in most CRC patients. A prospective study was conducted to determine the relationship between detection of these markers following surgery and risk for residual disease and for recurrence., Methods: ctDNA status with methylated BCAT1 and IKZF1 was determined within 12 months of surgical resection of CRC, and was related to presence of or risk for residual disease (margins involved, metastases present or nature of node involvement), and to recurrence-free survival., Results: Blood was collected from 172 CRC patients after surgery and 28 (16%) were ctDNA positive. Recurrence was diagnosed in 23 of the 138 with clinical follow-up after surgery (median follow-up 23.3 months, IQR 14.3-29.5). Multivariate modeling indicated that features suggestive of residual disease were an independent predictor of post-surgery ctDNA status: cases with any of three features (close resection margins, apical node involved, or distant metastases) were 5.3 times (95% CI 1.5-18.4, p = 0.008) more likely to be ctDNA positive. Multivariate analysis showed that post-surgery ctDNA positivity was independently associated with an increased risk of recurrence (HR 3.8, 1.5-9.5, p = 0.004)., Conclusions: CRC cases positive for methylated ctDNA after surgery are at increased risk of residual disease and subsequently recurrence. This could have implications for guiding recommendations for adjuvant therapy and surveillance strategies. Randomized studies are now indicated to determine if monitoring cases with these biomarkers leads to survival benefit.

Background: Cell-free circulating tumour-derived DNA (ctDNA) can be detected by testing for methylated BCAT1 and IKZF1 DNA, which has proven sensitivity for colorectal cancer (CRC). A prospective correlative biomarker study between presence of methylated BCAT1 and IKZF1 in tissue and blood was conducted in cases with CRC to explore how detection of such ctDNA biomarkers relates to cancer characteristics, methylation in tissue and surgical resection of the primary cancer., Methods: Enrolled patients with invasive CRC had blood collected at diagnosis, prior to any treatment or surgery (peri-diagnostic sample). A subgroup of patients also had cancer and adjacent non-neoplastic tissue collected at surgical resection, as well as a second blood sample collected within 12 months of surgery (post-surgery sample). DNA was extracted from all samples and assayed for methylated BCAT1 and IKZF1 to determine the degree of methylation in tissue and the presence of ctDNA in blood., Results: Of 187 cases providing peri-diagnostic blood samples, tissue was available in 91, and 93 provided at least one post-surgery blood sample for marker analysis. Significant methylation of either BCAT1 or IKZF1 was seen in 86/91 (94.5%) cancer tissues, with levels independent of stage and higher than that observed in adjacent non-neoplastic specimens ( P  < 0.001). ctDNA methylated in BCAT1 or IKZF1 was detected in 116 (62.0%) cases at diagnosis and was significantly more likely to be detected with later stage ( P  < 0.001) and distal tumour location ( P  = 0.004). Of the 91 patients who provided pre-and post-surgery blood samples, 47 patients were ctDNA-positive at diagnosis and 35 (74.5%) became negative after tumour resection., Conclusion: This study has shown that BCAT1 and IKZF1 methylation are common events in CRC with almost all cancer tissues showing significant levels of methylation in the two genes. The presence of ctDNA in blood is stage-related and show rapid reversion to negative following surgical resection. Monitoring methylated BCAT1 and IKZF1 levels could therefore inform adequacy of surgical resection., Trial Registration: Australian New Zealand Clinical Trial Registry number 12611000318987. Registered 25 March 2011., Competing Interests: The study was approved by the Southern Adelaide Clinical Human Research Ethics Committee (134.045). Written informed consent was obtained from all participants.GPY is a paid consultant to Clinical Genomics. SKP, DHM and RTB are paid employees of Clinical Genomics. The other authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: Colvera™ is a test that detects circulating tumor-derived DNA in patients with colorectal cancer by assaying for the presence of methylated BCAT1 and IKZF1 in blood. This study describes the analytical and clinical performance characteristics of the test., Methods: Validation was performed in accordance with ISO15189 and National Pathology Accreditation Advisory Council requirements. Spiked samples including 264 plasma and 120 buffer samples were randomized, divided into 8 batches of 48 samples, and processed over 8 days using 2 equipment lines (each line consisting of a QIAsymphony SP/AS, QIACube HT, and LC480); 2 reagent batches; and 2 operators to determine limit of detection, selectivity/specificity, precision, reproducibility, ruggedness, and susceptibility to commonly known interfering substances. Clinical performance was validated by assaying 222 archived plasma samples from subjects (n = 26 with cancer) enrolled in a previous prospective trial., Results: The limit of detection for Colvera was 12.6 pg/mL (95% CI, 8.6-23.9 pg/mL), which equates to 2 diploid genome copies per milliliter plasma. No statistically significant difference was determined between testing days (n = 8), instrumentation, operators, or reagent batches in precision studies for the methylation-specific assays. The assay performance was unaffected by 9 commonly known interference substances, variations in bisulfite conversion, or quantitative PCR settings (cycling temperatures, incubation times, and oligonucleotide concentrations). For this clinical cohort, sensitivity and specificity estimates for Colvera were 73.1% (19 of 26; 95% CI, 52.2-88.4) and 89.3% (175 of 196; 95% CI, 84.1-93.2), respectively., Conclusion: Colvera is a robust test and suitable for detection of circulating tumor-derived DNA by measuring levels of methylated BCAT1 and IKZF1 in human blood plasma., (© 2017 American Association for Clinical Chemistry.)

