Faculty Bibliography

OBJECTIVE: Telomere attrition may mediate some of the effects of aging on reproductive function in women. Mice null or haploinsufficient for telomerase phenocopy the profile of reproductive aging in women, with progressive infertility caused by numerous defects in their reproductive cells. Telomeropathies, such as Dyskeratosis Congenita, provide a natural experiment to test the Telomere Theory of Reproductive Aging in women. This study attempts to extensively characterize the reproductive function in women with telomeropathies for the first time. DESIGN: Blood samples, cumulus cells, and arrested embryos were collected following the cycle of A 30 year old woman with a precocious aging syndrome and aplastic anemia, attributed to a telomeropathy (AMH=0.3 and AFC=8). She underwent, controlled ovarian stimulation with E2 prime protocol and 600 IU/day of gonadotropin, using mixed protocol and GnRH antagonist for 18 days. MATERIALS AND METHODS: Monochrome multiplex quantitative polymerase chain reaction (qPCR) assay (Cawthon 2009) measured telomere length in leukocytes extracted from whole blood as well as, cumulus cells stripped from retrieved follicles. Telomere (T) amplification was normalized to a single copy gene (S), resulting in a T/S ratio proportional to average telomere length in the population. Single-Cell Amplification of Telomere Repeats (SCATR) PCR (Wang 2013) was used to measure telomere length in discarded embryo blastomeres. Telomere (T) amplification was normalized a reference gene (R), producing a T/R ratio . One-Way ANOVA test was used to determine statistical significance. RESULTS: Hyperstimulation resulted in only 7 oocytes and 1 euploid blastocyst. Over the treatment course, leukocyte telomere length increased from T/S ratio= 0.192+/-.0157 to 0.234+/-.0306 and there was a statistically significant (p= .0256) linear increase during the treatment. Further, telomere length in a retrieved parthenogenetic, 2-cell embryo was (T/R average= .169+/-.021) and that in cumulus cells (T/S= 0.586+/-.0147). Telomere lengths in all assayed cell types were shorter than those from age matched controls. CONCLUSIONS: Young woman with reduced ovarian reserve, poor response to ovarian stimulation and a high percentage of arrested embryos and aneuploid embryos was still able to generate one euploid blastocyst with high dose controlled ovarian stimulation, demonstrating the promise of ART for fertility preservation in women with telomeropathies. Intriguingly, controlled ovarian hyperstimulation increased her leukocyte telomere length. Presumably, the supraphysiologic levels of estrogen activated telomerase activity, consistent with prior studies reporting an estrogen response element in the TERT gene. Future studies should examine whether women with telomeropathies may benefit from estrogen supplementation

BACKGROUND: Patients with atopic dermatitis (AD) are prone to skin infections, with microbes such as Staphylococcus aureus suspected of contributing to pathogenesis. Bleach baths might improve AD by reducing skin microbial burden. OBJECTIVE: We sought to characterize the microbiota of lesional and nonlesional skin in young children with AD and control subjects and compare changes after treatment with a topical corticosteroid (TCS) alone or TCS + dilute bleach bath. METHODS: In a randomized, placebo-controlled, single-blinded clinical trial in 21 children with AD and 14 healthy children, lesional and nonlesional AD skin was examined at baseline and after 4-week treatment with TCS alone or TCS plus bleach bath. Microbial DNA was extracted for quantitative polymerase chain reaction of predominant genera and 16S rRNA sequencing. RESULTS: At baseline, densities of total bacteria and Staphylococcus, including Staphylococcus aureus, were significantly higher at the worst AD lesional site than nonlesional (P = .001) or control (P < .001) skin; bacterial communities on lesional and nonlesional AD skin significantly differed from each other (P = .04) and from control (P < .001). After TCS + bleach bath or TCS alone, bacterial compositions on lesional skin normalized (P < .0001), resembling nonlesional skin, with microbial diversity restored to control skin levels. LIMITATIONS: The 4-week time period and/or the twice-weekly baths may not have been sufficient for additional impact on the cutaneous microbiome. More detailed sequencing may allow better characterization of the distinguishing taxa with bleach bath treatment. CONCLUSIONS: Treatment with a TCS cream suffices to normalize the cutaneous microbiota on lesional AD; after treatment, bacterial communities on lesional skin resemble nonlesional skin but remain distinct from control.

