Extracellular hyaluronate pressure shaped by cellular tethers drives tissue morphogenesis.

How tissues acquire complex shapes is a fundamental question in biology and regenerative medicine. Zebrafish semicircular canals form from invaginations in the otic epithelium (buds) that extend and fuse to form the hubs of each canal. We find that conventional actomyosin-driven behaviors are not required. Instead, local secretion of hyaluronan, made by the enzymes uridine 5'-diphosphate dehydrogenase (ugdh) and hyaluronan synthase 3 (has3), drives canal morphogenesis. Charged hyaluronate polymers osmotically swell with water and generate isotropic extracellular pressure to deform the overlying epithelium into buds. The mechanical anisotropy needed to shape buds into tubes is conferred by a polarized distribution of actomyosin and E-cadherin-rich membrane tethers, which we term cytocinches. Most work on tissue morphogenesis ascribes actomyosin contractility as the driving force, while the extracellular matrix shapes tissues through differential stiffness. Our work inverts this expectation. Hyaluronate pressure shaped by anisotropic tissue stiffness may be a widespread mechanism for powering morphological change in organogenesis and tissue engineering.

Adhesion-Based Self-Organization in Tissue Patterning.

Since the proposal of the differential adhesion hypothesis, scientists have been fascinated by how cell adhesion mediates cellular self-organization to form spatial patterns during development. The search for molecular tool kits with homophilic binding specificity resulted in a diverse repertoire of adhesion molecules. Recent understanding of the dominant role of cortical tension over adhesion binding redirects the focus of differential adhesion studies to the signaling function of adhesion proteins to regulate actomyosin contractility. The broader framework of differential interfacial tension encompasses both adhesion and nonadhesion molecules, sharing the common function of modulating interfacial tension during cell sorting to generate diverse tissue patterns. Robust adhesion-based patterning requires close coordination between morphogen signaling, cell fate decisions, and changes in adhesion. Current advances in bridging theoretical and experimental approaches present exciting opportunities to understand molecular, cellular, and tissue dynamics during adhesion-based tissue patterning across multiple time and length scales.

Modeling the cell cycle: why do certain circuits oscillate?

Computational modeling and the theory of nonlinear dynamical systems allow one to not simply describe the events of the cell cycle, but also to understand why these events occur, just as the theory of gravitation allows one to understand why cannonballs fly in parabolic arcs. The simplest examples of the eukaryotic cell cycle operate like autonomous oscillators. Here, we present the basic theory of oscillatory biochemical circuits in the context of the Xenopus embryonic cell cycle. We examine Boolean models, delay differential equation models, and especially ordinary differential equation (ODE) models. For ODE models, we explore what it takes to get oscillations out of two simple types of circuits (negative feedback loops and coupled positive and negative feedback loops). Finally, we review the procedures of linear stability analysis, which allow one to determine whether a given ODE model and a particular set of kinetic parameters will produce oscillations.

Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

Impact of gonadotropins on oocyte maturation, fertilisation and developmental competence in vitro.

The aim of the present study was to evaluate the dose-dependent effects of gonadotropins, either singly (Bravelle (B), Luveris (L), Menupur (M), Repronex (R), Gonal-F (G), Follism (F) and Norvarel (N)) or in combination (Menupur+Bravelle; Repronext+Bravelle; and Bravelle+Norvarel), on rates of oocyte maturation, fertilisation and early embryo development in vitro in an animal model. Bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in TCM-199 with 10% fetal bovine serum supplemented with varying concentrations of gonadotropin (0, 5, 10, 20, 40IU or United States Pharmacopoeia (USP) mL-1) for 24 and 48h according to current IVF clinical stimulation protocols. All gonadotropins enhanced oocyte maturation in vitro in a dose-dependent manner. Individually, Gonal-F (Merck KGaA, Darmstadt, Germany), Follism (Merck Co, Whitehouse Station, NJ, USA) and Repronext (Ferring, Parsippany, NJ, USA) promoted oocyte maturation; in combination, they effectively enhanced COC expansion and increased the maturation competence of MII oocytes. However, high concentrations of gonadotropins may result in maturation arrest. Specific combinations of gonadotropins may change the rate of early embryonic development (8-16-cells) and morula-blastocyst formation. These data provide support for the responsiveness of bovine oocytes to gonadotropins in vitro and the need to consider variations in the relative concentrations and ratio of combinations (FSH/LH or human chorionic gonadotropin) for optimisation of oocyte developmental competence. The results of the present study could be applied to therapeutic clinical stimulation protocols and help improve IVF success rates.

