Selective Roles for Tumor Necrosis Factor α-converting Enzyme/ADAM17 in the Shedding of the Epidermal Growth Factor Receptor Ligand Family: THE JUXTAMEMBRANE STALK DETERMINES CLEAVAGE EFFICIENCY

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors

Clinical and radiologic factors associated with adnexal torsion in premenarchal and menarchal children and adolescents.

BACKGROUND: Adnexal torsion is a gynecologic emergency in children and adolescents but remains a challenging diagnosis, with no consistent clinical or radiologic diagnostic criteria. Our objective was to identify risk factors associated with adnexal torsion in premenarchal and menarchal patients with surgically confirmed torsion compared with those without torsion. METHODS: We conducted a retrospective chart review of all patients who underwent surgery between January 2016 and December 2019 for possible adnexal torsion. Data on demographics, clinical characteristics, radiologic variables, and operative findings were compared using descriptive statistics. Independent predictors of torsion were then examined in multivariate logistic regression models. RESULTS: Of the 291 patients who underwent surgery, 168 (57.7%) had torsion. Patients with torsion were younger than those without torsion (11.9 vs. 14.2 years, P \u3c .01). Vomiting was significantly associated with torsion for all patients (P \u3c .001). Large adnexal volume and absent arterial Doppler flow were associated with torsion for the total population and menarchal subgroup. A logistic regression model for the total population that controlled for age and menarchal status found that vomiting (adjusted odds ratio [aOR] 5.92, 95% confidence interval [CI] 2.87-12.22), highest adnexal volume category (aOR 4.92, 95% CI 2.25-10.75), and absent arterial Doppler flow (aOR 2.674, 95% CI 1.28-5.60) were associated with torsion. CONCLUSIONS: Vomiting, enlarged adnexal volume, and absent arterial Doppler flow were associated with adnexal torsion. However, no single risk factor accurately diagnosed torsion, and multiple factors should be interpreted together. LEVEL OF EVIDENCE: Study of Diagnostic Test, Level II

Ecological implications of a flower size/number trade-off in tropical forest trees

