First-Line Ipatasertib, Atezolizumab, and Taxane Triplet for Metastatic Triple-Negative Breast Cancer: Clinical and Biomarker Results.

PURPOSE: To evaluate a triplet regimen combining immune checkpoint blockade, AKT pathway inhibition, and (nab-) paclitaxel as first-line therapy for locally advanced/metastatic triple-negative breast cancer (mTNBC). PATIENTS AND METHODS: The single-arm CO40151 phase Ib study (NCT03800836), the single-arm signal-seeking cohort of IPATunity130 (NCT03337724), and the randomized phase III IPATunity170 trial (NCT04177108) enrolled patients with previously untreated mTNBC. Triplet therapy comprised intravenous atezolizumab 840 mg (days 1 and 15), oral ipatasertib 400 mg/day (days 1-21), and intravenous paclitaxel 80 mg/m2 (or nab-paclitaxel 100 mg/m2; days 1, 8, and 15) every 28 days. Exploratory translational research aimed to elucidate mechanisms and molecular markers of sensitivity and resistance. RESULTS: Among 317 patients treated with the triplet, efficacy ranged across studies as follows: median progression-free survival (PFS) 5.4 to 7.4 months, objective response rate 44% to 63%, median duration of response 5.6 to 11.1 months, and median overall survival 15.7 to 28.3 months. The safety profile was consistent with the known toxicities of each agent. Grade ≥3 adverse events were more frequent with the triplet than with doublets or single-agent paclitaxel. Patients with PFS >10 months were characterized by NF1, CCND3, and PIK3CA alterations and increased immune pathway activity. PFS <5 months was associated with CDKN2A/CDKN2B/MTAP alterations and lower predicted phosphorylated AKT-S473 levels. CONCLUSIONS: In patients with mTNBC receiving an ipatasertib/atezolizumab/taxane triplet regimen, molecular characteristics may identify those with particularly favorable or unfavorable outcomes, potentially guiding future research efforts.

Insights into the Mechanism for Vertical Graphene Growth by Plasma-Enhanced Chemical Vapor Deposition.

Vertically oriented graphene (VG) has attracted attention for years, but the growth mechanism is still not fully revealed. The electric field may play a role, but the direct evidence and exactly what role it plays remains unclear. Here, we conduct a systematic study and find that in plasma-enhanced chemical vapor deposition, the VG growth preferably occurs at spots where the local field is stronger, for example, at GaN nanowire tips. On almost round-shaped nanoparticles, instead of being perpendicular to the substrate, the VG grows along the field direction, that is, perpendicular to the particles' local surfaces. Even more convincingly, the sheath field is screened to different degrees, and a direct correlation between the field strength and the VG growth is observed. Numerical calculation suggests that during the growth, the field helps accumulate charges on graphene, which eventually changes the cohesive graphene layers into separate three-dimensional VG flakes. Furthermore, the field helps attract charged precursors to places sticking out from the substrate and makes them even sharper and turn into VG. Finally, we demonstrate that the VG-covered nanoparticles are benign to human blood leukocytes and could be considered for drug delivery. Our research may serve as a starting point for further vertical two-dimensional material growth mechanism studies.

Multicenter Evaluation of Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy-Based Method for Rapid Identification of Clinically Relevant Yeasts.

Fourier transform infrared (FTIR) spectroscopy has demonstrated applicability as a reagent-free whole-organism fingerprinting technique for both microbial identification and strain typing. For routine application of this technique in microbiology laboratories, acquisition of FTIR spectra in the attenuated total reflectance (ATR) mode simplifies the FTIR spectroscopy workflow, providing results within minutes after initial culture without prior sample preparation. In our previous central work, 99.7% correct species identification of clinically relevant yeasts was achieved by employing an ATR-FTIR-based method and spectral database developed by our group. In this study, ATR-FTIR spectrometers were placed in 6 clinical microbiology laboratories over a 16-month period and were used to collect spectra of routine yeast isolates for on-site identification to the species level. The identification results were compared to those obtained from conventional biochemical tests and/or matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Isolates producing discordant results were reanalyzed by routine identification methods, ATR-FTIR spectroscopy, and PCR gene sequencing of the D1/D2 and internal transcribed spacer (ITS) regions. Among the 573 routine clinical yeast isolates collected and identified by the ATR-FTIR-based method, 564 isolates (98.4%) were correctly identified at the species level, while the remaining isolates were inconclusive with no misidentifications. Due to the low prevalence of Candida auris in routine isolates, additional randomly selected C. auris (n = 24) isolates were obtained for evaluation and resulted in 100% correct identification. Overall, the data obtained in our multicenter evaluation study using multiple spectrometers and system operators indicate that ATR-FTIR spectroscopy is a reliable, cost-effective yeast identification technique that provides accurate and timely (∼3 min/sample) species identification promptly after the initial culture.

