Dry eye disease in mice activates adaptive corneal epithelial regeneration distinct from constitutive renewal in homeostasis.

Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.

Dysregulated CD200-CD200R signaling in early diabetes modulates microglia-mediated retinopathy.

Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.

Histamine-releasing factor in severe asthma and rhinovirus-associated asthma exacerbation.

BACKGROUND: Histamine-releasing factor (HRF) is implicated in allergic diseases. We previously showed its pathogenic role in murine models of asthma. OBJECTIVE: We aim to present data analysis from 3 separate human samples (sera samples from asthmatic patients, nasal washings from rhinovirus [RV]-infected individuals, and sera samples from patients with RV-induced asthma exacerbation) and 1 mouse sample to investigate correlates of HRF function in asthma and virus-induced asthma exacerbations. METHODS: Total IgE and HRF-reactive IgE/IgG as well as HRF in sera from patients with mild/moderate asthma or severe asthma (SA) and healthy controls (HCs) were quantified by ELISA. HRF secretion in culture media from RV-infected adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells and in nasal washings from experimentally RV-infected subjects was analyzed by Western blotting. HRF-reactive IgE/IgG levels in longitudinal serum samples from patients with asthma exacerbations were also quantified. RESULTS: HRF-reactive IgE and total IgE levels were higher in patients with SA than in HCs, whereas HRF-reactive IgG (and IgG1) level was lower in asthmatic patients versus HCs. In comparison with HRF-reactive IgElow asthmatic patients, HRF-reactive IgEhigh asthmatic patients had a tendency to release more tryptase and prostaglandin D2 on anti-IgE stimulation of bronchoalveolar lavage cells. RV infection induced HRF secretion from adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells, and intranasal RV infection of human subjects induced increased HRF secretion in nasal washes. Asthmatic patients had higher levels of HRF-reactive IgE at the time of asthma exacerbations associated with RV infection, compared with those after the resolution. This phenomenon was not seen in asthma exacerbations without viral infections. CONCLUSIONS: HRF-reactive IgE is higher in patients with SA. RV infection induces HRF secretion from respiratory epithelial cells both in vitro and in vivo. These results suggest the role of HRF in asthma severity and RV-induced asthma exacerbation.

Pandemic Recovery: Persistent Disparities in Access to Elective Surgical Procedures.

OBJECTIVE: To examine potential disparities in patient access to elective procedures during the recovery phase of the COVID-19 pandemic. SUMMARY OF BACKGROUND DATA: Elective surgeries during the pandemic were limited acutely. Access to surgical care was restored in a recovery phase but backlogs and societal shifts are hypothesized to impact surgical access. METHODS: Adults with electronic health record orders for procedures ("procedure requests"), from March 16 to August 25, 2019 and March 16 to August 25, 2020, were included. Logistic regression was performed for requested procedures that were not scheduled. Linear regression was performed for wait time from request to scheduled or completed procedure. RESULTS: The number of patients with procedure requests decreased 20.8%, from 26,789 in 2019 to 21,162 in 2020. Patients aged 36-50 and >65 years, those speaking non-English languages, those with Medicare or no insurance, and those living >100 miles away had disproportionately larger decreases. Requested procedures had significantly increased adjusted odds ratios (aORs) of not being scheduled for patients with primary languages other than English, Spanish, or Cantonese [aOR 1.60, 95% confidence interval (CI) 1.12-2.28]; unpartnered marital status (aOR 1.21, 95% CI 1.07-1.37); uninsured or self-pay (aOR 2.03, 95% CI 1.53-2.70). Significantly longer wait times were seen for patients aged 36-65 years; with Medi-Cal insurance; from ZIP codes with lower incomes; and from ZIP codes >100 miles away. CONCLUSIONS: Patient access to elective surgeries decreased during the pandemic recovery phase with disparities based on patient age, language, marital status, insurance, socioeconomic status, and distance from care. Steps to address modifiable disparities have been taken.

Accurate Identification of Mucinous Pancreatic Cystic Lesions Using Small-Volume Analytes.

