Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

BACKGROUND: Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS: To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS: Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS: Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

Transcriptomic profiling of Schlemm's canal cells reveals a lymphatic-biased identity and three major cell states.

Schlemm's canal (SC) is central in intraocular pressure regulation but requires much characterization. It has distinct inner and outer walls, each composed of Schlemm's canal endothelial cells (SECs) with different morphologies and functions. Recent transcriptomic studies of the anterior segment added important knowledge, but were limited in power by SEC numbers or did not focus on SC. To gain a more comprehensive understanding of SC biology, we performed bulk RNA sequencing on C57BL/6J SC, blood vessel, and lymphatic endothelial cells from limbal tissue (~4500 SECs). We also analyzed mouse limbal tissues by single-cell and single-nucleus RNA sequencing (C57BL/6J and 129/Sj strains), successfully sequencing 903 individual SECs. Together, these datasets confirm that SC has molecular characteristics of both blood and lymphatic endothelia with a lymphatic phenotype predominating. SECs are enriched in pathways that regulate cell-cell junction formation pointing to the importance of junctions in determining SC fluid permeability. Importantly, and for the first time, our analyses characterize 3 molecular classes of SECs, molecularly distinguishing inner wall from outer wall SECs and discovering two inner wall cell states that likely result from local environmental differences. Further, and based on ligand and receptor expression patterns, we document key interactions between SECs and cells of the adjacent trabecular meshwork (TM) drainage tissue. Also, we present cell type expression for a collection of human glaucoma genes. These data provide a new molecular foundation that will enable the functional dissection of key homeostatic processes mediated by SECs as well as the development of new glaucoma therapeutics.

FYN regulates aqueous humor outflow and IOP through the phosphorylation of VE-cadherin.

The exact sites and molecules that determine resistance to aqueous humor drainage and control intraocular pressure (IOP) need further elaboration. Proposed sites include the inner wall of Schlemms's canal and the juxtacanalicular trabecular meshwork ocular drainage tissues. The adherens junctions (AJs) of Schlemm's canal endothelial cells (SECs) must both preserve the blood-aqueous humor (AQH) barrier and be conducive to AQH drainage. How homeostatic control of AJ permeability in SC occurs and how such control impacts IOP is unclear. We hypothesized that mechano-responsive phosphorylation of the junctional molecule VE-CADHERIN (VEC) by SRC family kinases (SFKs) regulates the permeability of SEC AJs. We tested this by clamping IOP at either 16 mmHg, 25 mmHg, or 45 mmHg in mice and then measuring AJ permeability and VEC phosphorylation. We found that with increasing IOP: 1) SEC AJ permeability increased, 2) VEC phosphorylation was increased at tyrosine-658, and 3) SFKs were activated at the AJ. Among the two SFKs known to phosphorylate VEC, FYN, but not SRC, localizes to the SC. Furthermore, FYN mutant mice had decreased phosphorylation of VEC at SEC AJs, dysregulated IOP, and reduced AQH outflow. Together, our data demonstrate that increased IOP activates FYN in the inner wall of SC, leading to increased phosphorylation of AJ VEC and, thus, decreased resistance to AQH outflow. These findings support a crucial role of mechanotransduction signaling in IOP homeostasis within SC in response to IOP. These data strongly suggest that the inner wall of SC partially contributes to outflow resistance.

Highly size- and shape-controlled synthesis of silver nanoparticles via a templated Tollens reaction.

A mild, facile one-step synthetic strategy for the preparation of size- and shape-controlled silver nanoparticles (AgNPs) is presented. The high degree of size- and shape-control of these AgNPs is achieved by the use of triazole sugar ligands scaffolded by a central resorcinol ether core. Both the triazoles and the resorcinol ether core mediate the nucleation, growth, and passivation phases of the preparation of AgNP in the presence of the Tollens reagent as the silver source. Kinetic and (1)H NMR titration data is presented describing the nature of the interactions between the Tollens reagent and these ligands.

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10-6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP.