Blastocyst Vitrification and Trophectoderm Biopsy Cumulatively Alter Embryonic Gene Expression in a Mouse Model.

Although embryo vitrification has been used extensively in human assisted reproductive technology (ART) and animal models, epidemiologic evidence and randomized controlled trials suggest differences in pregnancy/perinatal outcomes (birthweight, risk for preterm birth, and pre-eclampsia) between babies born from fresh versus frozen embryo transfers. To address the uncertainty surrounding the effects of laboratory manipulations of embryos on clinical outcomes, we subjected mouse blastocysts to increasing levels of manipulation for transcriptome analysis. Blastocysts were randomly divided into four groups: no manipulation (control), single vitrification/thaw (1 vit), double vitrification/thaw (2 vit), and single vitrification/thaw plus trophectoderm biopsy and again vitrified/thawed (2 vit + bx). Three sets of 15 blastocysts in each group were pooled for RNA sequencing, and differentially expressed genes (DEGs) and pathways were determined by statistical analysis. Blastocysts were also stained for ZO-1 and F-actin to assess cytoskeletal integrity. Freeze/thaw and biopsy manipulation affected multiple biological pathways. The most significant differences were detected in genes related to innate immunity, apoptosis, and mitochondrial function, with the magnitude of change proportional to the extent to manipulation. Significant disruptions were also seen in cytoskeletal staining, with greater disruptions seen with greater of manipulation. Our data suggests that embryo vitrification and biopsy affect embryo gene transcription, with several identified DEGs that may have plausible mechanisms for the clinical outcomes seen in human offspring following ART. Further study is required to determine whether these alterations in gene expression are associated with clinical differences seen in children born from fresh or frozen embryo transfer.

A phase I study of pharmacokinetics of trastuzumab emtansine in Chinese patients with locally advanced inoperable or metastatic human epidermal growth factor receptor 2-positive breast cancer who have received prior trastuzumab-based therapy.

BACKGROUND: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that retains the antitumor effects of trastuzumab while also delivering the cytotoxic antimicrotubule agent, DM1, directly to tumor cells that overexpress human epidermal growth factor receptor 2. The pharmacokinetic (PK) profile of T-DM1 has been well characterized in Western, Asian, and Japanese patients; this single-center, phase I study (NCT03153163) examined the PK of T-DM1 and safety specifically in Chinese patients. METHODS: Patients with locally advanced or metastatic breast cancer, previously treated with trastuzumab and a taxane, received open-label T-DM1 at 3.6 mg/kg every 3 weeks. Serum T-DM1 and total trastuzumab, and plasma DM1 were evaluated, and PK parameters were calculated using standard noncompartmental approaches. Adverse events (AEs) were assessed, and immunogenicity was evaluated by measuring antidrug antibodies to T-DM1. RESULTS: Among 11 Chinese patients, mean (±standard deviation) PK parameters (maximum serum concentration, 77.6 ±â€Š17.4 µg/mL; clearance 11.0 ±â€Š2.6 mL/d/kg; terminal half-life 3.8 ±â€Š1.0 days) were similar to those previously reported in Western and Japanese patients. One patient transiently developed antidrug antibodies, which did not appear to influence safety or PK. T-DM1 was generally well tolerated. Grade 3-4 AEs occurred in 7 patients (63.6%) and serious AEs occurred in 4 patients (36.4%). Platelet count decrease was the most common all-grade AE (10/11; 90.9%), grade 3-4 AE (5/11; 45.5%), and serious AE (3/11; 27.3%), but did not appear to be associated with any clinically significant bleeding events. CONCLUSIONS: T-DM1 PK in Chinese patients was consistent with those in global and Asian populations, supporting its use in patients with advanced human epidermal growth factor receptor 2-positive breast cancer following progression on trastuzumab and a taxane. The safety profile of T-DM1 was consistent with prior experience.

Reagent-Free Identification of Clinical Yeasts by Use of Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy.

