Faculty Bibliography

Study question: What happens to telomere length during human meiotic maturation and pre-implantation development? What is the impact of DNA damage and telomere attrition on human development? Summary answer: DNA damage and telomere attrition limit oocyte maturation in vitro. Telomeres are short in oocytes, increase markedly, and develop increased heterogeneity during pre-implantation development. What is known already: Telomere length reflects aging in many cell types. Sperm, which emerge from spermatogonia throughout the life of the man, have long telomeres. Oocytes from mouse and women maintain short telomeres. Telomerase, the enzyme that lengthens telomeres, is minimally active in mouse and human oocytes and pre-implantation embryos until blastocyst stage. Lacking appreciable telomerase activity, early mouse embryos elongate telomeres via Alternative Lengthening of Telomeres (ALT), which provides robust telomere elongation, but increased genomic instability. We do not know whether ALT occurs during early human development, and if so, when it takes place. Study design, size, duration: 60 immature germinal vesicle (GV) and metaphase I (M1) human oocytes donated to research from women ages 18-45 were collected and in vitro matured (IVM) for up to 48 hours following the subjects' retrieval. Additionally, 28 cryopreserved human embryos donated to research from 7 couples (age 27-42 years old), at the New York University Langone Fertility Center. Participants/materials, setting, methods: Immature oocytes were in vitro matured to metaphase II (M2). Frozen embryos between the 2 pronuclear (2PN) and day 3 (8-10-cell) stage were thawed and dissociated into single blastomere, intact blastocysts were processed whole. Telomere length was evaluated using Single Cell Amplification of Telomere Repeats PCR (SCATRPCR), expressed as a telomere to reference gene ratio (T/R ratio). DNA damage was assessed by immunoflorescent staining. Statistical analysis was performed using one-way ANOVA or T-test where appropriate. Main results and the role of chance: During oocyte maturation, immunostaining revealed that oocytes which arrested at the GV stage and failed to mature, contained robust and abundant DNA damage signaling on their chromosomes compared with successfully matured M2s (mean total florescent units = 35073.4 +/- 10051.8 vs. 843.7 +/- 74.9). Telomere length however did not differ significantly between arrested GV and M2 oocytes (mean T/R ratio = 0.074 +/- 0.040 vs 0.105 +/- 0.067). Telomere length increased significantly (p < 0.05) between M2 oocytes and both 2PN embryos (mean T/R ratio = 0.837 +/- 0.546) and blastocysts (mean T/R ratio = 0.634 +/- 0.260). The most significant elongation (p < 0.0002) occurred by day 2 (2-4 cell) (mean T/ R ratio = 0.957 +/- 767). Between day 2 and day 3 (mean T/R ratio = 0.385 +/- 0.418) telomere length decreased. These data suggest early activation of a telomere lengthening mechanism, prior to zygotic genome activation. Additionally, Intra-embryo telomere length increased until its peak day 3 (Coefficient of Variation = 108.51%) and was at its lowest at the blastocyst stage (Coefficient of Variation = 40.97%) when telomerase becomes active. Together these data suggest that an Alternative Lengthening of Telomeres (ALT) mechanism may be responsible for both increases in telomere length and increased heterogeneity among blastomeres within early embryos. Limitations, reasons for caution: The limited sample size for some of the earliest embryonic stages in addition to the freezing method for the embryos may have affected quality, and an inability to infer developmental ability as the embryos were not cultured to blastocyst. Wider implications of the findings: Our study is the first to perform a molecular characterization of telomere dynamics and the role of DNA damage in human oocytes and embryos at the single cell level and provides the first evidence that ALT is part of normal embryonic development in humans

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 A X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-kappaB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4) in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN)-dependent histone deacetylase 1 (HDAC1) transcriptional repression of lanosterol-14alpha-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages in vitro, decreases IFNbeta-mediated signal transducer and activator of transcription (STAT)1-STAT2 activation and IFNbeta-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.

Pancreatic cancer is the third leading cause of cancer mortality in both men and women in the United States, with poor response to current standard of care, short progression-free and overall survival. Immunotherapies that target cytotoxic T lymphocyte antigen-4, programmed cell death protein-1, and programmed death-ligand 1 checkpoints have shown remarkable activities in several cancers such as melanoma, renal cell carcinoma, and non-small cell lung cancer due to high numbers of somatic mutations, combined with cytotoxic T-cell responses. However, single checkpoint blockade was ineffective in pancreatic cancer, highlighting the challenges including the poor antigenicity, a dense desmoplastic stroma, and a largely immunosuppressive microenvironment. In this review, we will summarize available clinical results and ongoing efforts of combining immune checkpoint therapies with other treatment modalities such as chemotherapy, radiotherapy, and targeted therapy. These combination therapies hold promise in unleashing the potential of immunotherapy in pancreatic cancer to achieve better and more durable clinical responses by enhancing cytotoxic T-cell responses.

Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders mstA cells hypersensitive to H2O2 Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders fur cells fully resistant to H2O2 Furthermore, the endogenous level of H2S is reduced in fur or sodA sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance, which enables disease to progress unabated by adaptive immunity. However, the drivers of this tolerogenic program are incompletely defined. In this study, we found that NLRP3 promotes expansion of immune-suppressive macrophages in PDA. NLRP3 signaling in macrophages drives the differentiation of CD4+ T cells into tumor-promoting T helper type 2 cell (Th2 cell), Th17 cell, and regulatory T cell populations while suppressing Th1 cell polarization and cytotoxic CD8+ T cell activation. The suppressive effects of NLRP3 signaling were IL-10 dependent. Pharmacological inhibition or deletion of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD complex), or caspase-1 protected against PDA and was associated with immunogenic reprogramming of innate and adaptive immunity within the TME. Similarly, transfer of PDA-entrained macrophages or T cells from NLRP3-/- hosts was protective. These data suggest that targeting NLRP3 holds the promise for the immunotherapy of PDA.

A fundamental challenge to our understanding of brown adipose tissue (BAT) is the lack of an animal model that faithfully represents human BAT. Such a model is essential for direct assessment of the function and therapeutic potential of BAT depots in humans. In human adults, most of the thermoactive BAT depots are located in the supraclavicular region of the neck, while mouse studies focus on depots located in the interscapular region of the torso. We recently discovered BAT depots that are located in a region analogous to that of human supraclavicular BAT (scBAT). Here, we report that the mouse scBAT depot has morphological characteristics of classical BAT, possesses the potential for high thermogenic activity, and expresses a gene signature that is similar to that of human scBAT. Taken together, our studies reveal a mouse BAT depot that represents human BAT and provides a unique tool for developing new translatable approaches for utilizing human scBAT.