INTRODUCTION: The accurate identification of mucinous pancreatic cystic lesions (PCLs) is paramount for cancer risk stratification. Cyst fluid carcinoembryonic antigen (CEA), the only routinely used test, requires high volumes and has low sensitivity. We aimed to compare the performance of two investigational small-volume biomarkers, glucose and the protease gastricsin, to CEA for PCL classification. METHODS: We obtained cyst fluid samples from 81 patients with pathologically confirmed PCLs from four institutions between 2003 and 2016. Gastricsin activity was measured using an internally quenched fluorescent substrate. Glucose levels were measured with a standard glucometer. CEA levels were obtained from the medical record. Models using Classification and Regression Trees were created to predict mucinous status. Model performance was evaluated using nested cross-validation. RESULTS: Gastricsin activity, CEA, and glucose levels from patients with mucinous (n = 50) and nonmucinous (n = 31) PCLs were analyzed. Area under the curve (AUC) was similar for individual classifiers (gastricsin volume normalized [GVN] 0.88; gastricsin protein concentration normalized [GPN] 0.95; glucose 0.83; CEA 0.84). The combination of two classifiers did not significantly improve AUC, with CEA + GVN (0.88) performing similarly to CEA + GPN (0.95), GVN + glucose (0.87), GPN + glucose (0.95), and CEA + glucose (0.84). The three-analyte combination performed similarly to single and dual classifiers (GPN + glucose + CEA AUC 0.95; GVN + glucose + CEA AUC 0.87). After multiple comparison corrections, there were no significant differences between the individual, dual, and triple classifiers. CONCLUSIONS: Gastricsin and glucose performed similarly to CEA and required <5% of the volume required for CEA; these classifiers may be useful in patients with limited cyst fluid. Future multicenter prospective studies are needed to validate and compare these novel small-volume biomarkers.

Enhancing robotic efficiency through the eyes of robotic surgeons: sub-analysis of the expertise in perception during robotic surgery (ExPeRtS) study.

BACKGROUND: Robotic technology affords surgeons many novel and useful features, but two stereotypes continue to prevail: robotic surgery is expensive and inefficient. To identify educational opportunities and improve operative efficiency, we analyzed expert commentary on videos of robotic surgery. METHODS: Expert robotic surgeons, identified through high case volumes and contributions to the surgical literature, reviewed eight anonymous video clips portraying key portions of two robotic general surgery procedures. While watching, surgeons commented on what they saw on the screen. All interactions with participants were in person, recorded, transcribed, and subsequently analyzed. Using content analysis, researchers double-coded each transcript applying a consensus developed codebook. RESULTS: Seventeen surgeons participated. The average participant was male (82.4%), 47 (SD = 6.6) years old, had 13.2 (SD = 8.23) years of teaching experience, worked in urban academic hospitals (64.7%) and had performed 643 (SD = 467) robotic operations at the time of interviews. Emphasis on efficiency (or lack thereof) surfaced across three main themes: overall case progression, robotic capabilities, and instrumentation. Experts verbally rewarded purposeful and "ergonomically sound" movements while language reflecting impatience with repetitive and indecisive movements was attributed to presumed inexperience. Efficient robotic capabilities included enhanced visualization, additional robotic arms to improve exposure, and wristed instruments. Finally, experts discussed instrument selection with regards to energy modality, safety features, cost, and versatility. CONCLUSION: This study highlights three areas for improved efficiency: case progression, robotic capabilities, and instrumentation. Development of education materials within these themes could help surgical educators overcome one of robotic technology's persistent challenges.

Determining Hospital Volume Threshold for Safety of Minimally Invasive Pancreaticoduodenectomy: A Contemporary Cutpoint Analysis.

BACKGROUND: Guidelines recommend limiting minimally invasive pancreaticoduodenectomy (MIPD) to high-volume centers. However, the definition of high-volume care remains unclear. We aimed to objectively define a minimum number of MIPD performed annually per hospital associated with improved outcomes in a contemporary patient cohort. PATIENTS AND METHODS: Resectable pancreatic adenocarcinoma patients undergoing MIPD were included from the National Cancer Database (2010-2017). Multivariable modeling with restricted cubic splines was employed to identify an MIPD annual hospital volume threshold associated with lower 90-day mortality. Outcomes were compared between patients treated at low-volume (≤ model-identified cutoff) and high-volume (> cutoff) centers. RESULTS: Among 3079 patients, 141 (5%) died within 90 days. Median hospital volume was 6 (range 1-73) cases/year. After adjustment, increasing hospital volume was associated with decreasing 90-day mortality for up to 19 (95% CI 16-25) cases/year, indicating a threshold of 20 cases/year. Most cases (82%) were done at low-volume (< 20 cases/year) centers. With adjustment, MIPD at low-volume centers was associated with increased 90-day mortality (OR 2.7; p = 0.002). Length of stay, positive surgical margins, 30-day readmission, and overall survival were similar. On analysis of the most recent two years (n = 1031), patients at low-volume centers (78.2%) were younger and had less advanced tumors but had longer length of stay (8 versus 7 days; p < 0.001) and increased 90-day mortality (7% versus 2%; p = 0.009). CONCLUSIONS: The cutpoint analysis identified a threshold of at least 20 MIPD cases/year associated with lower postoperative mortality. This threshold should inform national guidelines and institution-level protocols aimed at facilitating the safe implementation of this complex procedure.