Context: The burden of cardiovascular disease (CVD) continues to be a public health concern. Workplace interventions that focus on modifying lifestyle habits may reduce CVD risk factors in people at risk., Objective: To determine the effectiveness of a comprehensive lifestyle intervention program that integrated dietary modification, physical activity, stress management, and behavior modification counseling to reduce the risk of CVD in at-risk adults., Methods: Twelve 1-year cohorts who participated in a comprehensive employer-sponsored lifestyle intervention program targeting diet, exercise, behavior modification, and stress management between 2006 and 2010 at a rural university in Appalachian Ohio were studied. Body composition, fasting glucose and lipid levels, and maximal oxygen consumption were measured at baseline, after 100 days, and at 1 year. Repeated-measures analyses of variance were conducted to compare measures at baseline with measures at 100 days and at 1 year., Results: Seventy-four participants (57 women [77.1%], 17 men [22.9%]) of 97 completed the program (76.3% completion rate). Body weight (P<.001); percentage of body fat (P<.001); fat mass (P<.001); body mass index (P<.001); waist circumference (P<.001); blood levels of high-density lipoprotein (P=.035), low-density lipoprotein (P=.011), and glucose (P=.008); serum triglyceride levels (P=.019); blood pressure (systolic P=.028; diastolic, P=.001); and maximal oxygen consumption (P<.001) improved from baseline to 1 year after the intervention. Lean body mass (P=.111) and total cholesterol (P=.053) did not improve., Conclusions: This employer-based lifestyle intervention program was effective in reducing CVD risk factors after 1 year of treatment. Future studies should examine the effects of the intervention at 2 and 5 years to further assess long-term adoption of the lifestyle changes and maintenance of health promoted by this program.

Recurrence will develop in 30-50% of colorectal cancer (CRC) cases despite apparent clearance following treatment. Carcinoembryonic antigen (CEA) is the only guideline-recommended blood test for monitoring cases for recurrence, but its sensitivity and specificity are suboptimal. This observational study compared a novel 2-gene (methylated BCAT1 and IKZF1 DNA) blood test with CEA for detection of recurrent CRC. We conducted a paired comparison of the BCAT1/IKZF1 test with CEA (cut-off 5 ng/mL) in blood from patients in remission after treatment for primary CRC and undergoing surveillance. Blood collected in the 12 months prior to or 3 months after complete investigational assessment of recurrence status were assayed and the results compared by McNemar's test. Of 397 patients enrolled, 220 underwent satisfactory assessment for recurrence and 122 had blood testing performed within the prescribed period. In 28 cases with recurrent CRC, CEA was positive in 9 (32%; 95% CI 16-52%) compared to 19 (68%; 95% CI 48-84%) positive for methylated BCAT1/IKZF1 (P = 0.002). All samples that were CEA positive were also BCAT1/IKZF1 positive. In 94 patients without clinically detectable recurrence, CEA was positive in 6 (6%, 95% CI 2-13%) and BCAT1/IKZF1 in 12 (13%, 95% CI 7-21%), P = 0.210. The odds ratio of a positive CEA test for recurrence was 6.9 (95% CI 2-22) compared to 14.4 (5-39) for BCAT1/IKZF1. The BCAT1/IKZF1 test was more sensitive for recurrence than CEA and the odds of recurrence given a positive test was twice that of CEA. The BCAT1/IKZF1 test should be further considered for monitoring cases for recurrence., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion., Competing Interests: The authors declare no competing financial interests.

