Faculty Bibliography

The ovary contains oocytes within immature (primordial) follicles that are fixed in number at birth. Activation of follicles within this fixed pool causes an irreversible decline in reproductive capacity, known as the ovarian reserve, until menopause. Premenopausal women undergoing commonly used genotoxic (DNA-damaging) chemotherapy experience an accelerated loss of the ovarian reserve, leading to subfertility and infertility. Therefore, there is considerable interest but little effective progress in preserving ovarian function during chemotherapy. Here we show that blocking the kinase mammalian/mechanistic target of rapamycin (mTOR) with clinically available small-molecule inhibitors preserves ovarian function and fertility during chemotherapy. Using a clinically relevant mouse model of chemotherapy-induced gonadotoxicity by cyclophosphamide, and inhibition of mTOR complex 1 (mTORC1) with the clinically approved drug everolimus (RAD001) or inhibition of mTORC1/2 with the experimental drug INK128, we show that mTOR inhibition preserves the ovarian reserve, primordial follicle counts, serum anti-Mullerian hormone levels (a rigorous measure of the ovarian reserve), and fertility. Chemotherapy-treated animals had significantly fewer offspring compared with all other treatment groups, whereas cotreatment with mTOR inhibitors preserved normal fertility. Inhibition of mTORC1 or mTORC1/2 within ovaries was achieved during chemotherapy cotreatment, concomitant with preservation of primordial follicle counts. Importantly, our findings indicate that as little as a two- to fourfold reduction in mTOR activity preserves ovarian function and normal birth numbers. As everolimus is approved for tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer, these findings represent a potentially effective and readily accessible pharmacologic approach to fertility preservation during conventional chemotherapy.

BACKGROUND: The PGC-1alpha/PPAR axis has been proposed as a potential therapeutic target for several metabolic disorders. The aim was to evaluate the efficacy of the pan-PPAR agonist, bezafibrate, in tafazzin knockdown mice (TazKD), a mouse model of Barth syndrome that exhibits age-dependent dilated cardiomyopathy with left ventricular (LV) dysfunction. RESULTS: The effect of bezafibrate on cardiac function was evaluated by echocardiography in TazKD mice with or without beta-adrenergic stress. Adrenergic stress by chronic isoproterenol infusion exacerbates the cardiac phenotype in TazKD mice, significantly depressing LV systolic function by 4.5 months of age. Bezafibrate intake over 2 months substantially ameliorates the development of LV systolic dysfunction in isoproterenol-stressed TazKD mice. Without beta-adrenergic stress, TazKD mice develop dilated cardiomyopathy by 7 months of age. Prolonged treatment with suprapharmacological dose of bezafibrate (0.5% in rodent diet) over a 4-month period effectively prevented LV dilation in mice isoproterenol treatment. Bezafibrate increased mitochondrial biogenesis, however also promoted oxidative stress in cardiomyocytes. Surprisingly, improvement of systolic function in bezafibrate-treated mice was accompanied with simultaneous reduction of cardiolipin content and increase of monolysocardiolipin levels in cardiac muscle. CONCLUSIONS: Thus, we demonstrate that bezafibrate has a potent therapeutic effect on preventing cardiac dysfunction in a mouse model of Barth syndrome with obvious implications for treating the human disease. Additional studies are needed to assess the potential benefits of PPAR agonists in humans with Barth syndrome.

