Catalytic, Sulfur-Free Chain Transfer Agents That Alter the Mechanical Properties of Cross-Linked Photopolymers.

Thermosetting materials generated by photopolymerization frequently suffer from significant shrinkage stress, are often brittle, and have a limited range of mechanical properties. Various classes of chain transfer agents (CTAs) have been investigated and developed to reduce the cross-linking density of photopolymers by terminating chains and initiating new chains in situ. Although CTAs are successful in manipulating the mechanical properties of photopolymers, they are traditionally consumed during the polymerization and are therefore required in high loadings (up to 20 wt % of the total formulation). Moreover, traditional CTAs frequently contain sulfur, which is malodorous and can create unstable formulations. Presented here is a catalytic, sulfur-free CTA that can be added in ppm quantities to existing commercial monomer feedstocks to create photopolymers similar to those prepared using traditional CTAs, but at 10 000-fold lower loadings. These catalysts, which are based on macrocyclic cobaloximes, were found to tunably reduce the molecular weight of the chain proportional to catalyst loading. It was shown, using only commercial monomers, that this catalyst could reduce the glass-transition temperature (Tg), rubbery modulus (E'rubbery), and stiffness of a cross-linked photopolymer while utilizing identical processing conditions and keeping 99.99 wt % of the formulation the same.

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.

Pharmacist Practice Patterns Regarding Direct Oral Anticoagulants for Treatment of Venous Thromboembolism.

Background: Direct oral anticoagulants (DOACs) are increasingly prescribed for the treatment of venous thromboembolism (VTE). However, little is known regarding pharmacists' practice patterns and preferences in clinical areas of contention, such as initiation dosing, obesity, and renal impairment. Objective: To determine pharmacist trends in practice regarding DOACs for the treatment of VTE overall and within areas of clinical controversy. Methods: An electronic survey was distributed to pharmacists in the United States through national and state pharmacy organizations. Responses were collected for 30 days. Results: One hundred fifty-three complete responses were submitted. The majority of pharmacists preferred apixaban (90.2%) for the oral treatment of venous thromboembolism. When initiating apixaban or rivaroxaban for a new VTE, 76% and 64% of pharmacists surveyed, respectively, state the duration of the initiation dose phases are reduced if the patient received parenteral anticoagulation. Fifty-eight percent of pharmacists used body mass index to evaluate the appropriateness of DOACs in obese patients whereas 42% used total body weight. Preference for rivaroxaban (31.4%) was higher in this population compared to the global population (10%). Apixaban was preferred for patients with renal impairment (92.2%). However, as creatinine clearance as calculated by the Cockcroft-Gault equation (CrCl) reduced to ≤15 milliliters/minute (mL/min), preference for warfarin increased (36%). Conclusion: This national survey of pharmacists demonstrated an overall preference for apixaban and significant variability in practice patterns regarding DOACs for patients with new VTE, patients with obesity, and patients with renal impairment. Further research is warranted to evaluate the efficacy and safety of DOAC initiation dosing phase modifications. Prospective evaluations of DOACs in obese and renal dysfunction populations would confirm the safety and efficacy of DOACs in these populations.

Access to Substituted Indenol Ethers via a Regioselective Intermolecular Carbopalladation Cascade.

Alkyne carbopalladation reactions can rapidly generate multiple new C-C bonds; however, regioselectivity is challenging for intermolecular variants. Using ynol ethers, we observe complete regiocontrol of migratory insertion followed by a second migratory insertion with a pendant alkene to turn-over the catalytic cycle. The resulting products are oligosubstituted 1-indenol ethers with defined stereochemistry based on the initial alkene geometry. Blocking ß-hydride elimination allowed for C-H and C-C reductive elimination steps for catalyst turnover.

Effect of COVID-19 on patient access to health services for noncommunicable diseases in Latin America: a perspective from patient advocacy organizations.