Long-Term Outcomes of Laparoscopic Greater Curvature Plication and Laparoscopic Sleeve Gastrectomy: Critical Appraisal of the Role of Gastric Plication in Bariatric Surgery.

Background and Objectives: This single-center study aimed to assess the role of laparoscopic greater curvature plication (LGCP) in bariatric surgery. Materials and Methods: Using data from our institution's prospectively maintained database, we identified adult patients with obesity who underwent either laparoscopic sleeve gastrectomy (LSG) or LGCP between January 2012 and July 2017. In total, 280 patients were enrolled in this study. Results: The body mass index was higher in the LSG group than in the LGCP group (39.3 vs. 33.3, p < 0.001). Both groups achieved significant weight loss during the 3-year follow-up (p < 0.001). The weight-reduction rate was higher in the LSG group than in the LGCP group 6, 12, and 24 months postoperatively (p = 0.001, 0.001, and 0.012, respectively). The reoperation rate of the LGCP group was higher than that of the LSG group (p = 0.001). No deaths were recorded in either group. Conclusions: Although both the LGCP and LSG groups achieved significant weight loss over three years, the LGCP group demonstrated a lower weight-reduction rate and a higher reoperation rate than the LSG group. Thus, it is necessary to reassess the role of LGCP in bariatric surgery, particularly when LSG is a feasible alternative.

Development of AITC-induced dermal blood flow as a translational in vivo biomarker of TRPA1 activity in human and rodent skin.

BACKGROUND AND PURPOSE: Develop a translational assay of Transient Receptor Potential Ankyrin 1 (TRPA1) activity for use as a preclinical and clinical biomarker. EXPERIMENTAL APPROACH: Allyl isothiocyanate (AITC), capsaicin or citric acid were applied to ears of wildtype and Trpa1-knock out (Trpa1 KO) rats, and changes in dermal blood flow (DBF) were measured by laser speckle contrast imaging. In humans, the DBF, pain and itch responses to 5-20% AITC applied to the forearm were measured and safety was evaluated. Reproducibility of the DBF, pain and itch responses to topically applied 10% and 15% AITC were assessed at two visits separated by 13-15 days. DBF changes were summarized at 5-minute intervals as areas under the curve (AUC) and maxima. Intraclass correlation coefficient (ICC) was calculated to assess arm-arm and period-period reproducibility. KEY RESULTS: AITC- and citric acid-induced DBF were significantly reduced in Trpa1 KO rats compared to wildtype (90 ± 2% and 65 ± 11% reduction, respectively), whereas capsaicin response did not differ. In humans, each AITC concentration significantly increased DBF compared to vehicle with the maximal increase occurring 5 minutes post application. Ten percent and 15% AITC were selected as safe and effective stimuli. AUC from 0 to 5 minutes was the most reproducible metric of AITC-induced DBF across arms (ICC = 0.92) and periods (ICC = 0.85). Subject-reported pain was more reproducible than itch across visits (ICC = 0.76 vs 0.17, respectively). CONCLUSION AND IMPLICATIONS: AITC-induced DBF is a suitable target engagement biomarker of TRPA1 activity for preclinical and clinical studies of TRPA1 antagonists.

Long-term Lower Extremity and Cardiovascular Complications after Simultaneous Pancreas-Kidney Transplant.

Lower extremity (LE) vascular disease and adverse cardiovascular events (ACEs) cause significant long-term morbidity after simultaneous pancreas-kidney (SPK) transplantation. This study's purpose was to describe the incidence of, and risk factors associated with, LE vascular complications and related ACEs following SPK. All SPKs performed at the authors' institution from 2000 to 2019 were retrospectively analyzed. The primary outcome was any LE vascular event, defined as LE endovascular intervention, open surgery, amputation, or invasive podiatry intervention. Secondary outcomes included post-SPK ACE. A total of 363 patients were included, of whom 54 (14.9%) required at least one LE vascular intervention following SPK. Only 3 patients received pre-SPK ankle brachial indices (ABIs). A history of peripheral artery disease (PAD) (HR 2.95, CI 1.4-6.2) was a risk factor for post-SPK LE vascular intervention even after adjustment for other factors. Fifty-nine (16.3%) patients experienced an ACE in follow-up. Requiring a LE intervention post-SPK was associated with a subsequent ACE (HR 2.3, CI 1.2-4.5). LE vascular and cardiovascular complications continue to be significant sources of morbidity for SPK patients, especially for patients with preexisting PAD. The highest risk patients may benefit from more intensive pre- and post-SPK workup with ABIs and follow-up with a vascular surgeon.