Fundus image fusion in EYEPLAN software: an evaluation of a novel technique for ocular melanoma radiation treatment planning.

PURPOSE: The purpose of this study is to evaluate a novel approach for treatment planning using digital fundus image fusion in EYEPLAN for proton beam radiation therapy (PBRT) planning for ocular melanoma. The authors used a prototype version of EYEPLAN software, which allows for digital registration of high-resolution fundus photographs. The authors examined the improvement in tumor localization by replanning with the addition of fundus photo superimposition in patients with macular area tumors. METHODS: The new version of EYEPLAN (v3.05) software allows for the registration of fundus photographs as a background image. This is then used in conjunction with clinical examination, tantalum marker clips, surgeon's mapping, and ultrasound to draw the tumor contour accurately. In order to determine if the fundus image superimposition helps in tumor delineation and treatment planning, the authors identified 79 patients with choroidal melanoma in the macular location that were treated with PBRT. All patients were treated to a dose of 56 GyE in four fractions. The authors reviewed and replanned all 79 macular melanoma cases with superimposition of pretreatment and post-treatment fundus imaging in the new EYEPLAN software. For patients with no local failure, the authors analyzed whether fundus photograph fusion accurately depicted and confirmed tumor volumes as outlined in the original treatment plan. For patients with local failure, the authors determined whether the addition of the fundus photograph might have benefited in terms of more accurate tumor volume delineation. RESULTS: The mean follow-up of patients was 33.6 +/- 23 months. Tumor growth was seen in six eyes of the 79 macular lesions. All six patients were marginal failures or tumor miss in the region of dose fall-off, including one patient with both in-field recurrence as well as marginal. Among the six recurrences, three were managed by enucleation and one underwent retreatment with proton therapy. Three patients developed distant metastasis and all three patients have since died. The replanning of six patients with their original fundus photograph superimposed showed that in four cases, the treatment field adequately covered the tumor volume. In the other two patients, the overlaid fundus photographs indicated the area of marginal miss. The replanning with the fundus photograph showed improved tumor coverage in these two macular lesions. For the remaining patients without local failure, replanning with fundus photograph superimposition confirmed the tumor volume as drawn in the original treatment plan. CONCLUSIONS: Local control was excellent in patients receiving 56 GyE of PBRT for uveal melanomas in the macular region, which traditionally can be more difficult to control. Posterior lesions are better defined with the additional use of fundus image since they can be difficult to mark surgically. In one-third of treatment failing patients, the superposition of the fundus photograph would have clearly allowed improved localization of tumor. The current practice standard is to use the superimposition of the fundus photograph in addition to the surgeon's clinical and clip mapping of the tumor and ultrasound measurement to draw the tumor volume.

An adhesion code ensures robust pattern formation during tissue morphogenesis.

Animal development entails the organization of specific cell types in space and time, and spatial patterns must form in a robust manner. In the zebrafish spinal cord, neural progenitors form stereotypic patterns despite noisy morphogen signaling and large-scale cellular rearrangements during morphogenesis and growth. By directly measuring adhesion forces and preferences for three types of endogenous neural progenitors, we provide evidence for the differential adhesion model in which differences in intercellular adhesion mediate cell sorting. Cell type-specific combinatorial expression of different classes of cadherins (N-cadherin, cadherin 11, and protocadherin 19) results in homotypic preference ex vivo and patterning robustness in vivo. Furthermore, the differential adhesion code is regulated by the sonic hedgehog morphogen gradient. We propose that robust patterning during tissue morphogenesis results from interplay between adhesion-based self-organization and morphogen-directed patterning.