Poor glycemic control in patients with diabetes mellitus (DM) has long been considered a contraindication for dental implant therapy due to the increased risk of delayed healing, infection, and microvascular/macrovascular complications. Numerous animal and human studies have also suggested poor glycemic control as a contraindication to dental implant therapy. However, recent studies have assessed implant stability over the initial four to six months after implant placement in patients with Type II DM possessingHbA1C levels as high as 12% and reported high rates of implant survival regardless of the patients' level of glycemic control. Although these results are promising, the studies were of a short duration and primarily assessed implant-related outcomes prior to restoration of the implants. In contrast to these studies, the goal of our study was to assess the effects of elevated glycemic levels on post-loaded dental implant survival. We conducted a retrospective review of patients who completed dental implant therapy at a single institution from January 2013 through December 2015. Only patients who received stage I implant placement with delayed loading and documented HbA1C values obtained within 3-months pre- or postoperatively were included in our study. These values reflect the average level of glycemic control over 60-90 days, representing the critical time period for bone metabolism, healing and osseointegration. Additional inclusion criteria included a follow-up assessment of the restored dental implant at least 1-year postimplant placement and at least 6-months post-loading. We defined loading as implants restored with either a single- unit crown, multi-unit fixed partial denture, or a removable overdenture. We defined survival as an implant lacking any signs of clinical mobility or failure of the implant to osseointegrate, pain, infection, or periimplant radiolucency. Our study consisted a total of 42 male patients with 173 implants (83 maxillary, 90 mandibular). HbA1C levels ranged from 5.5 to 12.2%. At the time of implant placement, 27 patients (102 implants) in group 1 had no history of DM(and/or HbA1C<=5.9%), 10 patients (47 implants) in group 2 had well-controlled DM (HbA1C 6-8%) and 5 patients (24 implants) in group 3 had poorly-controlled DM (HbA1C >= 8.1%). There were a total of five implant failures (2.89%), with three implant failures (three patients) in group 1, two implant failures (one patient) in group 2, and zero implant failures in group 3. We found no statistically significant difference in the number of implant failures among the three groups (p = 0.42). We also found no statistically significant difference in the percentage of implant failures among the three groups (p = 0.34). Patients with poorly controlled DM are typically not considered appropriate candidates for dental implant therapy. However, recent studies demonstrate that the effects of hyperglycemia on implant therapy remain uncertain. Although further investigation of longer-termeffects of elevated HbA1C levels is warranted, the results of our study demonstrate favorable outcomes for post-loaded dental implant survival in patients with HbA1C levels as high as 12.2%. The clinical results of our study are consistent with those of previous studies, which reported similar rates of implant survival in patients with Type II DM and elevated HbA1C levels

In humans, creatinine is formed by a multistep process in liver and muscle and eliminated via the kidney by a combination of glomerular filtration and active transport. Based on current evidence, creatinine can be taken up into renal proximal tubule cells by the basolaterally localized organic cation transporter 2 (OCT2) and the organic anion transporter 2 (OAT2), and effluxed into the urine by the apically localized multidrug and toxin extrusion protein 1 (MATE1) and MATE2K. Drug induced elevation of serum creatinine (SCr) and/or reduced creatinine renal clearance (CLcr) is routinely used as a marker for acute kidney injury (AKI). Interpretation of elevated SCr can be complex, because such increases can be reversible and explained by inhibition of renal transporters involved in active secretion of creatinine or other secondary factors such as diet and disease state. Distinction between these possibilities is important from a drug development perspective as increases in SCr can result in the termination of otherwise efficacious drug candidates. In this review, we discuss the challenges associated with using creatinine as a marker for kidney damage. Furthermore, in order to evaluate whether reversible changes in SCr can be predicted prospectively based on in vitro transporter inhibition data, an in depth in vitro-in vivo correlation analysis was conducted for sixteen drugs with in house and literature in vitro transporter inhibition data for OCT2, MATE1 and MATE2K, as well as total and unbound maximum plasma concentration (Cmax and Cmax,u) data measured in the clinic.