Invasive fungal infections by opportunistic yeasts have increased concomitantly with the growth of an immunocompromised patient population. Misidentification of yeasts can lead to inappropriate antifungal treatment and complications. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy is a promising method for rapid and accurate identification of microorganisms. ATR-FTIR spectroscopy is a standalone, inexpensive, reagent-free technique that provides results within minutes after initial culture. In this study, a comprehensive spectral reference database of 65 clinically relevant yeast species was constructed and tested prospectively on spectra recorded (from colonies taken from culture plates) for 318 routine yeasts isolated from various body fluids and specimens received from 38 microbiology laboratories over a 4-month period in our clinical laboratory. ATR-FTIR spectroscopy attained comparable identification performance with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In a preliminary validation of the ATR-FTIR method, correct identification rates of 100% and 95.6% at the genus and species levels, respectively, were achieved, with 3.5% unidentified and 0.9% misidentified. By expanding the number of spectra in the spectral reference database for species for which isolates could not be identified or had been misidentified, we were able to improve identification at the species level to 99.7%. Thus, ATR-FTIR spectroscopy provides a new standalone method that can rival MALDI-TOF MS for the accurate identification of a broad range of medically important yeasts. The simplicity of the ATR-FTIR spectroscopy workflow favors its use in clinical laboratories for timely and low-cost identification of life-threatening yeast strains for appropriate treatment.

Trastuzumab Emtansine (T-DM1) in Patients with Previously Treated HER2-Overexpressing Metastatic Non-Small Cell Lung Cancer: Efficacy, Safety, and Biomarkers.

PURPOSE: HER2-targeted therapy is not standard of care for HER2-positive non-small cell lung cancer (NSCLC). This phase II study investigated efficacy and safety of the HER2-targeted antibody-drug conjugate trastuzumab emtansine (T-DM1) in patients with previously treated advanced HER2-overexpressing NSCLC. PATIENTS AND METHODS: Eligible patients had HER2-overexpressing NSCLC (centrally tested IHC) and received previous platinum-based chemotherapy and targeted therapy in the case of EGFR mutation or ALK gene rearrangement. Patients were divided into cohorts based on HER2 IHC (2+, 3+). All patients received T-DM1 3.6 mg/kg intravenously every 3 weeks until disease progression or unacceptable toxicity. The primary endpoint was investigator-determined overall response rate (ORR) using RECIST v1.1. RESULTS: Forty-nine patients received T-DM1 (29 IHC 2+, 20 IHC 3+). No treatment responses were observed in the IHC 2+ cohort. Four partial responses were observed in the IHC 3+ cohort (ORR, 20%; 95% confidence interval, 5.7%-43.7%). Clinical benefit rates were 7% and 30% in the IHC 2+ and 3+ cohorts, respectively. Response duration for the responders was 2.9, 7.3, 8.3, and 10.8 months. Median progression-free survival and overall survival were similar between cohorts. Three of 4 responders had HER2 gene amplification. No new safety signals were observed. CONCLUSIONS: T-DM1 showed a signal of activity in patients with HER2-overexpressing (IHC 3+) advanced NSCLC. Additional investigation into HER2 pathway alterations is needed to refine the target population for T-DM1 in NSCLC; however, HER2 IHC as a single parameter was an insufficient predictive biomarker.

Trastuzumab Emtansine for Residual Invasive HER2-Positive Breast Cancer.

BACKGROUND: Patients who have residual invasive breast cancer after receiving neoadjuvant chemotherapy plus human epidermal growth factor receptor 2 (HER2)-targeted therapy have a worse prognosis than those who have no residual cancer. Trastuzumab emtansine (T-DM1), an antibody-drug conjugate of trastuzumab and the cytotoxic agent emtansine (DM1), a maytansine derivative and microtubule inhibitor, provides benefit in patients with metastatic breast cancer that was previously treated with chemotherapy plus HER2-targeted therapy. METHODS: We conducted a phase 3, open-label trial involving patients with HER2-positive early breast cancer who were found to have residual invasive disease in the breast or axilla at surgery after receiving neoadjuvant therapy containing a taxane (with or without anthracycline) and trastuzumab. Patients were randomly assigned to receive adjuvant T-DM1 or trastuzumab for 14 cycles. The primary end point was invasive disease-free survival (defined as freedom from ipsilateral invasive breast tumor recurrence, ipsilateral locoregional invasive breast cancer recurrence, contralateral invasive breast cancer, distant recurrence, or death from any cause). RESULTS: At the interim analysis, among 1486 randomly assigned patients (743 in the T-DM1 group and 743 in the trastuzumab group), invasive disease or death had occurred in 91 patients in the T-DM1 group (12.2%) and 165 patients in the trastuzumab group (22.2%). The estimated percentage of patients who were free of invasive disease at 3 years was 88.3% in the T-DM1 group and 77.0% in the trastuzumab group. Invasive disease-free survival was significantly higher in the T-DM1 group than in the trastuzumab group (hazard ratio for invasive disease or death, 0.50; 95% confidence interval, 0.39 to 0.64; P<0.001). Distant recurrence as the first invasive-disease event occurred in 10.5% of patients in the T-DM1 group and 15.9% of those in the trastuzumab group. The safety data were consistent with the known safety profile of T-DM1, with more adverse events associated with T-DM1 than with trastuzumab alone. CONCLUSIONS: Among patients with HER2-positive early breast cancer who had residual invasive disease after completion of neoadjuvant therapy, the risk of recurrence of invasive breast cancer or death was 50% lower with adjuvant T-DM1 than with trastuzumab alone. (Funded by F. Hoffmann-La Roche/Genentech; KATHERINE ClinicalTrials.gov number, NCT01772472 .).