Objectives: To compare the performance of a new blood test for colorectal cancer (CRC) to an established fecal immunochemical test (FIT) in a study population with the full range of neoplastic and non-neoplastic pathologies encountered in the colon and rectum., Methods: Volunteers were asked to complete a FIT prior to colonoscopy. Blood was collected after bowel preparation but prior to colonoscopy, and plasma was assayed for the presence of methylated BCAT1 and IKZF1 DNA using a multiplex real-time PCR assay. Sensitivity and specificity estimates for the blood test were calculated from true- and false-positive rates for neoplasia and compared with FIT at a range of fecal hemoglobin (Hb) concentration positivity thresholds., Results: In total, 1,381 volunteers (median age 64 years; 49% male) completed both tests prior to colonoscopy. Estimated sensitivity of the BCAT1/IKZF1 blood test for CRC was 62% (41/66; 95% confidence interval 49-74%) with a specificity of 92% (1207/1315; 90-93%). FIT returned the same specificity at a cutoff of 60 μg Hb/g, at which its corresponding sensitivity for cancer was 64% (42/66; 51-75%). In the range of commonly used FIT cutoffs, respective cancer sensitivity and specificity estimates with FIT were: 59% (46-71%) and 93% (92-95%) at 80 μg Hb/g, and 79% (67-88%) and 81% (78-83%) at 10 μg Hb/g. Although estimated sensitivities were not significantly different between the two tests for any stage of cancer, FIT showed a significantly higher sensitivity for advanced adenoma at the lower cutoffs. Specificity of FIT, but not of the BCAT1/IKZF1 blood test, deteriorated substantially in people with overt blood in the feces. When combining FIT (cutoff 10 μg Hb/g) with the BCAT1/IKZF1 blood test, sensitivity for cancer was 89% (79-96%) at 74% (72-77%) specificity., Conclusions: A test based on detection of methylated BCAT1/IKZF1 DNA in blood has comparable sensitivity but better specificity for CRC than FIT at the commonly used positivity threshold of 10 μg Hb/g. Further evaluation of the new test relative to FIT in the population screening context is now required to fully understand the potential advantages and disadvantages of these biomarkers in screening.

Microbial pathogens induce or inhibit death of host cells during infection, with significant consequences for virulence and disease progression. Death of an infected host cell can either facilitate release and dissemination of intracellular pathogens or promote pathogen clearance. Histoplasma capsulatum is an intracellular fungal pathogen that replicates robustly within macrophages and triggers macrophage lysis by unknown means. To identify H. capsulatum effectors of macrophage lysis, we performed a genetic screen and discovered three mutants that grew to wild-type levels within macrophages but failed to elicit host-cell death. Each mutant was defective in production of the previously identified secreted protein Cbp1 (calcium-binding protein 1), whose role in intracellular growth had not been fully investigated. We found that Cbp1 was dispensable for high levels of intracellular growth but required to elicit a unique transcriptional signature in macrophages, including genes whose induction was previously associated with endoplasmic reticulum stress and host-cell death. Additionally, Cbp1 was required for activation of cell-death caspases-3/7, and macrophage death during H. capsulatum infection was dependent on the pro-apoptotic proteins Bax and Bak. Taken together, these findings strongly suggest that the ability of Cbp1 to actively program host-cell death is an essential step in H. capsulatum pathogenesis., (© 2015 John Wiley & Sons Ltd.)