Peer reviewedPublisher PD

Computational Modelling of Cardiac Trabecula Mechanics

Cardiac trabeculae are thin strips of muscle within the ventricles that can be readily excised and used to investigate contractile mechanics of cardiac muscle. Recently, the Auckland Bioengineering Institute has developed a novel cardiac myometer that simultaneously measures force, length and shape of actively contracting isolated cardiac trabeculae. Here we have developed a muscle-specific computational model based on optical coherence tomography geometric surface data that replicates passive mechanics of trabecula. We hypothesised that the muscle's surface geometry data, in addition to force-length data, would improve the fit between the model simulated mechanics and the experimental data. The trabecula model was optimised using two different objective functions (muscle length or shape) driven by a pressure boundary condition. For both objective functions, there was a region of optimal parameters the optimiser tended towards but, due to the coupling between parameters, the ability to find the true optimal parameters was hindered. Due to the limitations of the data, we found that the addition of surface data did not improve parameter estimation and that using only the force-length data provided sufficient information to produce an optimal fit. References A. Anderson. The Cardiac Myometer: Measuring Matters of the Heart. PhD thesis, University of Auckland, 2016. K. F. Augenstein, Brett R. Cowan, Ian J. LeGrice, Poul M. F. Nielsen, and Alistair A. Young. Method and apparatus for soft tissue material parameter estimation using tissue tagged Magnetic Resonance Imaging. Journal of Biomechanical Engineering, 127(1):148–157, February 2005. C. Bradley, Andy Bowery, Randall Britten, Vincent Budelmann, Oscar Camara, Richard Christie, Andrew Cookson, Alejandro F. Frangi, Thiranja Babarenda Gamage, Thomas Heidlauf, Sebastian Krittian, David Ladd, Caton Little, Kumar Mithraratne, Martyn Nash, David Nickerson, Poul Nielsen, Oyvind Nordbo, Stig Omholt, Ali Pashaei, David Paterson, Vijayaraghavan Rajagopal, Adam Reeve, Oliver Rohrle, Soroush Safaei, Rafael Sebastian, Martin Steghofer, Tim Wu, Ting Yu, Heye Zhang, and Peter Hunter. OpenCMISS: A multi-physics and multi-scale computational infrastructure for the VPH/Physiome project. Progress in Biophysics and Molecular Biology, 107(1):32–47, October 2011. doi:http://dx.doi.org/10.1016/j.pbiomolbio.2011.06.015 M. L. Cheuk, A. J. Anderson, J. C. Han, N. Lippok, F. Vanholsbeeck, B. P. Ruddy, D. S. Loiselle, P. M. F. Nielsen, and A. J. Taberner. Four-Dimensional Imaging of Cardiac Trabeculae Contracting In Vitro Using Gated OCT. IEEE Transactions on Biomedical Engineering, 64(1):218–224, January 2017. doi:http://dx.doi.org/10.1109/TBME.2016.2553154 M. L. Cheuk, N. Lippok, A. W. Dixon, B. P. Ruddy, F. Vanholsbeeck, P. M. F. Nielsen, and A. J. Taberner. Optical coherence tomography imaging of cardiac trabeculae. In 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, pages 182–185, August 2014. doi:http://dx.doi.org/10.1109/EMBC.2014.6943559 J. M Guccione, Andrew D McCulloch, and LK Waldman. Passive material properties of intact ventricular myocardium determined from a cylindrical model. J Biomech Eng, 113(1):42–55, 1991. J. C. Han, Andrew J. Taberner, Robert S. Kirton, Poul M. Nielsen, Nicholas P. Smith, and Denis S. Loiselle. A unique micromechanocalorimeter for simultaneous measurement of heat rate and force production of cardiac trabeculae carneae. Journal of Applied Physiology, 107(3):946–951, September 2009. doi:http://dx.doi.org/10.1152/japplphysiol.00549.2009 M. P. Nash and P. J. Hunter. Regional mechanics of the beating heart. In Cardiac Perfusion and Pumping Engineering, volume Volume 1 of Clinically-Oriented Biomedical Engineering, pages 83–127. WORLD SCIENTIFIC, July 2007. doi:http://dx.doi.org/10.1142/9789812775597_0004 J. H. Omens, D. A. MacKenna, and A. D. McCulloch. Measurement of strain and analysis of stress in resting rat left ventricular myocardium. Journal of Biomechanics, 26(6):665–676, June 1993. doi:http://dx.doi.org/10.1016/0021-9290(93)90030-I V. Y. Wang, H. I. Lam, Daniel B. Ennis, Brett R. Cowan, Alistair A. Young, and Martyn P. Nash. Modelling passive diastolic mechanics with quantitative MRI of cardiac structure and function. Medical Image Analysis, 13(5):773–784, October 2009. doi:http://dx.doi.org/10.1016/j.media.2009.07.00

Identification of an Allosteric Small-Molecule Inhibitor Selective for the Inducible Form of Heat Shock Protein 70

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i

High Genetic Diversity and Fine-Scale Spatial Structure in the Marine Flagellate Oxyrrhis marina (Dinophyceae) Uncovered by Microsatellite Loci

Free-living marine protists are often assumed to be broadly distributed and genetically homogeneous on large spatial scales. However, an increasing application of highly polymorphic genetic markers (e.g., microsatellites) has provided evidence for high genetic diversity and population structuring on small spatial scales in many free-living protists. Here we characterise a panel of new microsatellite markers for the common marine flagellate Oxyrrhis marina. Nine microsatellite loci were used to assess genotypic diversity at two spatial scales by genotyping 200 isolates of O. marina from 6 broad geographic regions around Great Britain and Ireland; in one region, a single 2 km shore line was sampled intensively to assess fine-scale genetic diversity. Microsatellite loci resolved between 1–6 and 7–23 distinct alleles per region in the least and most variable loci respectively, with corresponding variation in expected heterozygosities (He) of 0.00–0.30 and 0.81–0.93. Across the dataset, genotypic diversity was high with 183 genotypes detected from 200 isolates. Bayesian analysis of population structure supported two model populations. One population was distributed across all sampled regions; the other was confined to the intensively sampled shore, and thus two distinct populations co-occurred at this site. Whilst model-based analysis inferred a single UK-wide population, pairwise regional FST values indicated weak to moderate population sub-division (0.01–0.12), but no clear correlation between spatial and genetic distance was evident. Data presented in this study highlight extensive genetic diversity for O. marina; however, it remains a substantial challenge to uncover the mechanisms that drive genetic diversity in free-living microorganisms