Rapid frozen section immunostaining of melanocytes by microphthalmia-associated transcription factor.

Mohs micrographic surgery (MMS) has increasingly become an accepted therapy for melanoma in situ on chronically sun damaged skin (CSDS). However, melanocytes are difficult to locate in frozen material on hematoxylin and eosin. In addition, determining the cut-off between the melanoma and the "atypical melanocytic hyperplasia" in CSDS can be challenging in frozen or formalin-fixed paraffin-embedded sections, with or without immunohistochemistry (IHC). In this article, we report the use of a rapid, 35-minute protocol using microphthalmia-associated transcription factor (MITF) IHC for identifying melanocytes in frozen tissue for its potential use in MMS. In contrast to melanoma antigen recognized by T cells (MART-1), MITF is a nuclear stain, which simplifies identification of melanocytes and quantification of melanocytic parameters. In this study, MITF IHC in frozen sections yielded equivalent melanocyte nuclear diameter and density measurements compared with formalin-fixed paraffin-embedded sections. Nuclear diameter measurements obtained with MITF were similar to that previously reported with MART-1, but the melanocyte density figures were lower. Reliable labeling of melanocytes in frozen sections required the use of diaminobenzidine (DAB) chromogen with Giemsa counterstaining and a buffer devoid of surfactant. Our experience with MITF IHC indicates that it is a dependable immunostain in frozen sections, and may prove to be useful in MMS as an adjunct to hematoxylin and eosin and MART-1 IHC for interpretation of margins for melanoma in situ on CSDS.

Development and implementation of an e-visit objective structured clinical examination to evaluate student ability to provide care by telehealth.

ABSTRACT: The provision of care through telehealth is emerging as a strategy to meet the challenges of a changing health care system; however, educational experiences associated with telehealth are limited in nursing curricula. In 2018, the National Organization of Nurse Practitioner Faculties (NONPF) recommended incorporating telehealth modalities in nurse practitioner education, including clinical skills assessment. Objective structured clinical examinations (OSCEs) have been used as formative and summative evaluations of health science students since the 1970s to assess application of didactic learning in clinical practice. Although few studies have investigated integrating OSCEs, virtual simulation, or telehealth in distance education, those that exist demonstrate significant improvement in clinical decision making. This article describes the development and implementation of an OSCE at a large Midwestern university delivered via web conferencing to evaluate online family nurse practitioner (FNP) students' abilities to deliver care using telecommunication technology. An e-visit notification listing the types of symptoms available for treatment by telehealth served as a content list. Students completed a 15-minute e-visit assessing and managing the care of a standardized patient presenting with a recent onset of dysuria. The video conference included the FNP student, standardized patient, and faculty evaluating in real time using a rubric incorporating NONPF competencies for telehealth. Structured group debrief, an integral part of the learning process in clinical simulations, followed the e-visit OSCE using free conference calling. Faculty and student feedback and evaluations of this summative OSCE implemented for three cohort groups (103 students) positively reflect the importance of preparing students for practice in a transformed health care system.

Author Correction: Scalable and robust SARS-CoV-2 testing in an academic center.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Surgical management of melanoma-in-situ using a staged marginal and central excision technique.