The reactions of hydropersulfides (RSSH) with myoglobin.

Hydropersulfides are reported to be good biological reductants, superior to thiols and akin to selenols. As such, they have been previously shown to reduce metalloproteins such as ferric myoglobin and ferric cytochrome c to their ferrous forms under conditions where little or no reduction from corresponding thiols is observed. Not surprisingly, the reduction of ferric myoglobin to ferrous myoglobin under aerobic conditions results in the generation of oxymyoglobin (dioxygen bound ferrous myoglobin). Previous studies have demonstrated that oxymyoglobin can also act as an oxidant with highly reducing species such as hydroxylamine and ascorbate. Considering the reducing properties of hydropersulfides, it is possible that they can also react with oxymyoglobin similarly to hydroxylamine or ascorbate. Herein, this reaction is examined and indeed hydropersulfides are found to react with oxymyoglobin similarly to other reducing species leading to a fleeting ferric myoglobin which is rapidly reduced to the ferrous form also by hydropersulfide.

Association of surgical margins with local recurrence in patients undergoing breast-conserving surgery after neoadjuvant chemotherapy.

BACKGROUND: The aim of the current study was to report a single-institution experience using breast-conserving surgery after neoadjuvant chemotherapy (NACT), focusing on the association between microscopic resection margin status and locoregional recurrence (LRR). METHODS: Our institutional prospectively maintained database was reviewed to identify patients who were treated with NACT between January 2008 and April 2018. RESULTS: Among the main partial mastectomy specimens available for analysis (n = 161), 28 had margins < 1 mm, 21 had margin width of 1-2 mm and the remaining 112 had margins > 2 mm. LRR occurred in 16 patients (9.9%) and distant metastases were detected in 27 (16.8%) patients. There was no significant difference in the LRR between the > 2 mm margin group with a 60-month cumulative survival of 85.2% compared with 76.2% for the ≤2 mm group (P = 0.335) in the Kaplan-Meier analysis. When we stratified patients by margin widths of ≥1 mm or <  1 mm, there was no LRR-free survival benefit observed for the ≥1 mm pathologic excision margin group in the univariate analysis (hazard ratio = 0.443; 95% confidence interval = 0.142-1.383; P = 0.161) with a 60-month cumulative LRR-free survival of 84.9% compared with 69.5% for the < 1 mm margin cohort (P = 0.150). CONCLUSIONS: In the absence of multiple scattered microscopic tumour foci, a negative margin of no ink on tumour maybe sufficient for stage I-III invasive breast cancer treated with NACT and breast-conserving surgery.

Cysteine Trisulfide Protects E. coli from Electrophile-Induced Death through the Generation of Cysteine Hydropersulfide.

Hydropersulfide and polysulfide species have recently been shown to elicit a wide variety of biological and physiological responses. In this study, we examine the effects of cysteine trisulfide (Cys-SSS-Cys; also known as thiocystine) treatment on E. coli. Previous studies in mammalian cells have shown that Cys-SSS-Cys treatment results in protection from the electrophiles. Here, we show that the protective effect of Cys-SSS-Cys treatment against electrophile-induced cell death is conserved in E. coli. This protection correlates with the rapid generation of cysteine hydropersulfide (Cys-SSH) in the culture media. We go on to demonstrate that an exogenous phosphatase expressed in E. coli, containing only a single catalytic cysteine, is protected from electrophile-induced inactivation in the presence of hydropersulfides. These data together demonstrate that E. coli can utilize Cys-SSS-Cys to generate Cys-SSH and that the Cys-SSH can protect cellular thiols from reactivity with the electrophiles.

Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

The Uptake and Release of Polysulfur Cysteine Species by Cells: Physiological and Toxicological Implications.

Hydropersulfides and related polysulfides have recently become topics of significant interest due to their physiological prevalence and proposed biological functions. Currently, examination of the effects of hydropersulfide treatment on cells is difficult due to their lack of inherent stability with respect to disproportionation. Herein, it is reported that the treatment of a variety of cell types with cysteine trisulfide (also known as thiocystine; Cys-SSS-Cys), results in an increase in intracellular hydropersulfide levels (e.g., cysteine hydropersulfide; Cys-SSH, and glutathione hydropersulfide; GSSH). Thus, Cys-SSS-Cys represents a possible pharmacological agent for examining the effects of hydropersulfides on cell function/viability. It has also been found that cells with increased intracellular hydropersulfide levels can export Cys-SSH into the extracellular media. Interestingly, the Cys-SSH is the major hydropersulfide exported by cells, although GSSH is the predominant intracellular species. The possible implications of cellular export are discussed.