OBJECTIVE: The architecture and structure of the meiotic spindle influence embryo development(1) and risk of aneuploidy in women(2). The factors that disrupt the meiotic spindle remain incompletely understood. Dysfunctional mitochondria produce reactive oxygen species (ROS), which may directly perturb spindles(3). ROS also can disrupt spindles by inducing telomere attrition, since telomeres are essential for spindle formation and are especially susceptible to ROS(4). We studied the impact of ROS, produced by uncoupling mitochondria, on the area and retardance (measure ofmolecular order) ofmeiotic spindles and on mean telomere length of individual human oocytes. DESIGN: Prospective, randomized, paired research laboratory intervention. MATERIALS AND METHODS: 44 germinal vesicle (GV) stage oocytes were accessioned from 16 patients undergoing IVF/ICSI. GVs from each patient were randomly assigned to control or treatment group. Oocytes in the treatment group were cultured in media containing Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM) for one hour to induce mitochondrial stress. Control oocytes were cultured in media without FCCP. GVoocytes from both groups were further cultured to permit meiotic maturation, as indicated by extrusion of the first polar body. Spindles were imaged non-invasively with an orientation independent polarized light microscope (Oosight, Hamilton Thorne, MA, USA). Spindle area and mean retardance were measured with software from the Oosight Imaging System. Mean oocyte telomere length was measured by single cell qPCR. Data were analyzed by Chi-square test and independent t test. RESULTS: Maturation rates of oocytes in the control and FCCP groups were 66.67% and 56.52% respectively. 71.4% of MII oocytes in the control group and 61.5% in the FCCP group had a detectable birefrigent spindle. FCCP decreased the area of the spindles compared to controls (19.84+/-2.11 sq. microns versus 37.77+/-4.79, P=0.011). Mean spindle retardance in the FCCP group also was significantly lower than that of controls (1.45+/-0.11 nm versus 2.19+/-0.16 nm, P=0.003). Telomere length (T/R ratio) did not differ between treatment and control groups (1.11+/-1.56 versus 1.29+/-0.19, P>0.05). Telomere length of oocytes with and without birefringence spindle within the treatment group also did not differ significantly (P>0.05). CONCLUSIONS: Meiotic maturation itself is relatively resistant to ROS, consistent with prior studies showing limited cell cycle check point control during oogenesis. However, ROS produced by acute mitochondrial stress disrupts spindle retardance and size. Reactive oxygen, at least acutely, does not induce telomere attrition in human oocytes

OBJECTIVE: Netrin-1 is a chemorepulsant and matrix protein expressed during and required for osteoclast differentiation, which also plays a role in inflammation by preventing macrophage egress. Because wear particle-induced osteolysis requires osteoclast-mediated destruction of bone, we hypothesised that blockade of Netrin-1 or Unc5b, a receptor for Netrin-1, may diminish this pathological condition. METHODS: C57BL/6 mice, 6-8 weeks old, had 3 mg of ultrahigh-molecular-weight polyethylene particles implanted over the calvaria and then received 10 microg of monoclonal antibodies for Netrin-1 or its receptors, Unc5b and deleted in colon cancer (DCC), injected intraperitoneally on a weekly basis. After 2 weeks, micro-computed tomography and histology analysis were performed. Netrin-1 expression was analysed in human tissue obtained following primary prosthesis implantation or after prosthesis revision for peri-implant osteolysis and aseptic implant loosening. RESULTS: Weekly injection of anti-Netrin-1 or anti-Unc5b-antibodies significantly reduced particle-induced bone pitting in calvaria exposed to wear particles (46+/-4% and 49+/-3% of control bone pitting, respectively, p<0.001) but anti-DCC antibody did not affect inflammatory osteolysis (80+/-7% of control bone pitting, p=ns). Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibody treatment markedly reduced the inflammatory infiltrate and the number of tartrate resistance acid phosphatase (TRAP)-positive osteoclasts (7+/-1, 4+/-1 and 14+/-1 cells/high power field (hpf), respectively, vs 12+/-1 cells/hpf for control, p<0.001), with no significant changes in alkaline phosphatase-positive osteoblasts on bone-forming surfaces in any antibody-treated group. Netrin-1 immunostaining colocalised with CD68 staining for macrophages. The peri-implant tissues of patients undergoing prosthesis revision surgery showed an increase in Netrin-1 expression, whereas there was little Netrin-1 expression in soft tissues removed at the time of primary joint replacement. CONCLUSIONS: These results demonstrate a unique role for Netrin-1 in osteoclast biology and inflammation and may be a novel target for prevention/treatment of inflammatory osteolysis.