Oral isotretinoin and topical retinoid use in a series of young patients with ocular melanoma.

PURPOSE: To describe the first series of six young uveal melanoma (UM) patients with oral isotretinoin and/or topical retinoid therapy prior to diagnosis. OBSERVATIONS: The case series is based on clinical observations at our UM quaternary referral center. Six UM patient cases are reported, ages 16-44 years old. All had been using either oral (isotretinoin) and/or topical (tretinoin or tazarotene) retinoid treatment (3 months-~10 years) prior to or at the time of diagnosis (3 of 6 cases). All patients had ocular complaints on presentation, and the onset of certain symptoms corresponded with the course of retinoids. Other potential risk factors or relevant history included Caucasian background, cone-rod dystrophy and active smoker status (Case 2), family history of UM and pregnancy at time of diagnosis (Case 3), past smoking and possible secondary Chernobyl exposure as a baby (Case 5). All patients were treated with proton beam radiotherapy and currently have no sign of recurrent or metastatic disease. CONCLUSIONS AND IMPORTANCE: Retinoid therapy has been linked to various benign and/or reversible effects on the anterior and posterior eye, though pathophysiology remains not well understood. Uveal melanoma (UM) is a rare cancer diagnosis in young adults. We report here the first case series of young UM patients with a history of retinoid use and ocular complaints. No causal link is claimed and further systematic epidemiologic and biologic study of retinoid therapy and ocular impact may provide additional relevant data, particularly in young ocular melanoma patients.

Gene Expression Profiling in Uveal Melanoma: Five-Year Prospective Outcomes and Meta-Analysis.

INTRODUCTION: The prognostic 15-gene expression profile (15-GEP) test for uveal melanoma (UM) predicts metastatic risk based on primary tumor biology. Here we report outcomes from a prospective registry of 15-GEP-tested patients, and a meta-analysis with published cohorts. OBJECTIVES: Management and 5-year clinical outcomes following 15-GEP testing were evaluated. METHODS: Eighty-nine patients with 15-GEP results were prospectively enrolled at four centers. Physician-recommended management plans were collected, and clinical outcomes tracked every 6 months. RESULTS: Eighty percent of Class 1 (low-risk) patients underwent low-intensity management; all Class 2 (high-risk) patients underwent high-intensity management (p < 0.0001). Median follow-up for event-free patients was 4.9 years. Five Class 1 (10%) and 23 Class 2 (58%) tumors metastasized (p < 0.0001). Five-year Class 1 and 2 metastasis-free survival rates were 90% (81-100%) and 41% (27-62%; p < 0.0001), and melanoma-specific survival rates were 94% (87-100%) and 63% (49-82%; p = 0.0007). Class 2 was the only independent predictor of metastasis and was associated with increased risk for metastasis and mortality by meta-analysis. CONCLUSIONS: UM patient management is guided by 15-GEP testing. Class 2 patients were managed more intensely, in accordance with an observed metastatic rate of >50%; Class 1 patients were safely spared intensive surveillance, resulting in appropriate utilization of healthcare resources.

A Student-Led National Conference on Leadership: Broadening the Medical Student Role.

This article was migrated. The article was marked as recommended. Students have traditionally held a singular role in medical education - the learner. This narrow view neglects students unique perspective and ability to shape the future of medical education. In recognizing the need for deliberate leadership skill development and networking opportunities for medical student leaders, the American Medical Association (AMA) supported the first AMA Accelerating Change in Medical Education Student-Led Conference on Leadership in Medical Education. A planning committee of 19 students from seven medical schools collaborated to develop this conference, which took place on August 4-5, 2017 at the University of Michigan, Ann Arbor. The primary goal of the conference was for students to learn about leadership skills, connect with other student leaders, feel empowered to lead change, and continue to lead from their roles as students. Attendees participated in a variety of workshops and presentations focused on developing practical leadership skills. In addition, students formed multi-institutional teams to participate on in the MedEd Impact Challenge, attempting to address issues in medical education such as leadership curriculum development, wellness, and culture change. Post-conference surveys showed an overwhelming majority of students connected with other student leaders, shared ideas, developed collaborations, and felt empowered to enact change. Looking forward, we believe that similar student-led conferences focused on broadening the medical student role would provide avenues for positive change in medical education.