Background: Specific genes, such as BCAT1 and IKZF1, are methylated with high frequency in colorectal cancer (CRC) tissue compared to normal colon tissue specimens. Such DNA may leak into blood and be present as cell-free circulating DNA. We have evaluated the accuracy of a novel blood test for these two markers across the spectrum of benign and neoplastic conditions encountered in the colon and rectum., Methods: Circulating DNA was extracted from plasma obtained from volunteers scheduled for colonoscopy for any reason, or for colonic surgery, at Australian and Dutch hospitals. The extracted DNA was bisulphite converted and analysed by methylation specific real-time quantitative PCR (qPCR). A specimen was deemed positive if one or more qPCR replicates were positive for either methylated BCAT1 or IKZF1 DNA. Sensitivity and specificity for CRC were estimated as the primary outcome measures., Results: Plasma samples were collected from 2105 enrolled volunteers (mean age 62 years, 54 % male), including 26 additional samples taken after surgical removal of cancers. The two-marker blood test was run successfully on 2127 samples. The test identified 85 of 129 CRC cases (sensitivity of 66 %, 95 % CI: 57-74). For CRC stages I-IV, respective positivity rates were 38 % (95 % CI: 21-58), 69 % (95 % CI: 53-82), 73 % (95 % CI: 56-85) and 94 % (95 % CI: 70-100). A positive trend was observed between positivity rate and degree of invasiveness. The colonic location of cancer did not influence assay positivity rates. Gender, age, smoking and family history were not significant predictors of marker positivity. Twelve methylation-positive cancer cases with paired pre- and post-surgery plasma showed reduction in methylation signal after surgery, with complete disappearance of signal in 10 subjects. Sensitivity for advanced adenoma (n = 338) was 6 % (95 % CI: 4-9). Specificity was 94 % (95 % CI: 92-95) in all 838 non-neoplastic pathology cases and 95 % (95 % CI: 92-97) in those with no colonic pathology detected (n = 450)., Conclusions: The sensitivity for cancer of this two-marker blood test justifies prospective evaluation in a true screening population relative to a proven screening test. Given the high rate of marker disappearance after cancer resection, this blood test might also be useful to monitor tumour recurrence., Trial Registration: ACTRN12611000318987 .

Background: Specific genes are methylated with high frequency in colorectal neoplasia, and may leak into blood. Detection of multiple methylated DNA biomarkers in blood may improve assay sensitivity for colorectal cancer (CRC) relative to a single marker. We undertook a case-control study evaluating the presence of two methylation DNA markers, BCAT1 and IKZF1, in circulation to determine if they were complementary for detection of CRC., Methods: Methylation-specific PCR assays were developed to measure the level of methylated BCAT1 and IKZF1 in DNA extracted from plasma obtained from colonoscopy-confirmed 144 healthy controls and 74 CRC cases., Results: DNA yields ranged from 2 to 730 ng/mL plasma (mean 18.6ng/mL; 95% CI 11-26 ng/mL) and did not correlate with gender, age or CRC status. Methylated BCAT1 and IKZF1 DNA were detected in respectively 48 (65%) and 50 (68%) of the 74 cancers. In contrast, only 5 (4%) and 7 (5%) controls were positive for BCAT1 and IKZF1 DNA methylation, respectively. A two-gene classifier model ("either or" rule) improved segregation of CRC from controls, with 57 of 74 cancers (77%) compared to only 11 of 144 (7.6%) controls being positive for BCAT1 and/or IKZF1 DNA methylation. Increasing levels of methylated DNA were observed as CRC stage progressed., Conclusions: Detection of methylated BCAT1 and/or IKZF1 DNA in plasma may have clinical application as a novel blood test for CRC. Combining the results from the two methylation-specific PCR assays improved CRC detection with minimal change in specificity. Further validation of this two-gene blood test with a view to application in screening is now indicated.