BACKGROUND: The COVID-19 pandemic has been felt acutely in Latin America with several countries having among the highest numbers of SARS-CoV-2 cases and related deaths. Individuals living with underlying health conditions have an increased risk of severe disease or death from COVID-19. Patient advocacy organizations often provide supportive services to these individuals and can offer a unique perspective of the patient experience. The objective of this study was to assess the effects of COVID-19 on access to health services in Latin America, as reported by patient advocacy organizations representing individuals living with autoimmune, chronic, and noncommunicable diseases. METHODS: A cross-sectional study was conducted in August 2020 with patient advocacy organizations in Latin America to measure perceived effects from COVID-19 and reported access to health services among individuals living with autoimmune, chronic, and noncommunicable diseases. An original, online survey was developed and deployed in Spanish and Portuguese. Univariate and bivariate analysis was conducted across two main subject areas: perceived patient effects from COVID-19 and patient access to health services. The main outcomes of analysis considered patient access to care during COVID-19 based on type of chronic illness and geographical region in Latin America. RESULTS: A total of 81 survey responses were analyzed. A majority (83%) of patient advocacy organizations reported their patients experienced delays receiving their treatment and care services; 52% experienced delays of 30 days or more. Telemedicine was considered available, but not accessible to patients (37%) and a majority (76%) of patients faced challenges with electronic prescriptions. Patients were not likely to receive a multi-month prescription from their doctor (38%) or successfully fill it at the pharmacy (26%). CONCLUSIONS: According to responses from patient advocacy organizations, individuals living with noncommunicable diseases in Latin America have faced unique challenges during the COVID-19 pandemic. As countries re-evaluate their health systems, it is critical that chronic diseases are considered so that all can fully realize the right to health.

Onset of Mechanochromic Response in the High Strain Rate Uniaxial Compression of Spiropyran Embedded Silicone Elastomers.

The molecular processes that accompany dynamic mechanical response to large deformations at high strain rate (≈1000 s-1 or higher) underlie the early stages of damage in materials, but understanding of material response in this regime is typically limited to macroscopic constitutive equations. Here, spiropyran mechanophores are embedded in very short, stress-bearing strands in silicone elastomers, and their mechanochromic response to uniaxial compression is explored in a Split Hopkinson Pressure (or Kolsky) Bar. At strain rates of 1000 s-1 , the onset of mechanochromism occurs at lower strains, but higher stresses, than in the same materials under quasi-static loading. Similar to quasi-static loading, however, a negligible effect of mechanophore structure on the critical strain for colorimetric onset is observed. The results suggest that nonequilibrium, inhomogeneous local tension distributions in the elastomers lead to greater stress in individual strands than at the same strains under equilibrium loading, but that within the regions of force concentration, mechanochromic onset is determined primarily by a limiting local strain threshold.

Anterior Cruciate Ligament Injury at the Time of Anterior Tibial Spine Fracture in Young Patients: An Observational Cohort Study.

BACKGROUND: Anterior tibial spine fractures (ATSF) in the skeletally immature parallel anterior cruciate ligament (ACL) tears in adult patients, yet these injuries are generally regarded as mutually exclusive. Biomechanical analysis suggests that intrinsic ACL damage occurs during ATSF, and long-term clinical studies demonstrate residual anteroposterior knee laxity following ATSF. We aim to describe prevalence, demographics, and characteristics of pediatric patients who sustained ATSF with concomitant ACL injury. METHODS: We included 129 patients with ATSF over a 16-year period. Age, sex, injury mechanism, ATSF type, magnetic resonance imaging (MRI) evaluation, treatment modality, ACL injury, and concomitant meniscal/chondral injuries were analyzed. Concurrent ACL injury was confirmed either from MRI or intraoperatively. RESULTS: Nineteen percent (n=25) of ATSF patients had concomitant ACL injury, with ACL injury significantly more likely in type II or type III ATSF compared with type I ATSF (P=0.03). Patients with combined ATSF/ACL injury were significantly older (P=0.02) and more likely to be male (P=0.01). Mechanism of ATSF injury was not associated with ACL injury (P=0.83). Preoperative MRI had low sensitivity (0.09) for recognizing ACL injury at the time of ATSF relative to intraoperative assessment. Half of ATSF/ACL-injured patients had additional meniscal or chondral injury, with meniscal repair or debridement required in 37.5% of the type II ATSF/ACL injury. CONCLUSIONS: There are demographic characteristics, such as age (older) and sex (male), associated with a higher risk of concomitant ACL injury at the time of ATSF. Type II and type III ATSF patterns had a higher prevalence of ACL injury. MRI failed to correctly identify ACL injury at the time of ATSF. Concomitant ACL injury at the time of ATSF is highly prevalent in the skeletally immature, occurring in 19.4% of patients with ATSF. LEVEL OF EVIDENCE: Level IV-case series.