Melanoma-in-situ (MIS) represents 45% of all melanomas. The margins of MIS are often poorly defined with extensive subclinical disease. Standard fusiform excision with 5-mm margins results in positive margins in up to a third of cases. To decrease the incidence of involved margins, we use a staged excision approach for MIS. First, patients undergo excision under local anesthesia of a 2- to 3-mm "contoured" rim of tissue optimally 5 mm beyond the visible extent of the lesion. Formalin-fixed paraffin-embedded en face sections from this excision are then evaluated, if necessary with the aid of immunohistochemical stains. Any positive margins are further excised. When all margins are negative, the central area is then excised and reconstructed. A total of 61 patients with MIS or lentigo maligna melanoma underwent staged contoured excisions from 2004 to 2007 at Moffitt Cancer Center. We analyzed data only from patients with MIS of the head and neck. Patients with known invasive melanoma or non-head and neck primary disease were excluded. Demographics, tumor characteristics, margin status, number of stages, and type of reconstruction and recurrences were evaluated. Forty-nine patients with MIS of the head and neck, 28 (57%) male and 21 (43%) female, 42 to 88-years-old (median 72; mean 70), underwent staged contoured margin excision before definitive central tumor excision and reconstruction. The final surgical defect size ranged from 2 to 130 cm(2) (median 16 cm(2)). Twelve patients (24%) required reexcision of at least one margin; the median number of reexcisions was 1 (range 1-2). There seemed to be a positive association between lesion size and margin status (as well as number of excisions needed to clear the margin). Unsuspected invasive melanoma was found in the central specimen in six patients (12%). Even small tumors could have unsuspected invasive melanoma: invasive cancer was seen in 4 (21%) of 19 tumors < or =2 cm in greatest dimension and 2 (7%) of 30 > 2 cm, respectively. Surgical defects were reconstructed with flaps in 18 (37%), full-thickness grafts in 20 (41%), and split-thickness grafts in 10 patients (20%). Median time from first margin excision to completion/final reconstruction was 7 days (range 7-63 days). No local recurrences have been reported at a median follow-up of 14 months (range 1-36 months). This technique allows for careful margin analysis and subsequent central tumor excision with simultaneous reconstruction. This approach minimizes the need for a second major operation, which would have been necessary in 24% of our patients if treated by a one-stage excisional approach. It is noteworthy that 12% of MIS patients had invasive melanoma in the final excision specimen. This reinforces the importance of adequate full-thickness biopsies of suspicious pigmented lesions before any type of surgical management. With short follow-up, local control has been achieved by this technique in 100% of cases.

Understanding and reducing the risk of supply chain disruptions.

Natural disasters can wreck havoc on business operations. When civil unrest swept the UK in August 2011, the effect on business was stark, losing the retail sector £300m in unexpected costs and lost revenues. On the other side of the world, the natural disaster that hit Japan in early 2011 is estimated to have run up costs in the region of £189bn in repairs. Beyond this, the earthquake and its aftermath shattered supply chains, with technology companies expecting delays of up to six months before business could resume fully. It is impossible to predict incidents like these, but businesses can help mitigate disruption in the supply chain by undertaking business continuity management (BCM). A flexible supply chain is essential when it comes to BCM - whether it means being able to cope with altering transport routes at short notice, or finding or replacing a supplier at the last minute. Understanding the supply chain is critical when responding to major impacts that affect supply chains in multiple points - like IT system failures and country-wide fuel strikes. Businesses should carry out detailed business impact assessments and risk assessments right across the end-to-end supply chain and not just at key single points of failure. It is an intensive process that needs dedicated resources and ownership at the highest level. Recognising this, DHL has designed a 10-step process, which it has implemented across its global supply chain business. This paper provides an overview of what a supply chain really looks like, what can cause disruptions and how far up/down the supply chain companies need to go with their BCM planning.

Surgical management of melanoma in situ on chronically sun-damaged skin.

BACKGROUND: Lentigo maligna (LM) commonly presents as a slow-growing pigmented macular lesion in chronically sun-damaged skin and may progress to invasive melanoma. Many regard it as a subtype of melanoma in situ (MIS), and surgical excision remains the preferred treatment, but standard 5-mm surgical margins recommended for typical MIS are often insufficient for LM due to its indistinct borders both clinically and histologically. METHODS: A search of the literature was conducted to review specialized surgical techniques for the treatment of LM, focusing on methods that employ total peripheral margin assessment prior to definitive closure, using either frozen or permanent histologic sections. RESULTS: Many investigators have reported surgical modalities utilizing permanent sections for margin control, including variations of the "square" procedure and "perimeter" technique. Recurrence rates are low with these methods, but only short-term data have been reported. Similarly, several studies have demonstrated the efficacy of Mohs micrographic surgery (MMS) for treatment of MIS, with recurrence rates generally less than 1% over 3 to 5 years of follow-up. Many investigators have had success with immunohistochemical stains to identify melanocytes on frozen sections, aiding margin assessment in MMS. CONCLUSIONS: Compared to standard excision, methods that employ surgical margin control offer superior cure rates for LM and should be utilized when available. Total peripheral margin assessment using staged excisions and permanent sections is a simple and effective alternative to MMS for institutions that lack the resources for intraoperative frozen section analysis.