Objectives: Stable fixation of displaced type II distal clavicle fractures presents a challenge to the surgeon because of distal fragment comminution and the large deforming forces created by the weight of the arm. We hypothesized that suture fixation around the coracoid and interfragmentary suture fixation would adequately counteract these forces and lead to a high rate of union and restoration of function., Design: This is a retrospective study of 20 sequential patients treated between 1997 and 2009., Setting: In-patient operating room followed by the clinic., Patients/participants: All patients presenting with acute displaced distal clavicle fractures to 2 surgeons were included. All patients completed the study without loss to follow-up., Intervention: Open repair was performed by placing two #5 Fiberwire sutures around the coracoid and through drill holes in the clavicular shaft. Interfragmentary fixation was performed with figure-of-eight #2 Fiberwire sutures., Main Outcome Measures: Patients evaluation included the American Shoulder and Elbow Surgeons scale, range of motion, radiographic evaluation, and patient satisfaction., Results: All fractures healed without loss of reduction at an average of 2.6 months, and all patients were satisfied. American Shoulder and Elbow Surgeons score was 98.5 out of 100. Average follow-up was 7.1 years (range, 3.1-14.3 years). One postoperative wound hematoma and one superficial wound infection were treated nonoperatively., Conclusions: Interfragmentary and coracoclavicular suture fixation for displaced acute distal clavicle fractures is a safe and effective treatment with an excellent union rate, low complication rate, and high patient satisfaction., Level of Evidence: Therapeutic level IV. See instructions for authors for a complete description of levels of evidence.

A monoclonal gammopathy is defined by the detection a monoclonal immunoglobulin (M-protein). In clinical practice, the M-protein is detected by protein gel electrophoresis (PEL) and immunofixation electrophoresis (IFE). We theorized that molecular mass could be used instead of electrophoretic patterns to identify and quantify the M-protein because each light and heavy chain has a unique amino acid sequence and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background. In addition, we surmised that top-down MS could be used to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. Our method first enriches serum for immunoglobulins followed by reduction using DTT to separate light chains from heavy chains and then by microflow LC-ESI-Q-TOF MS. The multiply charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Using this method, we demonstrate how the light chain portion of an M-protein can be monitored by molecular mass, and we also show that in sequential samples from a patient with multiple myeloma the light chain portion of the M-protein was detected in all samples, even those negative by PEL, IFE, and quantitative FLC. We also present top-down MS isotyping of M-protein light chains using a unique isotype-specific fragmentation pattern allowing for quantification and isotype identification in the same run. Our results show that microLC-ESI-Q-TOF MS provides superior sensitivity and specificity compared to conventional methods and shows promise as a viable method of detecting and isotyping an M-protein.

Introduction: Ebullism is the spontaneous evolution of liquid water in tissues to water vapor at body temperature when the ambient pressure is 47 mmHg or less. While injuries secondary to ebullism are generally considered fatal, some reports have described recovery after exposure to near vacuum for several minutes. The objectives of this article are to review the current literature on ebullism and to present prevention and treatment recommendations that can be used to enhance the safety of high altitude activities and space operations., Methods: A systematic review was conducted on currently available information and published literature of human and animal studies involving rapid decompression to vacuum and ebullism, with subsequent development of an applicable treatment protocol., Results: Available research on ebullism in human and animal subjects is extremely limited. Literature available identified key pathophysiologic processes and mitigation strategies that were used for treatment protocol design and outlining appropriate interventions using current best medical practices and technologies., Discussion: Available literature suggests that the pathophysiology of ebullism leads to predictable and often treatable injuries, and that many exposures may be survivable. With the growing number of high altitude and space-related activities, more individuals will be at risk for ebullism. An integrated medical protocol can provide guidance for the prevention and treatment of ebullism and help to mitigate this risk in the future.