Evaluation of the effect of new formulation, food, or a proton pump inhibitor on the relative bioavailability of the smoothened inhibitor glasdegib (PF-04449913) in healthy volunteers.

PURPOSE: This phase I open-label study investigated the oral bioavailability of two novel maleate salt-based glasdegib (PF-04449913) tablet formulations (small- and large-particle size) relative to the current clinical formulation (diHCl salt-based). In addition, the effect of a gastric pH-altering agent (rabeprazole) and food on the pharmacokinetics of the large-particle size formulation of glasdegib were evaluated. The pharmacokinetics of glasdegib oral solution was also assessed. METHODS: Thirty-four healthy subjects received glasdegib 100 mg as three different formulations in the fasted state (diHCl salt or small- or large-particle size maleate formulation); 13 received the large-particle maleate formulation (fed), and 14 concurrently with rabeprazole (fasted); six subjects received glasdegib 50 mg oral solution (fasted). RESULTS: For both new tablet formulations of glasdegib, ratios (Test:Reference) of adjusted geometric means (90% confidence interval) of area under the concentration-time curve from 0 to infinity and maximum plasma concentration were within 80-125% compared with the diHCl formulation (fasted). For the large-particle size formulation (fed), these ratios were 86.3% (81.0-92.0%) and 75.7% (65.3-87.7%), respectively, compared with fasted. When the large-particle maleate formulation was administered concurrently with rabeprazole versus alone (fasted), these ratios were 111.9% (102.8-121.9%) and 87.2% (75.9-100.3%), respectively. The pharmacokinetics of oral solution was similar to the tablet. CONCLUSIONS: The maleate salt-based tablet formulations were bioequivalent to the diHCl tablet formulation. The extent of the observed effect of a high-fat, high-calorie meal or concurrent rabeprazole treatment on glasdegib exposure is not considered clinically meaningful.

Adrenal Suppression With Chronic Topical Corticosteroid Use in Psoriasis Patients.

BACKGROUND: Topical corticosteroids (TCS) are typically used for extended periods of time for chronic skin conditions, including psoriasis. Chronic TCS use may result in side effects similar to those of systemic corticosteroids. Patients may have subclinical adrenal suppression and be unaware of their risk in the case of serious trauma.
OBJECTIVE: The objective of this study was to investigate the real world effects of chronic TCS use and its effects on adrenal suppression in a chronic disease such as psoriasis.
MATERIALS: This retrospective study utilized data from screening visits of a psoriasis clinical trial in which subjects had been on chronic TCS.
RESULTS: In this study, subjects with moderate to severe psoriasis affecting 16-20% of total body surface area (BSA) and using high-potency TCS at screening had a lower post-cosyntropin cortisol level (18.83 mcg/dL) compared to those with moderate psoriasis involving 10-15% of total BSA and using lower potency TCS at screening (23.22 mcg/dL; P=0.03). Both subject groups had lower post-cosyntropin cortisol levels compared to normal, healthy adults (P<0.001 for both).
CONCLUSION: This suggests that real world chronic use of high potency TCS over a larger BSA may result in silent adrenal suppression.

J Drugs Dermatol. 2016;15(8):945-948.

Association of concurrent acid-suppression therapy with survival outcomes and adverse event incidence in oncology patients receiving erlotinib.