Epidemiology of Anterior Tibial Spine Fractures in Young Patients: A Retrospective Cohort Study of 122 Cases.

BACKGROUND: Historically, bicycle accidents were described as the most common mechanism for pediatric anterior tibial spine fractures (ATSFs). There is a paucity of current literature examining the demographic factors associated with these injuries. The purpose of this cohort study was to characterize the epidemiology of ATSFs presenting to a single tertiary referral pediatric hospital. METHODS: A consecutive cohort of 122 pediatric patients with ATSFs between 1996 and 2014 were reviewed. Radiographic variables, classification of fractures (Meyers and McKeever type), age, sex, height, weight, body mass index, and mechanism of injury were retrieved. Categories of mechanism of injury included organized sports (football, soccer, basketball, lacrosse, wrestling, and gymnastics), bicycling, outdoor sports (skiing, skateboarding, and sledding), fall, motor vehicle collision/pedestrian versus motor vehicle, and trampoline. RESULTS: Organized sports-related injuries represented the most common cause of ATSFs (36%). Other common mechanisms of injury included bicycle accidents (25%), outdoor sports (18%), and falls (11%). There was a higher proportion of males (69%) compared with females (31%). Males (mean age, 11.6 y) were significantly older than females (mean age, 9.8 y) (P=0.004). Younger patients (aged 11.5 y and below) were more likely to have displaced fractures (type III), whereas type I and type II were more common in patients above 11.5 years (P=0.02). Patients with fracture type I were significantly taller than patients with fracture type III. No other variables were found to differ significantly according to fracture severity, including sex, weight, and body mass index. CONCLUSIONS: To our knowledge, our study represents both the largest (n=122) and most up-to-date epidemiological ATSF study in pediatric patients. A higher rate of ATSF occurs due to organized sports rather than bicycling or motor vehicle collision. This 18-year data collection represents a change in the paradigm, and is likely multifactorial, including increased participation in youth sports and early sport specialization. LEVEL OF EVIDENCE: Level IV-retrospective, cohort study.

'Seeing' Strain in Soft Materials.

Several technologies can be used for measuring strains of soft materials under high rate impact conditions. These technologies include high speed tensile test, split Hopkinson pressure bar test, digital image correlation and high speed X-ray imaging. However, none of these existing technologies can produce a continuous 3D spatial strain distribution in the test specimen. Here we report a novel passive strain sensor based on poly(dimethyl siloxane) (PDMS) elastomer with covalently incorporated spiropyran (SP) mechanophore to measure impact induced strains. We have shown that the incorporation of SP into PDMS at 0.25 wt% level can adequately measure impact strains via color change under a high strain rate of 1500 s-1 within a fraction of a millisecond. Further, the color change is fully reversible and thus can be used repeatedly. This technology has a high potential to be used for quantifying brain strain for traumatic brain injury applications.

Regiochemical Effects on Mechanophore Activation in Bulk Materials.