The neuronal acceptor SNARE complex that functions as the receptor for synaptic vesicle docking and fusion at the presynaptic membrane is composed of the single-span transmembrane protein syntaxin-1A and the palmitoylated soluble protein SNAP-25. Previously, we explored interactions that promote the formation of syntaxin-1A clusters in membranes. Cholesterol activates clustering in native and model membranes, and its depletion in neuroendocrine cells results in a homogeneous distribution of the protein. However, as little as 1 mol % phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)) or 20 mol % phosphatidylserine was found to disperse syntaxin-1A clusters [Murray, D. H., and Tamm, L. K. (2009) Biochemistry 48, 4617-4625]. Strong evidence suggests that syntaxin-1A and its synaptic vesicle cognate synaptobrevin both interact directly with PI-4,5-P(2) and that this interaction activates fusion. However, the molecular details of this interaction and its relationship to the partial dispersion of syntaxin-1A clusters remain largely unexplored. Hence, we mutated the polybasic juxtamembrane motif of syntaxin-1A and found several residues that partially or fully abrogate the electrostatic interaction with PI-4,5-P(2). We further show that even in the presence of physiological concentrations of phosphatidylserine, the PI-4,5-P(2)-syntaxin interaction is sufficiently strong to disrupt syntaxin-1A clustering. The stereochemistry of PI-4,5-P(2) is not critical for this interaction as other polyphosphoinositides have similar effects. Forming an acceptor SNARE complex between syntaxin-1A and SNAP-25 weakens but does not abrogate cholesterol/PI-4,5-P(2)-controlled cluster formation. Potential consequences of these interactions with respect to synaptic vesicle fusion are discussed.

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL-1β and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2-photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a "smooth" membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL-1β or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno-histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL-1β-associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r(2) = 0.988) between the number of processes/cell (0-5) and IL-1β, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL-1β and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration., (© 2010 Orthopaedic Research Society.)

Many organophosphorus (OP) based compounds are highly toxic and powerful inhibitors of cholinesterases that generate serious environmental and human health concerns. Organothiophosphates with a thiophosphoryl (P=S) functional group constitute a broad class of these widely used pesticides. They are related to the more reactive phosphoryl (P=O) organophosphates, which include very lethal nerve agents and chemical warfare agents, such as, VX, Soman and Sarin. Unfortunately, widespread and frequent commercial use of OP-based compounds in agricultural lands has resulted in their presence as residues in crops, livestock, and poultry products and also led to their migration into aquifers. Thus, the design of new sensors with improved analyte selectivity and sensitivity is of paramount importance in this area. Herein, we review recent advances in the development of fluorescent chemosensors for toxic OP pesticides and related compounds. We also discuss challenges and progress towards the design of future chemosensors with dual modes for signal transduction.

Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biological functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biological processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) experiments that these vesicle are mobile above the plane of the supported membrane. At higher concentrations, the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was determined by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT experiments with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.

Bone strength is, in part, dependent on a mechanical input that regulates the (re)modelling of skeletal elements to an appropriate size and architecture to resist fracture during habitual use. The rate of longitudinal bone growth in juveniles can also affect fracture incidence in adulthood, suggesting an influence of growth rate on later bone quality. We have compared the effects of fast and slow growth on bone strength and architecture in the tibiotarsi of embryonic and juvenile birds. The loading-related biochemical responses (intracellular G6PD activity and NO release) to mechanical load were also determined. Further, we have analysed the proliferation and differentiation characteristics of primary tibiotarsal osteoblasts from fast and slow-growing strains. We found that bones from chicks with divergent growth rates display equal resistance to applied loads, but weight-correction revealed that the bones from juvenile fast growth birds are weaker, with reduced stiffness and lower resistance to fracture. Primary osteoblasts from slow-growing juvenile birds proliferated more rapidly and had lower alkaline phosphatase activity. Bones from fast-growing embryonic chicks display rapid radial expansion and incomplete osteonal infilling but, importantly, lack mechanical responsiveness. These findings are further evidence that the ability to respond to mechanical inputs is crucial to adapt skeletal architecture to generate a functionally appropriate bone structure and that fast embryonic and juvenile growth rates may predispose bone to particular architectures with increased fragility in the adult.

