Gefei Alex Zhu

Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors.

Functional reconstitution of transmembrane proteins remains a significant barrier to their biochemical, biophysical, and structural characterization. Studies of seven-transmembrane G-protein coupled receptors (GPCRs) in vitro are particularly challenging because, ideally, they require access to the receptor on both sides of the membrane as well as within the plane of the membrane. However, understanding the structure and function of these receptors at the molecular level within a native-like environment will have a large impact both on basic knowledge of cell signaling and on pharmacological research. The goal of this article is to review the main classes of membrane mimics that have been, or could be, used for functional reconstitution of GPCRs. These include the use of micelles, bicelles, lipid vesicles, nanodiscs, lipidic cubic phases, and planar lipid membranes. Each of these approaches is evaluated with respect to its fundamental advantages and limitations and its applications in the field of GPCR research. This article is part of a Special Issue entitled: Membrane protein structure and function.

Two Different Scenarios of Squamous Cell Carcinoma Within Advanced Basal Cell Carcinomas: Cases Illustrating the Importance of Serial Biopsy During Vismodegib Usage

IMPORTANCE Vismodegib is a Hedgehog signaling pathway inhibitor recently approved by the US Food and Drug Administration for advanced basal cell carcinoma. We present 2 cases of clinically significant squamous cell carcinoma within the tumor bed of locally advanced basal cell carcinoma found during vismodegib treatment. OBSERVATIONS The first case is that of a patient with locally advanced basal cell carcinoma responsive to vismodegib but with an enlarging papule within the tumor bed. On biopsy, this papule was an invasive acantholytic squamous cell carcinoma. The second case is that of a patient with Gorlin syndrome with a locally advanced basal cell carcinoma that was stable while the patient was receiving therapy with vismodegib for 2.5 years but subsequently increased in size. Biopsy specimens from this tumor showed invasive squamous cell carcinoma, spindle cell subtype. In both cases, the squamous cell carcinomas were surgically resected. CONCLUSIONS AND RELEVANCE These cases highlight the importance of repeated biopsy in locally advanced basal cell carcinomas in 2 clinical situations: (1) when an area within the tumor responds differentially to vismodegib, and (2) when a tumor stops being suppressed by vismodegib. Timely diagnosis of non-basal cell histologic characteristics is critical to institution of effective therapy.

a pIlot study examInIng food InsecurIty and hIv rIsk BehavIors among IndIvIduals recently released from prIson

Annually 700,000 individuals are released from U.S. prison, many at risk for food insecurity and HIV. The association between food insecurity and HIV risk behaviors has been established but not in this population. To investigate this association, we recruited 110 recently released prisoners to participate in a survey. Ninety-one percent of our sample was food insecure; 37% did not eat for an entire day in the past month. Those who did not eat for an entire day were more likely to report using alcohol, heroin, or cocaine before sex or exchanging sex for money compared to those who had at least a meal each day. From this pilot study, released prisoners appear to be at risk for food insecurity, and not eating for an entire day is associated with certain HIV risk behaviors. HIV prevention efforts should include longitudinal studies on the relationship between food insecurity and HIV risk behaviors among recently released prisoners.

Chemical Kinetic Analysis of Thermal Decay of Rhodopsin Reveals Unusual Energetics of Thermal Isomerization and Hydrolysis of Schiff Base

The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55 °C, as well as WT rhodopsin in H2O and D2O at 59 °C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments.

Association Between Programmed Death Ligand 1 Expression in Patients With Basal Cell Carcinomas and the Number of Treatment Modalities