OBJECTIVE: Low to moderate inorganic arsenic (iAs) exposure is independently associated with cardiovascular disease (CVD), particularly for patients with diabetes mellitus (DM). The mechanism of increased CVD risk from iAs exposure in DM has not been adequately characterized. We evaluated whether increasing concentrations of glucose enhance the effects of iAs on platelet and megakaryocyte activity, key steps in atherothrombosis. METHODS: Healthy donor whole blood was prepared in a standard fashion and incubated with sodium arsenite in a range from 0 to 10 microM. iAs-induced platelet activation was assessed by platelet receptor CD62P (P-selectin) expression and monocyte-platelet and leukocyte-platelet aggregation (MPA and LPA, respectively) in the presence of increasing sodium arsenite and glucose concentrations. Megakaryocyte (Meg-01) cell adhesion and gene expression was assessed after incubation with or without iAs and increasing concentrations of D-glucose. RESULTS: Platelet activity markers increased significantly with 10 vs. 0 microM iAs (P < 0.05 for all) and with higher D-glucose concentrations. Platelet activity increased significantly following co incubation of 1 and 5 microM iAs concentrations with hyperglycemic D-glucose (P < 0.01 for both) but not after incubation with euglycemic D-glucose. Megakaryocyte adhesion was more pronounced after co incubation with iAs and hyperglycemic than euglycemic D-glucose, while gene expression increased significantly to iAs only after co incubation with hyperglycemic D-glucose. CONCLUSION: We demonstrate that glucose concentrations common in DM potentiate the effect of inorganic arsenic exposure on markers of platelet and megakaryocyte activity. Our results support recent observational cohort data that DM enhances the vasculotoxic effects of arsenic exposure, and suggest that activation of the platelet-megakaryocyte hemostatic axis is a pathway through which inorganic arsenic confers atherothrombotic risk, particularly for patients with DM.

Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8,706 proteins and 28,186 phosphopeptides, including 7,394 proteins and 23,739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 >/= 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide data sets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta, and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2 > 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics.

BACKGROUND: To some degree, acne vulgaris affects nearly every individual worldwide. Oral antibiotic therapy is routinely prescribed for the treatment of moderate to severe inflammatory acne; however, long-term use of oral antibiotics for acne may have unintended consequences. OBJECTIVE: The aim of this study was to provide a systematic evaluation of the scientific evidence on the efficacy and appropriate use of oral antibiotics in the treatment of acne. METHODS: A systematic search of MEDLINE was conducted to identify randomized controlled clinical trials, systematic reviews, and meta-analyses evaluating the efficacy of oral antibiotics for acne. Overall, 41 articles that examined oral antibiotics compared with placebo, another oral therapy, topical therapy, alternate dose, or duration were included in this study. RESULTS: Tetracyclines, macrolides, and trimethoprim/sulfamethoxazole are effective and safe in the treatment of moderate to severe inflammatory acne. Superior efficacy of one type or class of antibiotic could not be determined, therefore the choice of antibiotic is generally based on the side-effect profile. Although different dosing regimens have been studied, there is a lack of standardized comparator trials to determine optimal dosing and duration of each oral antibiotic used in acne. The combination of oral antibiotics with a topical therapy is superior to oral antibiotics alone. CONCLUSION: This article provides a systematic evaluation of the scientific evidence of the efficacy of oral antibiotics for acne. Due to heterogeneity in the design of the trials, there is insufficient evidence to support one type, dose, or duration of oral antibiotic over another in terms of efficacy; however, due to increasing resistance to antibiotics, dermatologists should heed consensus guidelines for their appropriate use.

Atrial and ventricular cardiac chambers behave as distinct subunits with unique morphological, electrophysiological, and contractile properties. Despite the importance of chamber-specific features, chamber fate assignments remain relatively plastic, even after differentiation is underway. In zebrafish, Nkx transcription factors are essential for the maintenance of ventricular characteristics, but the signaling pathways that operate upstream of Nkx factors in this context are not well understood. Here, we show that FGF signaling plays an essential part in enforcing ventricular identity. Loss of FGF signaling results in a gradual accumulation of atrial cells, a corresponding loss of ventricular cells, and the appearance of ectopic atrial gene expression within the ventricle. These phenotypes reflect important roles for FGF signaling in promoting ventricular traits, both in early-differentiating cells that form the initial ventricle and in late-differentiating cells that append to its arterial pole. Moreover, we find that FGF signaling functions upstream of nkx genes to inhibit ectopic atrial gene expression. Together, our data suggest a model in which sustained FGF signaling acts to suppress cardiomyocyte plasticity and to preserve the integrity of the ventricular chamber.