Syntaxin-1A is part of the SNARE complex that forms in membrane fusion in neuronal exocytosis of synaptic vesicles. Together with SNAP-25 the single-span transmembrane protein syntaxin-1A forms the receptor complex on the plasma membrane of neuroendocrine cells. Previous studies have shown that syntaxin-1A occurs in clusters that are different from lipid rafts in neuroendocrine plasma membranes. However, the interactions that promote these clusters have been largely unexplored. Here, we have reconstituted syntaxin-1A into lipid model membranes, and we show that syntaxin cluster formation depends on cholesterol in a lipid system that lacks sphingomyelin and therefore does not form liquid-ordered phases that are commonly believed to represent lipid rafts in cell membranes. Rather, the cholesterol-induced clustering of syntaxin is found to be reversed by as little as 1-5 mol % of the regulatory lipid phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)), and PI-4,5-P(2) is shown to bind electrostatically to syntaxin, presumably mediated by the highly positively charged juxtamembrane domain of syntaxin. Possible implications of these results to the regulation of SNARE-mediated membrane fusion are discussed.

Control of chondrocyte pH (pH(i)) determines articular cartilage matrix metabolism. However, the transporters of chondrocytes in situ throughout cartilage zones are unclear, and we tested the hypothesis that chondocytes within the superficial zone (SZ) utilise a HCO(3) (-)-dependent system absent from other zones. Imaging of single BCECF-labelled cells was used to monitor the pH(i) of in situ chondrocytes within the cartilage zones, and also that of cells isolated from the SZ or full depth (FD) explants. Resting pH(i) and intrinsic buffering power (beta(i)) in HEPES-buffered saline was not different between SZ and DZ cells, however the pH(i) of SZ chondrocytes was lower in HCO(3) (-) saline. Ammonium pre-pulse was used to acid-load cells and pH(i) recovery by in situ or isolated SZ chondrocytes shown to be totally dependent on HCO(3) (-). pH(i) recovery rate was significantly (P < 0.05) greater for in situ cells, suggesting that isolation damaged the HCO(3) (-)-dependent system. Recovery of pH(i) by in situ cells was blocked by the anion transport inhibitor DIDS, and partially inhibited by EIPA probably non-specifically. Recovery of pH(i) by acidified MZ or DZ cells or those isolated from FD explants was not affected by HCO(3) (-) (P > 0.05). Na(+)-dependent HCO(3) (-)-(NBC) transporters were identified in SZ chondrocytes by fluorescence immunohistochemistry suggesting that this system might account for the HCO(3) (-)-dependent recovery of pH(i). Bovine articular cartilage chondrocytes possess a HCO(3) (-)-dependent transporter which plays a key role in pH(i) regulation in cells in the SZ, but not in chondrocytes within deeper cartilage zones.

This animal study evaluated the healing of supraspinatus tendon tears by use of a cartilage-derived morphogenetic protein 2 growth factor (CDMP-2) delivered to the repair. Forty-eight rats had bilateral, surgically created complete tears repaired by sutures with the growth factor introduced on one side. They were killed at 2, 3, 4, and 6 weeks, and the strength of the repairs was determined and histologic analysis performed. At 4 and 6 weeks, the CDMP-2-treated repairs were significantly stronger than the untreated repairs and histologic analysis showed more organized healing. The use of growth factors introduced at the time of rotator cuff repair might promote more rapid healing and subsequent, rapid patient rehabilitation.

This study compares the results of arthroscopic and arthroscopically assisted mini-open rotator cuff repair in a series of 84 patients who underwent repair of small, medium, or large tears between March 1997 and September 2001 with at least 2 years of follow-up. There were 42 arthroscopic repairs and 42 mini-open repairs. Of the patients, 81 (96.4%) had good or excellent UCLA (University of California, Los Angeles) scores (40 arthroscopic repairs [95.2%] and 41 mini-open repairs [97.6%]); there were 2 fair results and 1 poor outcome. The ASES (American Shoulder and Elbow Surgeons) scores averaged 91.1 for the arthroscopic group and 90.2 for the mini-open group (P > .05). Six patients required further surgery (three from the arthroscopic group and three from the mini-open group). Of 84 patients, 83 (98.8%) reported being satisfied with the procedure. At greater than 2 years of follow-up, arthroscopic and mini-open rotator cuff repairs produced similar results for small, medium, and large rotator cuff tears with equivalent patient satisfaction rates.