Mechanochromic force probes, including spiropyran derivatives, have proven to be useful in visualizing the stress/strain distribution and fracture behavior in polymeric materials. Here, we report the macroscopic response of silicone elastomers including cross-links made up of three spiropyran (SP) regioisomers. The SP derivatives SP( o), SP( m), and SP( p) are connected to the network through an identical attachment point on the indoline fragment and regioisomeric attachments ortho, meta, and para to the spirocyclic C-O bond on the benzaldehyde fragment, respectively. The relative colorimetric response of these regioisomers under quasi-static uniaxial tensile load is SP( o) > SP( m) > SP( p), consistent with the expected mechanical sensitivity of the regioisomers obtained from molecular modeling. The extrapolated strain onset for detectable activation of all three regioisomers, however, is indistinguishable and occurs at ∼90% uniaxial strain. Finally, the ratiometric response of the three isomers is constant across the strains investigated (90-135% uniaxial strain), in contrast to expectations based on simulations of strained intact polymer networks.

Substituent Effects and Mechanism in a Mechanochemical Reaction.

We report the effect of substituents on the force-induced reactivity of a spiropyran mechanophore. Using single molecule force spectroscopy, force-rate behavior was determined for a series of spiropyran derivatives substituted with H, Br, or NO2 para to the breaking spirocyclic C-O bond. The force required to achieve the rate constants of ∼10 s-1 necessary to observe transitions in the force spectroscopy experiments depends on the substituent, with the more electron withdrawing substituent requiring less force. Rate constants at 375 pN were determined for all three derivatives, and the force-coupled rate dependence on substituent identity is well explained by a Hammett linear free energy relationship with a value of ρ = 2.9, consistent with a highly polar transition state with heterolytic, dissociative character. The methodology paves the way for further application of linear free energy relationships and physical organic methodologies to mechanochemical reactions, and the characterization of new force probes should enable additional, quantitative studies of force-coupled molecular behavior in polymeric materials.

Nesprins anchor kinesin-1 motors to the nucleus to drive nuclear distribution in muscle cells.

During skeletal muscle development, nuclei move dynamically through myotubes in a microtubule-dependent manner, driven by the microtubule motor protein kinesin-1. Loss of kinesin-1 leads to improperly positioned nuclei in culture and in vivo. Two models have been proposed to explain how kinesin-1 functions to move nuclei in myotubes. In the cargo model, kinesin-1 acts directly from the surface of the nucleus, whereas in an alternative model, kinesin-1 moves nuclei indirectly by sliding anti-parallel microtubules. Here, we test the hypothesis that an ensemble of Kif5B motors acts from the nuclear envelope to distribute nuclei throughout the length of syncytial myotubes. First, using an inducible dimerization system, we show that controlled recruitment of truncated, constitutively active kinesin-1 motors to the nuclear envelope is sufficient to prevent the nuclear aggregation resulting from depletion of endogenous kinesin-1. Second, we identify a conserved kinesin light chain (KLC)-binding motif in the nuclear envelope proteins nesprin-1 and nesprin-2, and show that recruitment of the motor complex to the nucleus via this LEWD motif is essential for nuclear distribution. Together, our findings demonstrate that the nucleus is a kinesin-1 cargo in myotubes and that nesprins function as nuclear cargo adaptors. The importance of achieving and maintaining proper nuclear position is not restricted to muscle fibers, suggesting that the nesprin-dependent recruitment of kinesin-1 to the nuclear envelope through the interaction of a conserved LEWD motif with kinesin light chain might be a general mechanism for cell-type-specific nuclear positioning during development.

TM6SF2 rs58542926 impacts lipid processing in liver and small intestine.

The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).

Impulse Control in Negative Mood States, Emotional Eating, and Food Addiction are Associated with Lower Quality of Life in Adolescents with Severe Obesity.