Cytochrome P450 27A1 Deficiency and Regional Differences in Brain Sterol Metabolism Cause Preferential Cholestanol Accumulation in the Cerebellum.

Cytochrome P450 27A1 (CYP27A1 or sterol 27-hydroxylase) is a ubiquitous, multifunctional enzyme catalyzing regio- and stereospecific hydroxylation of different sterols. In humans, complete CYP27A1 deficiency leads to cerebrotendinous xanthomatosis or nodule formation in tendons and brain (preferentially in the cerebellum) rich in cholesterol and cholestanol, the 5α-saturated analog of cholesterol. In Cyp27a1-/- mice, xanthomas are not formed, despite a significant cholestanol increase in the brain and cerebellum. The mechanism behind cholestanol production has been clarified, yet little is known about its metabolism, except that CYP27A1 might metabolize cholestanol. It also is unclear why CYP27A1 deficiency results in preferential cholestanol accumulation in the cerebellum. We hypothesized that cholestanol might be metabolized by CYP46A1, the principal cholesterol 24-hydroxylase in the brain. We quantified sterols along with CYP27A1 and CYP46A1 in mouse models (Cyp27a1-/-, Cyp46a1-/-, Cyp27a1-/-Cyp46a1-/-, and two wild type strains) and human brain specimens. In vitro experiments with purified P450s were conducted as well. We demonstrate that CYP46A1 is involved in cholestanol removal from the brain and that several factors contribute to the preferential increase in cholestanol in the cerebellum arising from CYP27A1 deficiency. These factors include (i) low cerebellar abundance of CYP46A1 and high cerebellar abundance of CYP27A1, the lack of which probably selectively increases the cerebellar cholestanol production; (ii) spatial separation in the cerebellum of cholesterol/cholestanol-metabolizing P450s from a pool of metabolically available cholestanol; and (iii) weak cerebellar regulation of cholesterol biosynthesis. We identified a new physiological role of CYP46A1, an important brain enzyme and cytochrome P450 that could be activated pharmacologically.

WNT7A/B promote choroidal neovascularization.

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

Mapping of the Allosteric Site in Cholesterol Hydroxylase CYP46A1 for Efavirenz, a Drug That Stimulates Enzyme Activity.

Cytochrome P450 46A1 (CYP46A1) is a microsomal enzyme and cholesterol 24-hydroxylase that controls cholesterol elimination from the brain. This P450 is also a potential target for Alzheimer disease because it can be activated pharmacologically by some marketed drugs, as exemplified by efavirenz, the anti-HIV medication. Previously, we suggested that pharmaceuticals activate CYP46A1 allosterically through binding to a site on the cytosolic protein surface, which is different from the enzyme active site facing the membrane. Here we identified this allosteric site for efavirenz on CYP46A1 by using a combination of hydrogen-deuterium exchange coupled to MS, computational modeling, site-directed mutagenesis, and analysis of the CYP46A1 crystal structure. We also mapped the binding region for the CYP46A1 redox partner oxidoreductase and found that the allosteric and redox partner binding sites share a common border. On the basis of the data obtained, we propose the mechanism of CYP46A1 allostery and the pathway for the signal transmission from the P450 allosteric site to the active site.

Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.

The retina, a thin tissue in the back of the eye, has two apparent sources of cholesterol: in situ biosynthesis and cholesterol available from the systemic circulation. The quantitative contributions of these two cholesterol sources to the retinal cholesterol pool are unknown and have been determined in the present work. A new methodology was used. Mice were given separately deuterium-labeled drinking water and chow containing 0.3% deuterium-labeled cholesterol. In the retina, the rate of total cholesterol input was 21 µg of cholesterol/g retina • day, of which 15 µg of cholesterol/g retina • day was provided by local biosynthesis and 6 µg of cholesterol/g retina • day was uptaken from the systemic circulation. Thus, local cholesterol biosynthesis accounts for the majority (72%) of retinal cholesterol input. We also quantified cholesterol input to mouse brain, the organ sharing important similarities with the retina. The rate of total cerebral cholesterol input was 121 µg of cholesterol/g brain • day with local biosynthesis providing 97% of total cholesterol input. Our work addresses a long-standing question in eye research and adds new knowledge to the potential use of statins (drugs that inhibit cholesterol biosynthesis) as therapeutics for age-related macular degeneration, a common blinding disease.