Faculty Bibliography

Many military veterans who experienced blast-related traumatic brain injuries (TBI) in the conflicts in Iraq and Afghanistan currently suffer from chronic cognitive and mental health problems including post-traumatic stress disorder (PTSD). Besides static symptoms, new symptoms may emerge or existing symptoms may worsen. TBI is also a risk factor for the later development of neurodegenerative diseases. Rats exposed to repetitive low-level blast overpressure (BOP) develop robust and enduring cognitive and PTSD-related behavioral traits that are present for at least one year after blast exposure. Here we determined the time-course of these traits appearance by testing rats in the immediate post-blast period. Three cohorts of rats examined within the first eight weeks exhibited no behavioral phenotype, or in one cohort features of anxiety. None showed the altered cued fear responses, or impaired novel object recognition characteristic of the fully developed phenotype. Two cohorts retested 36 to 42 weeks after blast exposure exhibited the expanded behavioral phenotype including anxiety as well as altered cued fear learning and impaired novel object recognition. Combined with previous work the chronic behavioral phenotype has been observed in six cohorts of blast-exposed rats studied at 3-4 months or longer after blast injury and the three cohorts studied here document the progressive nature of the cognitive/behavioral phenotype. These studies suggest the existence of a latent, delayed emerging, and progressive blast-induced cognitive and behavioral phenotype. The delayed onset has implications for the evolution of post-blast neurobehavioral syndromes in military veterans and its modeling in experimental animals.

BACKGROUND:Appropriate passive-active immunoprophylaxis effectively reduces mother-to-child transmission (MTCT) of hepatitis B virus (HBV), but the immunoprophylaxis failure was still more than 5% under the current strategy. The study objective was to investigate the effects of high dose of HB vaccine on MTCT and immune response for infants born to hepatitis B surface antigen (HBsAg)-positive mothers. METHODS:This was a prospective, multicenter, large-sample cohort study in four sites of China, and 955 pairs of HBsAg-positive mothers and their infants were enrolled in our investigation. The infants were given 10 μg or 20 μg HB vaccine (at age 0, 1, and 6 months) plus HB immunoglobulin (at age 0 and 1 month). Serum HBsAg, antibody to HBsAg (anti-HBs), and/or HBV DNA levels in the infants were determined at age 12 months. The safety of 20 μg HB vaccine was evaluated by adverse events and observing the growth indexes of infants. RESULTS:IU/mL, 20 μg HB vaccine did not present these above response advantages. The 20 μg HB vaccine showed good safety for infants. CONCLUSIONS:IU/mL. TRIAL REGISTRATION/BACKGROUND:Chinese Clinical Trial Registry, ChiCTR-PRC-09000459.

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.

Hepatic uptake and biosynthesis of fatty acids (FA), as well as the partitioning of FA into oxidative, storage, and secretory pathways are tightly regulated processes. Dysregulation of one or more of these processes can promote excess hepatic lipid accumulation, ultimately leading to systemic metabolic dysfunction. Angiopoietin-like-4 (ANGPTL4) is a secretory protein that inhibits lipoprotein lipase (LPL) and modulates triacylglycerol (TAG) homeostasis. To understand the role of ANGPTL4 in liver lipid metabolism under normal and high-fat fed conditions, we generated hepatocyte specific Angptl4 mutant mice (Hmut). Using metabolic turnover studies, we demonstrate that hepatic Angptl4 deficiency facilitates catabolism of TAG-rich lipoprotein (TRL) remnants in the liver via increased hepatic lipase (HL) activity, which results in a significant reduction in circulating TAG and cholesterol levels. Consequently, depletion of hepatocyte Angptl4 protects against diet-induce obesity, glucose intolerance, liver steatosis, and atherogenesis. Mechanistically, we demonstrate that loss of Angptl4 in hepatocytes promotes FA uptake which results in increased FA oxidation, ROS production, and AMPK activation. Finally, we demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the liver and protects against diet-induced obesity, dyslipidemia, glucose intolerance, and liver damage, which likely occurs via increased HL activity. Notably, this novel inhibition strategy does not cause any of the deleterious effects previously observed with neutralizing antibodies.