Although it is well accepted that bone architecture adapts to withstand the loads placed on it, the manner in which this occurs in the immature growing skeleton is not fully understood. To investigate the possible mechanisms, we have compared morphometric differences between tibiae from chickens with fast and those with slow growth potential and also distinguished between the effects of genetic potential and growth rate on their impact on bone quality. Two different fast-growing (ad lib modern) strains, one additionally feed-restricted and one slow-growing (control) strain of chicken, were compared at 15 and 42 days of age. The ad lib modern strains had similar final body weights and were approximately twice the weight of the control and restricted-fed birds. Tibiae from the control and restricted birds had a higher ash content and lower porosity than the ad lib modern strain at 42 days. The porosity was a result of rapid primary osteon formation at the periosteal surface and incomplete infilling of the resultant canal by osteoblasts. When adjusted to average body weight of contemporaries, bones from the control strain and the restricted-fed modern birds were stiffer and at least as strong as those from the fast growing ad lib-fed birds. In conclusion, rapid bone deposition at the periosteal surface was associated with decreased mineralization, increased cortical porosity, and altered biomechanical properties. Our results also indicate that growth rate, and not genetic potential, of the fast growing birds was responsible for the rapid periosteal bone deposition.

A study of motor vehicle accident deaths occurring in Napa County, California, from 1979 through 1983 showed that there was a preventable death rate of 42% for deaths that were not related to central nervous system injuries. After developing a Level II trauma center at our hospital, the preventable death rate decreased to 14%. This was statistically significant (total chi-square, 0.01 less than p less than 0.025). There was a significant increase in the average Injury Severity Score (34 versus 45, p less than 0.005) as well as significant improvements in the surgeon's response time (32 minutes versus 11 minutes, p less than 0.005) and in the time from hospital arrival to the start of surgery (3.6 hours versus 1.9 hours, 0.01 less than p less than 0.025). We conclude that these changes are indicative of improved trauma care and reflect favorably upon the effectiveness of a rural trauma center that meets Level II trauma center guidelines established by the American College of Surgeons Committee on Trauma.

One hundred eleven patients with 130 emboli treated in nonteaching private hospitals have been evaluated with respect to cause, mortality rate, and amputation rate. For surgically treated patients with balloon embolectomy, the mortality rate was 13.7 percent overall, with a limb salvage rate of 94.5 percent. The operative mortality was 8.3 percent. The mortality for late embolectomy was 3 percent with a limb salvage rate of 100 percent. The most critical factor in predicting mortality was age of the patient, with a significantly higher mortality in patients of advanced years. In this series, a prolonged duration of embolus before embolectomy did not have an adverse effect on the mortality rate.

The advent of synthetic collagen suture (polyglycolic acid and polyglacin 910) has provided a new dimension in surgical suturing. However, surgeons in general have not yet taken full advantage of the potential that this new material offers. Associated evisceration and wound dehiscence rates as well as the incidence rate of wound infection were assessed since these represent standards by which the suture material can be evaluated. In an entire series of 650 cases, there has been one case of wound dehiscence with evisceration. The incidence of infection has been less than 1%, which is comparable to previous experience with interrupted, nonabsorbable sutures. The time required for closure using the new material has been approximately one third of that associated with the use of conventional interrupted closure sutures. This implies a substantial cost reduction for the patient. As a result of this experience, the synthetic absorbable sutures seem to represent the material of choice for routine abdominal or chest wound closure.

Many methods have been described for control of bleeding from the liver. We have found that the technique of simply securing a 2 1/2 cm (1-in) Penrose drain as a tourniquet around the left or right lobe of the liver is a rapid and effective method of obtaining hemostasis. The tourniquet can be used to temporaily control bleeding so individual points can be secured, or tied down onto the liver and removed later. This method has been used without complications in five patients with severe liver trauma. Serial liver function studies obtained on all patients found no significant alterations in any.