Efficient Front-Rear Coupling in Neutrophil Chemotaxis by Dynamic Myosin II Localization.

Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability.

Single-cell zeroth-order protein degradation enhances the robustness of synthetic oscillator.

In Escherichia coli, protein degradation in synthetic circuits is commonly achieved by the ssrA-tagged degradation system. In this work, we show that the degradation kinetics for the green fluorescent protein fused with the native ssrA tag in each cell exhibits the zeroth-order limit of the Michaelis-Menten kinetics, rather than the commonly assumed first-order. When measured in a population, the wide distribution of protein levels in the cells distorts the true kinetics and results in a first-order protein degradation kinetics as a population average. Using the synthetic gene-metabolic oscillator constructed previously, we demonstrated theoretically that the zeroth-order kinetics significantly enlarges the parameter space for oscillation and thus enhances the robustness of the design under parametric uncertainty.

Thoracic paravertebral block for implantable cardioverter-defibrillator and laser lead extraction.

Implantable cardioverter defibrillators (ICDs) are routinely placed in the cardiac electrophysiology laboratory. Previously, these procedures were performed in the operating room during general anesthesia. In recent years, electrophysiologists have been performing these procedures using local anesthetics in conjunction with intravenous sedation. We report a case in which thoracic paravertebral blocks with mild sedation were successful for infected ICD and laser lead extraction. Randomized, controlled trials are required to determine whether thoracic paravertebral block with sedation is more effective than local anesthesia with sedation in providing adequate anesthesia for ICD placement and extraction.

Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure.

The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities.

The Accuracy of Web Sites and Cellular Phone Applications in Predicting the Fertile Window.

OBJECTIVE: To evaluate the validity of fertility web sites and applications (apps) by comparing the predicted fertile window of these modalities to the actual fertile window of a standard 28-day cycle. METHODS: This was a descriptive study. The top resulting free web sites and electronic apps downloadable to a cellular phone that provide calendars for fertility and ovulation prediction were assessed. Cycles were standardized to 28 days in length, 4 days of menses, and the last menstrual period was set to January 1, 2015. The predicted date of ovulation and fertility window generated were compared with an actual estimated date of ovulation on cycle day 15, January 15, and a fertile window consisting of cycle day 10 to cycle day 15, the day of ovulation plus the preceding 5 cycle days, January 10-15. RESULTS: Data from 20 web sites and 33 apps were collected. Of all the web sites and apps used, one web site and three apps predicted the precise fertile window. CONCLUSION: Web sites and electronic apps used by the general public to predict fertile windows are generally inaccurate, although the clinical effect of this inaccuracy is unknown. Although they all include the most fertile cycle day, the range of the fertility window varies widely. Patients who are trying to conceive with the assistance of calendars generated from web sites and electronic apps should be counseled on the inaccuracy of these modalities.

Clinical Performance and Management Outcomes with the DecisionDx-UM Gene Expression Profile Test in a Prospective Multicenter Study.

Uveal melanoma management is challenging due to its metastatic propensity. DecisionDx-UM is a prospectively validated molecular test that interrogates primary tumor biology to provide objective information about metastatic potential that can be used in determining appropriate patient care. To evaluate the continued clinical validity and utility of DecisionDx-UM, beginning March 2010, 70 patients were enrolled in a prospective, multicenter, IRB-approved study to document patient management differences and clinical outcomes associated with low-risk Class 1 and high-risk Class 2 results indicated by DecisionDx-UM testing. Thirty-seven patients in the prospective study were Class 1 and 33 were Class 2. Class 1 patients had 100% 3-year metastasis-free survival compared to 63% for Class 2 (log rank test p = 0.003) with 27.3 median follow-up months in this interim analysis. Class 2 patients received significantly higher-intensity monitoring and more oncology/clinical trial referrals compared to Class 1 patients (Fisher's exact test p = 2.1 × 10(-13) and p = 0.04, resp.). The results of this study provide additional, prospective evidence in an independent cohort of patients that Class 1 and Class 2 patients are managed according to the differential metastatic risk indicated by DecisionDx-UM. The trial is registered with Clinical Application of DecisionDx-UM Gene Expression Assay Results (NCT02376920).

