Faculty Bibliography

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.

BACKGROUND:Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS:We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO") 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS:homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS:handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Motivation/UNASSIGNED:Optimal growth temperature is a fundamental characteristic of all living organisms. Knowledge of this temperature is central to the study of a prokaryote, the thermal stability and temperature dependent activity of its genes, and the bioprospecting of its genome for thermally adapted proteins. While high throughput sequencing methods have dramatically increased the availability of genomic information, the growth temperatures of the source organisms are often unknown. This limits the study and technological application of these species and their genomes. Here, we present a novel method for the prediction of growth temperatures of prokaryotes using only genomic sequences. Results/UNASSIGNED:By applying the reverse ecology principle that an organism's genome includes identifiable adaptations to its native environment, we can predict a species' optimal growth temperature with an accuracy of 5.17 °C root-mean-square error and a coefficient of determination of 0.835. The accuracy can be further improved for specific taxonomic clades or by excluding psychrophiles. This method provides a valuable tool for the rapid calculation of organism growth temperature when only the genome sequence is known. Availability and implementation/UNASSIGNED:Source code, genomes analyzed, and features calculated are available at: https://github.com/DavidBSauer/OGT_prediction. Supplementary information/UNASSIGNED:Supplementary data are available at Bioinformatics online.

Translating advances in cancer research to clinical applications requires better insight into the metabolism of normal cells and tumour cells in vivo. Much effort has focused on understanding how glycolysis and oxidative phosphorylation (OxPhos) support proliferation, while their impact on other aspects of development and tumourigenesis remain largely unexplored. We found that inhibition of OxPhos in neural stem cells (NSCs) or tumours in the Drosophila brain not only decreases proliferation, but also affects many different aspects of stem cell behaviour. In NSCs, OxPhos dysfunction leads to a protracted G₁/S-phase and results in delayed temporal patterning and reduced neuronal diversity. As a consequence, NSCs fail to undergo terminal differentiation, leading to prolonged neurogenesis into adulthood. Similarly, in brain tumours inhibition of OxPhos slows proliferation and prevents differentiation, resulting in reduced tumour heterogeneity. Thus, in vivo, highly proliferative stem cells and tumour cells require OxPhos for efficient growth and generation of diversity.

Regenerative paradigms exhibit nerve dependency, including regeneration of the mouse digit tip and salamander limb. Denervation impairs regeneration and produces morphological aberrancy in these contexts, but the direct effect of innervation on the stem and progenitor cells enacting these processes is unknown. We devised a model to examine nerve dependency of the mouse skeletal stem cell (mSSC), the progenitor responsible for skeletal development and repair. We show that after inferior alveolar denervation, mandibular bone repair is compromised because of functional defects in mSSCs. We present mSSC reliance on paracrine factors secreted by Schwann cells as the underlying mechanism, with partial rescue of the denervated phenotype by Schwann cell transplantation and by Schwann-derived growth factors. This work sheds light on the nerve dependency of mSSCs and has implications for clinical treatment of mandibular defects.

An example of the peer review process for "Distinct molecular trajectories converge to induce naive pluripotency" (Stuart et al., 2019) is presented here.