Importance Response to programmed death 1 (PD-1) inhibitors has been associated with programmed death ligand 1 (PD-L1) expression levels in several cancers, but PD-L1 expression and its clinical significance in basal cell carcinoma (BCC) are unknown to date. Objective To assess PD-L1 expression in treatment-naive and treated BCCs. Design, Setting, and Participants This investigation was a cross-sectional study at a single academic tertiary referral center. Immunohistochemical staining on formalin-fixed BCCs from a dermatology clinic were examined in masked fashion by a dermatopathologist and a dermatologist. The study dates were March 31, 2014, to June 7, 2016. Exposures Treated BCCs (including those recurrent after surgery, radiotherapy, systemic chemotherapy, or topical chemotherapy) vs treatment-naive BCCs. Main Outcomes and Measures Percentage of tumor cells and tumor-infiltrating lymphocytes (TILs) with PD-L1 expression, intensities of expression, and association with treatment modalities. Results Among 138 BCCs from 62 patients (43 males and 19 females; mean [SD] age at biopsy, 61.6 [13.7] years), 89.9% (124 of 138) were positive for PD-L1 expression in tumor cells, and 94.9% (131 of 138) were positive for PD-L1 expression in TILs, defined as greater than 5% positive immunohistochemical staining in the respective cell populations. The PD-L1 immunohistochemical staining intensity of 78 treated BCCs compared with 60 treatment-naive BCCs was significantly different in tumor cells (32% vs 7%, P = .003) and TILs (47% vs 18%, P = .008) after adjusting for the age at diagnosis. In a multivariable model adjusting for age, sex, and BCC location, PD-L1 staining intensity in tumor cells increased with the number of distinct prior treatment modalities (median, 0.12; interquartile range, 0.03-0.20; P = .007). Conclusions and Relevance Our data suggest that PD-1 immunotherapy may have activity against BCCs, including in those that have been previously treated. This hypothesis needs to be tested in future clinical trials.

Patient With Gorlin Syndrome and Metastatic Basal Cell Carcinoma Refractory to Smoothened Inhibitors

IMPORTANCE Basal cell carcinomas (BCCs) in patients with Gorlin syndrome have been reported to be extremely sensitive to Smoothened (SMO) inhibitors, a novel targeted therapy against the Hedgehog pathway, because of characteristic mutations in these patients. A few cases of disease refractory to oral therapy with SMO inhibitors have been reported in patients with Gorlin syndrome and nonmetastatic BCCs, but refractory disease in distantly metastatic tumors has not been documented in this high-risk group. OBSERVATIONS A man with Gorlin syndrome and innumerable cutaneous BCCs presented with biopsy-proven BCC in his lungs. After SMO inhibitor therapy, almost all of his cutaneous tumors shrank, but his lung metastases did not. These lung metastases remained refractory to treatment despite institution of a second SMO inhibitor. CONCLUSIONS AND RELEVANCE We report a case of Gorlin syndrome in a patient with metastatic BCC refractory to SMO inhibitors. Furthermore, clinical responses in this patients cutaneous tumors did not parallel the responses in the distant site. However, serial imaging after diagnosis of metastatic disease can be critical to monitor for response to therapy.

Discordance in routine second opinion pathology review of head and neck oncology specimens: A single-center five year retrospective review

OBJECTIVES Second opinion review of pathology specimens is a common institutional practice, supported by large retrospective studies demonstrating significant histologic discordance. Since the most recent study of head and neck-specific pathology review was conducted, routine HPV and EBV testing is now recommended for certain specimens. We describe the frequency of and reasons for discordant reports and their potential impact on treatment recommendations and prognosis in a five-year retrospective cohort study at a single academic referral institution from 2005 to 2010. MATERIALS AND METHODS Following institutional review board review, 1003 cases referred to the Head and Neck Oncology Service were identified using a retrospective database search. Discordance between outside and second review pathology report was assessed by a board-certified medical oncologist. RESULTS 667 cases were included, of which 22% were discordant. Discordance was associated with adenocarcinomas (AOR [adjusted odds ratio] 0.09, 95% CI 0.03-0.31; p<0.001), poorly differentiated carcinomas (AOR 0.14, 95% CI 0.06-0.39; p<0.001), and specimens of uncommon histology (AOR 0.18, 95% CI 0.07-0.45; p<0.001) but not biopsy site in a multivariate model. The most common reasons for discordance included histology (61%), followed by the results of special studies (36%), and the presence or absence of stromal invasion (14%). Differences in tumor HPV status comprised 16% of discordant cases and were associated with better prognosis (p<0.001) following second opinion review. CONCLUSIONS Routine second opinion pathology review may lead to clinically significant differences in treatment recommendations and prognosis.