Gold nanoparticles decorated with oligo(ethylene glycol) thiols: kinetics of colloid aggregation driven by depletion forces.

We have studied the kinetics of the phase-separation process of mixtures of colloid and protein in solutions by real-time UV-vis spectroscopy. Complementary small-angle X-ray scattering (SAXS) was employed to determine the structures involved. The colloids used are gold nanoparticles functionalized with protein resistant oligo(ethylene glycol) (OEG) thiol, HS(CH2)11(OCH2CH2)6OMe (EG6OMe). After mixing with protein solution above a critical concentration, c*, SAXS measurements show that a scattering maximum appears after a short induction time at q = 0.0322 A-1, which increases its intensity with time but the peak position does not change with time, protein concentration and salt addition. The peak corresponds to the distance of the nearest neighbor in the aggregates. The upturn of scattering intensities in the low q-range developed with time indicating the formation of aggregates. No Bragg peaks corresponding to the formation of colloidal crystallites could be observed before the clusters dropped out from the solution. The growth kinetics of aggregates is followed in detail by real-time UV-vis spectroscopy, using the flocculation parameter defined as the integral of the absorption in the range of 600-800 nm wavelengths. At low salt addition (<0.5 M), a kinetic crossover from reaction-limited cluster aggregation (RLCA) to diffusion-limited cluster aggregation (DLCA) growth model is observed, and interpreted as being due to the effective repulsive interaction barrier between colloids within the depletion potential. Above 0.5 M NaCl, the surface charge of proteins is screened significantly, and the repulsive potential barrier disappeared, thus the growth kinetics can be described by a DLCA model only.

Gold nanoparticles decorated with oligo(ethylene glycol) thiols: protein resistance and colloidal stability.

The interactions between proteins and gold colloids functionalized with protein-resistant oligo(ethylene glycol) (OEG) thiol, HS(CH2)11(OCH2CH2)6OMe (EG6OMe), in aqueous solution have been studied by small-angle X-ray scattering (SAXS) and UV-vis spectroscopy. The mean size, 2R, and the size distribution of the decorated gold colloids have been characterized by SAXS. The monolayer-protected gold colloids have no correlations due to the low volume fraction in solution and are stable in a wide range of temperatures (5-70 degrees C), pH (1.3-12.4), and ionic strength (0-1.0 M). In contrast, protein (bovine serum albumin) solutions with concentrations in the range of 60-200 mg/mL (4.6-14.5 vol %) show a pronounced correlation peak in SAXS, which results from the repulsive electrostatic interaction between charged proteins. These protein interactions show significant dependence on ionic strength, as would be expected for an electrostatic interaction (Zhang et al. J. Phys. Chem. B 2007, 111, 251). For a mixture of proteins and gold colloids, the protein-protein interaction changes little upon mixing with OEG-decorated gold colloids. In contrast, the colloid-colloid interaction is found to be strongly dependent on the protein concentration and the size of the colloid itself. Adding protein to a colloidal solution results in an attractive depletion interaction between functionalized gold colloids, and above a critical protein concentration, c*, the colloids form aggregates and flocculate. Adding salt to such mixtures enhances the depletion effect and decreases the critical protein concentration. The aggregation is a reversible process (i.e., diluting the solution leads to dissolution of aggregates). The results also indicate that the charge of the OEG self-assembled monolayer at a curved interface has a rather limited effect on the colloidal stabilization and the repulsive interaction with proteins.