Objective: Retrotransposons are a group of abundant, repetitive sequences, which originated from ancient retroviral infections of our ancestral genomes. They are silenced by epigenetic marks throughout most of life, but become activated during early embryo development, with reprogramming of the epigenome. Some retrotransposons play regulatory roles during early development. Non-Long Terminal Repeat (LTR) retrotransposons (e.g. LINE-1) regulate gene expression during early mouse embryo development. LTR retrotransposons, such as the human endogenous retrovirus HERV-W and HERV-FRD (Syncytin-1 and Syncytin-2), mediate placentation¹. We hypothesized that aneuploidy, which disrupts implantation, would affect expression of retrotransposons during early human development.
Design(s): Prospective laboratory study.
Material(s) and Method(s): Blastocysts donated by patients who underwent IVF/PGT-A at NYU Langone FC were thawed, stripped of zona pellucidae by laser (to remove sperm or cumulus) and their genomic DNA and mRNA separated by the G&Tseq protocol² with modifications. mRNA expression and gene copy number were measured by RT-qPCR and qPCR using Bio-Rad CFX96 thermocycler and iQ SYBY Green mix, and the DELTADELTACq method was used to express their levels relative to GAPDH and 5S RNA, respectively. Data were analyzed by Mann Whitney U test and Student's T-test with GraphPad Prism 8 software.
Result(s): Syncytin-1 was expressed in all human embryos, but its expression in euploid embryos (n=2) was significantly higher than in aneuploid embryos (n=6) (median 1.943 vs 0.316, P= 0.0019). Expression of Syncytin-2 also was extremely higher in euploid compared to aneuploid embryos (median 2.155 vs. 0.1124, P= 0.0003). The copy number of ALU sequences in aneuploid embryos was greater than in euploid embryos (mean 0.943+/-0.067 vs 0.807+/-0.0716, T-test P=0.0486), but LINE1 copy number or expression did not differ between euploid and aneuploid embryos (mean 0.787+/-0.069 vs 0.904+/-0.094, T-test P= 0.1778).
Conclusion(s): We compared expression of a number of retrotransposons between euploid and aneuploid human blastocysts, and discovered that the human endogenous retrovirus, Syncytin-1 and Syncytine-2, are markedly decreased in aneuploid compared to euploid embryos. Given the crucial role of Syncytins in formation of human placenta, our data provide a possible mechanism of implantation failure in euploid embryos, and suggest a possible biomarker for implantation. References: 1. Soygur B, Moore H. Expression of Syncytin 1 (HERV-W), in the preimplantation human blastocyst, embryonic stem cells and trophoblast cells derived in vitro. Hum Reprod. 2016 Jul; 31(7):1455-61. 2. Macaulay IC, Teng MJ, Haerty W, Kumar P, Ponting CP, Voet T. Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq. Nat Protoc. 2016 Nov; 11(11):2081-103.
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Objective: Clinical guidelines on the optimal duration of controlled ovarian stimulation and ideal follicle size were developed for fresh embryo transfer cycles. Whether these apply to freeze all cycles remain unclear. We evaluated the impact of lead follicle size and duration of stimulation on the probability of euploid embryos in women undergoing IVF/PGT-A Design: Cross-sectional study
Material(s) and Method(s): Data from 721 patients undergoing at least two cycles of COS for IVF with preimplantation genetic testing for aneuploidy (PGT-A) via Next Generation Sequencing (NGS) (1859 cycles). Mixed-effect logistic regression, which can account for correlations among repeated outcomes within sample patients, was used to evaluate the association between independent variables and probability of achieving euploid embryos. We first conducted a mixed-effect logistic regression in a univariate manner. All variables then were evaluated in a multivariate model to control for confounding effects. Significant variables to p<0.05 were retained in the final model. p-values <0.05 were considered significant. Statistical analyses were performed using "nlme" and "lme4" package from R project. Results are reported as odds ratios (OR) with 95% confidence intervals (CI).
Result(s): Increasing sum (1.034 [1.022 1.046]/p<0.001) and mean diameter (1.129 [1.046 1.219] p=0.002) of the 5 largest follicles increased the probability of forming euploid embryos. Increasing days of stimulation showed a non-significant trend toward lower chance of forming euploid embryos (0.976 [0.923 1.031]/p=0.382) (Table 1).
Conclusion(s): Allowing the lead follicles to exceed 18mm increases the total number of euploid embryos formed per cycle, presumably by enabling retrieval of additional mature oocytes. Evidence of a detrimental effect of excessive follicle size was not evident in our study, though the number of cycles with follicles exceeding 24 mm was limited. The non-significant trend toward decreased euploid embryos following prolonged stimulation may reflect the effects of poor responders. [Figure presented]
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Objective: Posthumous assisted reproduction (PAR) raises complicated ethical and legal issues. ASRM recommends that assisted reproductive technology (ART) and fertility preservation (FP) programs develop written policies regarding cases of PAR, though little is known about adoption of such policies and how they have been implemented. Our objective was to assess the presence and content of policies toward PAR using oocytes and embryos amongSociety for Assisted Reproductive Technology (SART) member clinics in the U.S.
Design(s): Cross-sectional questionnaire-based study.