PURPOSE: Acid-suppression therapy is known to decrease the systemic exposure of erlotinib. The erlotinib prescribing information recommends staggering dosing with a histamine-2 receptor antagonist (H2RA) and avoiding concurrent use of a proton pump inhibitor (PPI). This retrospective analysis evaluated the frequency of concurrent acid-suppression therapy in oncology patients receiving erlotinib and its association with outcomes. METHODS: All patients prescribed erlotinib within UC San Diego Health System between February 26, 2011, and February 28, 2014, were assessed for eligibility, survival outcomes and adverse events. RESULTS: Of the 76 patients in the analysis, 24 were prescribed both a PPI and an H2RA with erlotinib therapy (31.6 %). The two patient groups, with (n = 24) and without PPI/H2RA (n = 52), were similar in clinical characteristics and erlotinib dose. One patient received an H2RA therapy alone and was excluded from the analysis; no one received PPI therapy alone. Patients receiving erlotinib alone had a longer median progression-free survival (PFS) compared to patients with concurrent PPI/H2RA therapy (11.0 months vs. 5.3 months; P = 0.029). Overall survival (OS) and incidence of rash and/or diarrhea did not correlate with use of acid-suppression therapy. CONCLUSION: Nearly one-third of patients received acid-suppression therapy. Patients treated with erlotinib and PPI/H2RA therapy had shorter PFS, but similar OS and adverse event profile compared to those who did not receive acid-suppression.

A Case-Based Approach to Integrating Opioid Pharmacokinetic and Pharmacodynamic Concepts in Cancer Pain Management.

Opioids are prescribed for cancer pain. Over the past decade, the annual increase in opioid prescriptions has been accompanied by an increase in opioid-associated deaths. Health care professionals must be proficient in proper dosing, titrating, and monitoring of opioid medications. With the numerous opioid medications and formulations available, an understanding of pharmacokinetic (PK) and pharmacodynamic (PD) concepts is necessary to appropriately individualize opioid-based cancer pain regimens. The purpose of this review is to highlight PK/PD concepts that are clinically relevant to the use of opioids. By way of a cancer pain patient case scenario, PK/PD concepts that are relevant in the initiation, titration, and rotation of an opioid regimen are discussed.

ANTIOXIDANT CAPACITY OF WYOMING BIG SAGEBRUSH (ARTEMISIA TRIDENTATA SSP. WYOMINGENSIS) VARIES SPATIALLY AND IS NOT RELATED TO THE PRESENCE OF A SAGEBRUSH DIETARY SPECIALIST.

Sagebrush (Artemisia spp.) in North America is an abundant native plant species that is ecologically and evolutionarily adapted to have a diverse array of biologically active chemicals. Several of these chemicals, specifically polyphenols, have antioxidant activity that may act as biomarkers of biotic or abiotic stress. This study investigated the spatial variation of antioxidant capacity, as well as the relationship between a mammalian herbivore and antioxidant capacity in Wyoming big sagebrush (Artemisia tridentata wyomingensis). We quantified and compared total polyphenols and antioxidant capacity of leaf extracts from sagebrush plants from different spatial scales and at different levels of browsing by a specialist mammalian herbivore, the pygmy rabbit (Brachylagus idahoensis). We found that antioxidant capacity of sagebrush extracts was positively correlated with total polyphenol content. Antioxidant capacity varied spatially within and among plants. Antioxidant capacity in sagebrush was not related to either browsing intensity or duration of association with rabbits. We propose that the patterns of antioxidant capacity observed in sagebrush may be a result of spatial variation in abiotic stress experienced by sagebrush. Antioxidants could therefore provide a biomarker of environmental stress for sagebrush that could aid in management and conservation of this plant in the threatened sagebrush steppe.

Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice.

This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.

Intravital imaging of podocyte calcium in glomerular injury and disease.

Intracellular calcium ([Ca²âº]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²âº]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²âº]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²âº]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²âº]i around the injury site and promoted cell-to-cell propagating podocyte [Ca²âº]i waves along capillary loops. [Ca²âº]i wave propagation was ameliorated by inhibitors of purinergic [Ca²âº]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²âº]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²âº]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²âº]i in glomerular pathology and suggest that purinergic [Ca²âº]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags.

Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PECs) in vivo. In podocin-GFP mice, podocytes formed sporadic multicellular clusters after unilateral ureteral ligation and migrated into the parietal Bowman's capsule. The tracking of single cells in podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP or RFP revealed the simultaneous migration of multiple podocytes. In phosphoenolpyruvate carboxykinase (PEPCK)-GFP mice, serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support a highly dynamic rather than a static nature of the glomerular environment and cellular composition. Future application of this new approach should advance our understanding of the mechanisms of glomerular injury and regeneration.

Localization and proliferation of lymphatic vessels in the tympanic membrane in normal state and regeneration.

We clarified the localization of lymphatic vessels in the tympanic membrane and proliferation of lymphatic vessels during regeneration after perforation of the tympanic membrane by using whole-mount imaging of the tympanic membrane of Prox1 GFP mice. In the pars tensa, lymphatic vessel loops surrounded the malleus handle and annulus tympanicus. Apart from these locations, lymphatic vessel loops were not observed in the pars tensa in the normal tympanic membrane. Lymphatic vessel loops surrounding the malleus handle were connected to the lymphatic vessel loops in the pars flaccida and around the tensor tympani muscle. Many lymphatic vessel loops were detected in the pars flaccida. After perforation of the tympanic membrane, abundant lymphatic regeneration was observed in the pars tensa, and these regenerated lymphatic vessels extended from the lymphatic vessels surrounding the malleus at day 7. These results suggest that site-specific lymphatic vessels play an important role in the tympanic membrane.

Angiotensin receptor-mediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats.

Activation of angiotensin receptor type 1 (AT1) contributes to NADPH oxidase (Nox)-derived oxidative stress during metabolic syndrome. However, the specific role of AT1 in modulating redox signaling, mitochondrial function, and oxidative stress in the heart remains more elusive. To test the hypothesis that AT1 activation increases oxidative stress while impairing redox signaling and mitochondrial function in the heart during diet-induced insulin resistance in obese animals, Otsuka Long Evans Tokushima Fatty (OLETF) rats (n = 8/group) were treated with the AT1 blocker (ARB) olmesartan for 6 wk. Cardiac Nox2 protein expression increased 40% in OLETF compared with age-matched, lean, strain-control Long Evans Tokushima Otsuka (LETO) rats, while mRNA and protein expression of the H2O2-producing Nox4 increased 40-100%. ARB treatment prevented the increase in Nox2 without altering Nox4. ARB treatment also normalized the increased levels of protein and lipid oxidation (nitrotyrosine, 4-hydroxynonenal) and increased the redox-sensitive transcription factor Nrf2 by 30% and the activity of antioxidant enzymes (SOD, catalase, GPx) by 50-70%. Citrate synthase (CS) and succinate dehydrogenase (SDH) activities decreased 60-70%, whereas cardiac succinate levels decreased 35% in OLETF compared with LETO, suggesting that mitochondrial function in the heart is impaired during obesity-induced insulin resistance. ARB treatment normalized CS and SDH activities, as well as succinate levels, while increasing AMPK and normalizing Akt, suggesting that AT1 activation also impairs cellular metabolism in the diabetic heart. These data suggest that the cardiovascular complications associated with metabolic syndrome may result from AT1 receptor-mediated Nox2 activation leading to impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart.

Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins.

Budding yeast septins assemble into hetero-octamers and filaments required for cytokinesis. Solvent-exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site-directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid-expressed, each Cys-less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys-less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys-less septins were co-expressed as the sole source of these proteins. Cells grew less well when three Cys-less septins were co-expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys-less Cdc10, Cdc11, and Cdc12, and expressing Cys-less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys-less septins--or where one of the Cys-less septins contained a single Cys introduced at a new site--displayed assembly properties in vitro indistinguishable from wild-type.

Metabolic control of renin secretion.

One emerging topic in renin-angiotensin system (RAS) research is the direct local control of renin synthesis and release by endogenous metabolic intermediates. During the past few years, our laboratory has characterized the localization and signaling of the novel metabolic receptor GPR91 in the normal and diabetic kidney and established GPR91 as a new, direct link between high glucose and RAS activation in diabetes. GPR91 (also called SUCNR1) binds tricarboxylic acid (TCA) cycle intermediate succinate which can rapidly accumulate in the local tissue environment when energy supply and demand are out of balance. In a variety of physiological and pathological conditions associated with metabolic stress, succinate signaling via GPR91 appears to be an important mediator or modulator of renin secretion. This review summarizes our current knowledge on the control of renin release by molecules of endogenous metabolic pathways with the main focus on succinate/GPR91.