Retinoblastoma and hypochondroplasia: a case report of two germline mutations arising simultaneously.

PURPOSE: To report a rare case of a patient with two germline mutations arising de novo resulting in bilateral retinoblastoma and hypochondroplasia. DESIGN: A brief review about retinoblastoma and hypochondroplasia; a case report with genetic mutational analysis results. CASE REPORT: We report a patient manifesting the clinical features of both bilateral retinoblastoma and hypochondroplasia. Genetic analysis revealed two germline mutations, a seven base-pair deletion in exon 12 (G70313-703129del) in one allele of the retinoblastoma gene (RB1) and the N540K (C1620C > A) mutation in one allele of the fibroblast growth factor 3 (FGFR3) gene, a frequent mutation in hypochondroplasia. Neither parent has a personal or family history of cancer or ocular tumors. Only the patient's mother is short in stature, and her genetic analysis revealed no FGFR3 mutations. CONCLUSIONS: Although the probability of both germline mutations occurring in a single individual is exceedingly low, the etiology and mechanism are unknown in this patient. To the best of our knowledge, this is the first report of two clinically distinct heritable germline mutations arising de novo in an individual.

Rapid identification of germline mutations in retinoblastoma by protein truncation testing.

OBJECTIVE: To demonstrate the utility of protein truncation testing (PTT) for rapid detection and sequencing of germline mutations in the retinoblastoma tumor suppressor gene (RB1). METHODS: We performed PTT, a technique based on the in vitro synthesis of protein from amplified RNA, on 27 probands from 27 kindreds with hereditary retinoblastoma. In 4 kindreds, PTT was also performed on 1 additional affected relative. Ten unrelated patients without retinoblastoma were included as negative control subjects. All PTT-detected mutations were further analyzed by focused sequencing of genomic DNA. When no mutation was detected by PTT, we performed exon-by-exon sequencing, as well as cytogenetic analysis by Giemsa-trypsin-Giemsa banding and by fluorescent in situ hybridization for RB1. The results of proband testing were used for direct genetic testing by polymerase chain reaction and sequencing in 11 relatives from 7 of the 27 kindreds. RESULTS: Of the probands tested, 19 (70%) of 27 tested positive for germline mutations by PTT. In 1 kindred, the proband had negative PTT results but an additional affected relative had positive PTT results. Focused DNA sequencing of 1 patient with positive PTT results from each of the 20 kindreds with positive PTT results revealed truncating mutations in 19 kindreds. Four demonstrated frameshift deletions, 6 had splice site mutations, and 9 showed nonsense mutations. Further analysis by genomic exon-by-exon sequencing and karyotype analysis of the 8 probands who tested negative for germline mutations by PTT revealed 1 splice site mutation, 2 missense mutations, and 1 chromosomal deletion. Focused sequencing based on positive PTT results was successfully used to confirm shared truncating mutations in additional affected family members in 2 kindreds. Using a multitiered approach to genetic testing, 23 (85%) of 27 kindreds had mutations identified and those detected by PTT received a positive result in as few as 7 days. In control subjects, PTT produced no false-positive results. CONCLUSIONS: Protein truncation testing is an effective, rapid single-modality screen for germline mutations in patients with retinoblastoma. When used as an initial screen, PTT can increase the yield of additional testing modalities, such as sequencing and chromosomal analysis, providing a timely and cost-effective approach for the diagnosis of heritable germline mutations in patients with retinoblastoma.Clinical Relevance The clinical application of PTT in retinoblastoma will improve detection of germline retinoblastoma mutations, which will supply critical information for prognosis, treatment planning, follow-up care, and genetic counseling.