Material(s) and Method(s): Our study consists of three phases of communication: email-, postal mail-, and phone-based survey. We report on the first phase of anonymous email survey responses. Surveys were emailed to ASRM-member medical directors of all SART member clinics (n=332) during March and April 2019 using a modified Dillman Method; contact information was acquired from SART and ASRM membership data. The survey included 23 multiple-choice and 3 opened-ended questions assessing practice characteristics (practice type, location, IVF cycle volume), presence of a clinic policy towards PAR, and the content of such policy. Descriptive data are presented as %, with Fisher's exact test used where appropriate, and thematic content analysis was applied to open-ended responses.
Result(s): The first phase of the study received 39 clinic responses (12% response rate). Respondents were distributed across the U.S.; average volume of IVF cycles per year ranged from < 250 to > 1500. More than one-third (35.9%, n=14) of clinics reported participating in any cases of PAR over the past five years, and 5.1% (n=2) reported participation in more than five cases. Participation in cases of PAR was not significantly associated with practice type or IVF cycle volume (p>0.05). 57.9% (n=22) had written policies towards PAR using oocytes or embryos, while 36.8% (n=14) reported they did not have a policy. Practice type, IVF cycle volume, FP volume, and prior participation in cases of PAR were not significantly associated with the presence of a policy (p>0.05). Of those with a policy, 52.4% (n=11) reported they had used that policy, 66.7% (n=10) without a policy reported they had considered adopting one, and 60.0% (n=9) reported they had received a request for PAR services. Only 44% (n=15) of clinics specified that patients not expected to survive to use oocytes due to terminal illness were eligible for oocyte cryopreservation, while 50.0% (n=17) did not specify. Open-ended comments suggested need for case-by-case appraisal and firm consent polices regarding gamete disposition.
Conclusion(s): Our preliminary results suggest that SART programs are receiving an increasing number of requests for PAR services, but many SART programs lack PAR policies, and those with policies do not always follow ASRM recommendations. As PAR cases become more common, clinics should be equipped to manage the complexities of PAR. More data are needed as this study continues, and future research is needed to understand barriers to the creation and implementation of these increasingly needed policies.
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Objective: Treatment of chronic endometritis (CE) improves implantation rates in patients undergoing assisted reproductive technology (ART), but causative organisms are difficult to identify and the most effective treatment regimen remains undefined¹. Our objective was to identify the optimal duration and choice of antimicrobial agent(s) on clearance of CE.
Design(s): Retrospective cohort study of patients between 1/2017 and 12/2018 at a single academic center with an endometrial biopsy (EMB) showing CE.
Material(s) and Method(s): All patients diagnosed with CE (defined as >1 plasma cell/HPF, stained for CD138) on EMB followed by test of cure biopsy (TOC) were included. Antimicrobial agents prescribed and length of course were recorded. Regimens were classified as 14 days or less versus 15 days or more (up to 21 days), and by spectra of coverage: Gram positive, Gram negative, Anaerobe, Atypical and Anti-fungal. Primary outcome was presence or absence of CE on TOC. If a patient remained positive on TOC, subsequent treatment(s) were included as separate course(s) for analysis. Statistical analysis included chi square test of independence and a stepwise multiple logistic regression, with p < 0.05 significant.
Result(s): 144 women with an initial EMB positive for CE received a total of 225 treatment courses. 11 TOC results were unavailable, leaving 214 courses of treatment with known TOC outcomes. The most common indication for EMB was failed frozen embryo transfer(s) (FET) (mean 0.98+/-1.00, range 0-7), euploid pregnancy loss or recurrent pregnancy loss. The mean age of women in the cohort was 36.90+/-3.93 years (range 27-47). Mean number of courses required for clearance was 1.55+/-0.88 (range 0-6). All courses included antimicrobials providing gram positive and negative coverage. 62.6% (134/214) included anaerobic coverage and 66.3% (142/214) included atypical agent(s). 2 courses included anti-fungals. Including anaerobic coverage did not affect outcome (58.2% with vs 61.3% without, p = 0.67), nor did use of an atypical agent (59.2% with vs 59.67% without, p=1.00). Antibiotic regimens lasting 14 days or less (n=155) had lower rates of CE clearance when compared to those lasting 15 days or more (54.8% vs 71.2%, p < 0.03). Stepwise multiple logistic regression showed that only length of antimicrobial course retained a significant impact on TOC (B -0.762, p < 0.027, Omnibus test for variance p <0.024, Hosmer-Lemeshow test for fit p = 0.99).
Conclusion(s): CE is a treatable but poorly defined inflammatory process, which may affect ART success. Ideally, CE is cleared with the first course of antibiotics. Our results show that longer courses (15-21 days) are more effective, regardless of antimicrobial choice. This suggests that patients do not need to take less tolerable agents to achieve high clearance rates, and highlights the need for further, prospective analyses. References: ¹: Cicinelli E, et al. Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy. Human Reproduction 2015;30(2):323-330.
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