Objectives: Quality of life (QoL) is an important outcome to evaluate in adolescents with severe obesity, yet intrapersonal predictors of QoL are understudied. The current study assessed whether difficulty with impulse control when experiencing a negative mood (negative urgency) is associated with poorer QoL, mediated by more emotional eating and food addiction. Method: Participants consisted of 69 primarily female (71%), minority (76%) adolescents aged 13-21 (M age = 16.5, SD = 1.5) with severe obesity presenting for prebariatric surgery psychological evaluations. Structural Equation Modeling was used to appraise a model of the association of adolescent report of negative urgency with more emotional eating (Emotional Eating Scale for Children) and food addiction (Yale Food Addiction Scale) and poorer weight-related QoL (Impact of Weight on Quality of Life-Kids). Results: Greater difficulty controlling behavior when experiencing a negative mood was significantly associated with poorer weight-related QoL, and this relationship was mediated by an association with emotional eating and food addiction such that adolescents with severe obesity who reported more difficulties with impulse control in negative mood states were more likely to report more emotional eating and food addiction, which was in turn associated with lower QoL. Conclusions: Intrapersonal factors, including impulse control in negative mood states, are associated with lower QoL in adolescents with severe obesity. Interventions aimed at reducing frequency of negative affect, reducing impulsivity in negative mood states, and improving coping skills that are not eating based may contribute to improved QoL and merit further study.

An HPLC-CAD/fluorescence lipidomics platform using fluorescent fatty acids as metabolic tracers.

Fluorescent lipids are important tools for live imaging in cell culture and animal models, yet their metabolism has not been well-characterized. Here we describe a novel combined HPLC and LC-MS/MS method developed to characterize both total lipid profiles and the products of fluorescently labeled lipids. Using this approach, we found that lipids labeled with the fluorescent tags, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY FL), 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene [BODIPY(558/568)], and dipyrrometheneboron difluoride undecanoic acid (TopFluor) are all metabolized into varying arrays of polar and nonpolar fluorescent lipid products when they are fed to larval zebrafish. Quantitative metabolic labeling experiments performed in this system revealed significant effects of total dietary lipid composition on fluorescent lipid partitioning. We provide evidence that cholesterol metabolism in the intestine is important in determining the metabolic fates of dietary FAs. Using this method, we found that inhibitors of dietary cholesterol absorption and esterification both decreased incorporation of dietary fluorescent FAs into cholesterol esters (CEs), suggesting that CE synthesis in enterocytes is primarily responsive to the availability of dietary cholesterol. These results are the first to comprehensively characterize fluorescent FA metabolism and to demonstrate their utility as metabolic labeling reagents, effectively coupling quantitative biochemistry with live imaging studies.

Endoplasmic Reticulum Lipid Flux Influences Enterocyte Nuclear Morphology and Lipid-dependent Transcriptional Responses.

Responding to a high-fat meal requires an interplay between multiple digestive tissues, sympathetic response pathways, and the gut microbiome. The epithelial enterocytes of the intestine are responsible for absorbing dietary nutrients and preparing them for circulation to distal tissues, which requires significant changes in cellular activity, including both morphological and transcriptional responses. Following a high-fat meal, we observe morphological changes in the enterocytes of larval zebrafish, including elongation of mitochondria, formation and expansion of lipid droplets, and the rapid and transient ruffling of the nuclear periphery. Dietary and pharmacological manipulation of zebrafish larvae demonstrated that these subcellular changes are specific to triglyceride absorption. The transcriptional changes that occur simultaneously with these morphological changes were determined using RNA sequencing, revealing a cohort of up-regulated genes associated with lipid droplet formation and lipid transport via lipoprotein particles. Using a microsomal triglyceride transfer protein (MTP) inhibitor to block ß-lipoprotein particle formation, we demonstrate that the transcriptional response to a high-fat meal is associated with the transfer of ER triglyceride to nascent ß-lipoproteins, possibly through the activation of Creb3l3/cyclic AMP-responsive element-binding protein. These data suggest that a transient increase in ER lipids is the likely mediator of the initial physiological response of intestinal enterocytes to dietary lipid.

Structural Basis for the Regulation of the MmpL Transporters of Mycobacterium tuberculosis.

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the MmpL (mycobacterial membrane protein large) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816, we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

The majority of 9,729 group A streptococcus strains causing disease secrete SpeB cysteine protease: pathogenesis implications.