BACKGROUND: Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of beta-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. METHODS: Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. FINDINGS: Serum PGRN levels were significantly lower in GD patients (96.65+/-53.45ng/ml) than those in healthy controls of the general population (164.99+/-43.16ng/ml, p<0.0001) and of Ashkenazi Jews (150.64+/-33.99ng/ml, p<0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, "aged" and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients. INTERPRETATION: Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCase's lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD.

OBJECTIVE: Reactive oxygen species (ROS) are a major cause of aging in all tissues studied, including reproductive tissues. Recent studies demonstrate extensiveDNA damage repair capacity in oocytes (1), and genetic variation in DNA damage repair pathways is associated with reproductive lifespan in women (2). We hypothesized that MII oocytes are more resistant to oxidative stress than other stages of development. DESIGN: Prospective, randomized study of a biologic intervention on mouse oocytes and embryos. MATERIALS AND METHODS: 80 MII oocytes and 40 cleavage embryos from B6C3F1 mice (Embryotech Laboratories, Inc, USA) were thawed and exposed to oxidative stress induced by Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 750nM), which generates ROS by uncoupling mitochondrial electron transport and disrupting mitochondrial function. Oocytes and embryos were randomized into untreated controls, or 5, 10 or 20 hour exposure to FCCP. DNA damage was determined by immuno- fluorescent staining for gamma-H2AX, or by mean telomere length, measured by single-cell qPCR. Data were analyzed by chi-square test and one-way ANOVA. RESULTS: 20 hours of exposure to FCCP is lethal for all embryos. Embryos exposed to FCCP for 5 and 10 hours of FCCP show increased g- H2AX staining (5 hours- 87.5% vs. 25% positive cells P<0.05; 10 hours- 100% vs 25%, P<0.05). However, these same doses and durations of FCCP do not increase DNA damage in oocytes- there is no significant increase of gamma-H2AX positive MII oocytes compared to controls (30% in the control group, 40% in the 5-hour group and 30% in the 10-hour group, P>0.05). Significantly higher numbers of gamma-H2AX positive MII oocytes are found only in the 20-hour group (100%, P<0.05), a dose which is uniformly lethal to embryos. As previously demonstrated, 45 minutes of 750nM FCCP treatment shortens telomeres in cleavage stage mouse embryos (3). However, MII oocytes exposed to 750nM FCCP for 5, 10, or 20 hours show no statistically significant telomere shortening compared to controls (P>0.05). CONCLUSIONS: MIIoocytes are more resistant to oxidative stress than cleavage embryos

OBJECTIVES: Autoantibodies against tumor-associated antigens (TAAs) identified in patients with advanced lung cancer may be detected in subjects with early lung cancer or even predate the diagnosis. The purpose of this study is to address the temporal relationship between lung cancer development and serum autoantibody response. MATERIALS AND METHODS: Two cohorts of patients with newly diagnosed lung cancer were included. The first cohort included 90 sera from patients with lung cancer (Stages I-III) and 89 normal control sera. In the second cohort, 93 serial serum samples from 25 patients with CT-scan screen-detected stage I lung cancer were collected before the diagnosis of lung cancer (average 32 months) and 56 controls were matched on age, gender, and smoking. Autoantibody levels were measured by immunoassay. RESULTS: Measurement of autoantibodies against seven TAAs (14-3-3zeta, c-Myc, MDM2, NPM1, p16, p53 and cyclin B1) individually could discriminate lung cancer patients from normal individuals in the first cohort and the area under curve (AUC) was 0.863 based on a panel of seven autoantibodies, with sensitivity of 68.9% and specificity of 79.5%. Autoantibodies in serial pre-diagnostic serum samples against the same panel of seven TAAs were detected prior to lung cancer diagnosis with sensitivity of 76.0% and specificity of 73.2% (AUC) (95%CI): 0.885 (0.797-0.973)). Elevated autoantibody levels could be detected greater than four years prior to lung cancer diagnosis. CONCLUSION: A panel of seven TAAs may enhance the early detection of lung cancer, consistent with a humoral immune response to TAAs that can be detected months to years prior to the diagnosis.

This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.