Melanocyte stem cells (McSCs) reside in the hair follicle bulge/ secondary hair germ niche where they are essential for hair pigmentation and have the potential to also regulate epidermal pigmentation. A better understanding of the molecular mechanisms that govern these stem cells holds broad implications in pigmentation disorders including hair graying, vitiligo and melanoma. We show that Wnt signaling is temporarily activated in McSCs at the onset of hair follicle regeneration and is necessary and sufficient for their differentiation. Nonetheless, lineage tracing of Wnt-active differentiated McSCs demonstrates that McSCs can revert back to undifferentiated McSCs following withdrawal of Wnt signal activation. This suggests that McSC differentiation driven by Wnt signaling can be reversible, and temporal Wnt activation in McSCs does not deprive their self-renewing capacity. This ability of McSCs to oscillate between the differentiated and undifferentiated/stem cell states is not prevented when they differentiate into mature melanocytes after UVB irradiation. In aged mice, this process is compromised due to the failure to cease Wnt signaling, leading to ectopic McSC differentiation and a failure to return to their undifferentiated state, ultimately resulting in their loss. Our results show the critical regulation of Wnt signaling in governing maintenance as well as differentiation of McSCs in adult skin

OBJECTIVE: The purpose of this study was to determine whether the finding of failed fracture implants in association with lower extremity long bone fracture nonunion portends worse clinical or functional outcome following surgical nonunion repair. DESIGN: Retrospective analysis of prospectively collected data. SETTING: Academic Medical Center. PATIENTS: One hundred eighty-one patients who presented to our institution over a 10-year period and underwent surgical repair of a lower extremity fracture nonunion. INTERVENTION: Surgical repair of lower extremity fracture nonunion. MAIN OUTCOME MEASUREMENTS: Time to union, postoperative complications, VAS pain scores, and Short Musculoskeletal Function Assessment (SMFA) scores following lower extremity nonunion repair. Data was analyzed to assess for differences in postoperative outcomes based on the integrity of fracture implants at the time of nonunion diagnosis. Implant integrity was defined using 3 groups: broken implants (BI), implants intact (II), and no implants (NI). RESULTS: There was no significant difference in time to union following surgery between the BI, II, or NI groups (mean 8.1 months vs 7.6 months vs 6.2 months, respectively). Fourteen patients (7.7%) failed to heal, including 5 BI patients, 7 II patients, and 2 NI patients. One tibial nonunion patient in each of the 3 groups underwent amputation for persistent nonunion following multiple failed revision attempts at a mean of 4.8 years after initial injury. There was no difference in postoperative pain scores, the rate of postoperative complications, or functional outcome scores identified between the 3 groups. CONCLUSION: The finding of failed fracture implants at the time of lower extremity long bone nonunion diagnosis does not portend worse clinical or functional outcome following surgical nonunion repair. Patients who present with failed fracture implants at the time of nonunion diagnosis can anticipate similar time to union, complication rates, and functional outcomes when compared to patients who present with intact implants or those with history of nonoperative management. LEVEL OF EVIDENCE: Prognostic Level IV.

Tafazzin is a mitochondrial enzyme that transfers fatty acids from phospholipids to lysophospholipids. Mutations in tafazzin cause abnormal molecular species of cardiolipin and the clinical phenotype of Barth syndrome. However, the mechanism by which tafazzin creates acyl specificity has been controversial. We have shown that the lipid phase state can produce acyl specificity in the tafazzin reaction but others have reported that tafazzin itself carries enzymatic specificity. To resolve this issue, we replicated and expanded the controversial experiments, i.e. the transfer of different acyl groups from phosphatidylcholine to monolyso-cardiolipin by yeast tafazzin. Our data show that this reaction requires the presence of detergent and does not take place in liposomes but in mixed micelles. In order to separate thermodynamic (lipid-dependent) from kinetic (enzyme-dependent) parameters, we followed the accumulation of cardiolipin during the reaction from the initial state to the equilibrium state. The transacylation rates of different acyl groups varied over 2 orders of magnitude and correlated tightly with the concentration of cardiolipin in the equilibrium state (lipid-dependent parameter). In contrast, the rates by which different transacylations approached the equilibrium state were very similar (enzyme-dependent parameter). Furthermore, we found that tafazzin catalyzes the remodeling of cardiolipin by combinations of forward and reverse transacylations, essentially creating an equilibrium distribution of acyl groups. These data strongly support the idea that the acyl specificity of the tafazzin reaction results from the physical properties of lipids.