Group A streptococcus (GAS), the causative agent of pharyngitis and necrotizing fasciitis, secretes the potent cysteine protease SpeB. Several lines of evidence suggest that SpeB is an important virulence factor. SpeB is expressed in human infections, protects mice from lethal challenge when used as a vaccine, and contributes significantly to tissue destruction and dissemination in animal models. However, recent descriptions of mutations in genes implicated in SpeB production have led to the idea that GAS may be under selective pressure to decrease secreted SpeB protease activity during infection. Thus, two divergent hypotheses have been proposed. One postulates that SpeB is a key contributor to pathogenesis; the other, that GAS is under selection to decrease SpeB during infection. In order to distinguish between these alternative hypotheses, we performed casein hydrolysis assays to measure the SpeB protease activity secreted by 6,775 GAS strains recovered from infected humans. The results demonstrated that 84.3% of the strains have a wild-type SpeB protease phenotype. The availability of whole-genome sequence data allowed us to determine the relative frequencies of mutations in genes implicated in SpeB production. The most abundantly mutated genes were direct transcription regulators. We also sequenced the genomes of 2,954 GAS isolates recovered from nonhuman primates with experimental necrotizing fasciitis. No mutations that would result in a SpeB-deficient phenotype were identified. Taken together, these data unambiguously demonstrate that the great majority of GAS strains recovered from infected humans secrete wild-type levels of SpeB protease activity. Our data confirm the important role of SpeB in GAS pathogenesis and help end a long-standing controversy.

Primary transpupillary thermotherapy for choroidal melanoma in 391 cases: importance of risk factors in tumor control.

PURPOSE: To report the long-term outcome of primary transpupillary thermotherapy (TTT) for choroidal melanoma. DESIGN: Retrospective review of medical records. PARTICIPANTS: We included 391 patients with choroidal melanoma treated between 1995 and 2012 at the Oncology Service, Wills Eye Hospital, Philadelphia. METHODS: We delivered TTT with an infrared diode laser. MAIN OUTCOME MEASURES: Local tumor recurrence, Snellen visual acuity after TTT, and distant metastasis. RESULTS: Of 391 patients, 311 (80%) were treated from 1995 to 2000 and 80 (20%) from 2001 to 2012. Tumors in the 2001 to 2012 group were ultrasonographically thinner (2.2 vs. 2.7 mm), more distant from the optic disc (3.2 vs. 2.5 mm) and foveola (4.0 vs. 2.0 mm), were less often located in the macular area (14% vs. 40%), and had lower rates of acoustic hollowness on B-scan ultrasonography (63% vs. 84%), subretinal fluid (58% vs. 90%), and orange pigment (50% vs. 70%). Kaplan-Meier estimates for tumor recurrence in the 1995 to 2000 group were 29% at 5 years and 42% at 10 years, whereas estimates for tumor recurrence in the 2001-2012 group were 11% at 5 years and 15% at 10 years. Of 108 recurrent tumors 20 were controlled with additional TTT and 62 required plaque radiation (n=60) or proton beam radiation (n=2), with enucleation necessary in 26 patients. Tumor recurrence correlated with the number of high-risk tumor features: 10-year recurrence was 18% in those with 1 or 2 risk factors, 35% in those with 3 to 5 factors, and 55% in those with 6 or 7 factors. On multivariate analysis, features predictive of tumor recurrence were presence of symptoms (P<0.001), shorter distance between the tumor and the optic disc (P=0.026), subretinal fluid (P=0.035), thickness of residual tumor scar (P<0.001), and elevation of residual tumor scar (P<0.001). The only factor predictive of extraocular tumor extension was intraocular tumor recurrence after TTT treated with additional TTT (P=0.007). Presence of orange pigment before TTT (P=0.019), tumor recurrence (P=0.002), and extraocular tumor extension (P=0.017) were predictive of distant metastasis. CONCLUSION: This study shows a direct correlation between a larger number of high-risk tumor features and higher rates of tumor recurrence after primary TTT of (small) choroidal melanoma. We advise that, when possible, small choroidal melanomas with multiple risk factors be treated with methods other than TTT.

Pulse-shaping based two-photon FRET stoichiometry.

Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.