Acquiring snapshots of the orientation of trans-membrane protein domains using a hybrid FRET pair

One challenge in studying the function of membrane‐embedded proteins is determining the orientation of key domains in the context of the changing and dynamic membrane environment. We describe a confocal microscopy setup that utilizes external electric field pulses to direct dipicrylamine (DPA) to a membrane leaflet. The detection of FRET between DPA and a fluorescent probe attributes it to the inner or outer leaflet of a membrane. By utilizing short acquisition times and confocal imaging, this attribution could be made even in changing membrane environments. Our setup adds versatility to the study of the biological activity of membrane‐embedded proteins.

Overall and progression-free survival of stage 4 cutaneous squamous cell carcinoma at a single large referral center

To the Editor: Cutaneous squamous cell carcinoma (SCC) is the second most common nonmelanoma skin cancer (NMSC) but accounts for the bulk of deaths from NMSCs due to its capacity to metastasize. Although incidence is increasing in the United States, outcomes data are not reported in national cancer registries and disease mortality is not well-described in the literature.We report theoverall survival (OS) and progression-free survival (PFS) in 36 patientswith American Joint Committee onCancer (AJCC) stage IV SCC in this retrospective cohort study at a large academic center. The Stanford Cancer Center Research Database was queried using the terms ‘‘metastatic,’’ ‘‘cutaneous,’’ ‘‘SCC,’’ and ‘‘squamous cell carcinoma’’ for potential cases from 1997 to 2013 following institutional review board approval. Cases were included if there was documentation of a cutaneous primary site and biopsy-proven stage IV disease under the current AJCC TNM staging guidelines (7th ed, 2010).* Clinical and demographic data were analyzed using STATA 12 (Statacorp LP). OS and PFS were estimated by Kaplan-Meier analysis with date of stage IV disease diagnosis as the start of follow-up. The endpoint for OS was the all-cause date of death ascertained through medical records or public records including the Social Security Death Index; the endpoint for PFS was the earliest date of disease progression, defined as development of new metastases, enlargement of existing tumors, recurrence, or all-cause death. Patients not meeting the endpoint before the data cutoff date of October 1, 2014, were right-censored. Thirty-six patients met inclusion criteria, of whom 31 were male and 33 were Caucasian. At the time of stage IV diagnosis, 14 had M1, 23 had N2/N3,

Gender differences in HIV risk behaviors in individuals recently released from prison: results of a pilot study

BackgroundIndividuals recently released from prison engage in risky behaviors that predispose them to contracting HIV. Women may be at increased risk in the immediate period post-release, given higher rates of poverty, food insecurity, and substance dependence and lower educational attainment compared with men.MethodsWe describe gender differences in HIV risk behaviors using validated measures and assess potential mediators of this relationship using data from a cross-sectional study of 109 individuals recently released from prison.ResultsWomen had higher rates of HIV drug-related risk behaviors compared with men (mean score 2.72 vs. 0.068; p = .003) and HIV sex-related risk behaviors (mean score 4.32 vs. 2.31; p = .016). Women also had higher mean incomes and severity of drug and alcohol use compared with men, but equally high rates of food insecurity and low levels of AIDS knowledge. In multivariate analysis, the relationship between gender and HIV drug-related and sex-related risk behaviors was attenuated by a greater monthly income ([drug] adjusted β 0.82, 95% CI -1.02 – 2.66, p = 0.38; [sex] adjusted β 0.75, 95% CI -1.04 – 2.54, p = 0.41), as well as severity of drug use ([drug] adjusted β 0.79, 95% CI -0.55 – 2.13, p = 0.24; [sex] adjusted β 0.09, 95% CI -1.17 – 1.35, p = 0.89).ConclusionsWomen had higher rates of HIV risk behaviors compared with men post-release. Gender specific interventions may be useful in reducing risky drug-related and sex-related behaviors in the period immediately following release.