Osteoarthritis (OA), the most prevalent joint disease, is a major cause of disability worldwide. Growth hormone (GH) has been suggested to play significant roles in maintaining articular chondrocyte function and ultimately articular cartilage (AC) homeostasis. In humans, the age-associated decline in GH levels was hypothesized to play a role in the etiology of OA. We studied the impact of adult-onset isolated GH deficiency (AOiGHD) on the life span and skeletal integrity including the AC, in 23- to 30-month-old male and female mice on C57/BL6 genetic background. Reductions in GH during adulthood were associated with extended life span and reductions in body temperature in female mice only. However, end-of-life pathology revealed high levels of lymphomas in both sexes, independent of GH status. Skeletal characterization revealed increases in OA severity in AOiGHD mice, evidenced by AC degradation in both femur and tibia, and significantly increased osteophyte formation in AOiGHD females. AOiGHD males showed significant increases in the thickness of the synovial lining cell layer that was associated with increased markers of inflammation (IL-6, iNOS). Furthermore, male AOiGHD showed significant increases in matrix metalloproteinase-13 (MMP-13), p16, and β-galactosidase immunoreactivity in the AC as compared to controls, indicating increased cell senescence. In conclusion, while the life span of AOiGHD females increased, their health span was compromised by high-grade lymphomas and the development of severe OA. In contrast, AOiGHD males, which did not show extended life span, showed an overall low grade of lymphomas but exhibited significantly decreased health span, evidenced by increased OA severity.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM^1,2. The most common disease-causing mutation (DOK7^{1124_1127 dup}) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7^CM mice) and a mouse with point mutations in the two tyrosine residues (Dok7^2YF). We show that Dok7^CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7^CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Study question: Do young women with idiopathic early ovarian ageing have changes in telomere length and epigenetic age indicating accelerated biological aging? Summary answer: The telomere length and epigenetic age were comparable to those in young women with normal ovarian ageing. What is known already: Increased risk of several health events usually considered to be age-related such as cardiovascular disease, osteoporosis, over-all morbidity and mortality have been associated with premature and early menopause when compared to the risk in women with normal menopausal age suggesting an accelerated general ageing process associated to early ovarian ageing. It is unclear whether the onset of this process may start before menopause. Study design, size, duration: A prospective cohort study. Young women (<= 37 years) having ART at two Danish Public fertility clinics during the period 2016 to 2018 were divided into two groups dependent on their ovarian reserve status: early ovarian ageing (EOA) (N=55) and normal ovarian ageing (NOA)( N=52). Number of oocytes harvested in first and subsequent cycles was used as a marker of ovarian reserve. Blood samples was drawn at time of oocyte retrieval to assess biological age. Participants/materials, setting, methods: EOA was defined as >= 2 IVF cycles with <= 5 harvested oocytes despite sufficient stimulation with FSH and NOA as >=8 oocytes harvested in minimum 1 cycle. Known causes influencing the ovarian reserve (endometriosis, ovarian surgery, etc.) was reason for exclusion. Relative telomere length (qPCR) and epigenetic age acceleration (DNA methylation levels) were measured in white blood cells as markers of accelerated biological ageing. Main results and the role of chance: Relative telomere length was comparable with a mean of 0.46 (+/- sd 0.12) in the EOA group and 0.47 (0.14) in the normal ovarian ageing group (p=0.64). The difference of predicted mean epigenetic age and mean chronological age (i.e. epigenetic age acceleration) was, insignificantly, 0.5 years older in the EOA group when compared to the NOA group( (-1.02 years (2.62) and -1.57 years (2.56), respectively, p=0.27)), but this difference disappeared when adjusting for chronological age. Limitations, reasons for caution: Discrete changes in epigenetic age acceleration may not have been captured as the study only had power to detect an age acceleration of >= 2 years. Wider implications of the findings: By analysis of biomarkers for ageing in whole blood, we did not find any indications of a premenopausal accelerated aging in young women with idiopathic EOA. Further investigations in a similar cohort of premenopausal women is needed to fully elucidate the potential relationship between premenopausal accelerated biological ageing and EOA

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment^1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis^3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions¹. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8⁺ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.

Knockout of the chloride channel protein 2 (CLC-2; CLCN2) results in fast progressing blindness in mice. Retinal Pigment Epithelium (RPE) and photoreceptors undergo, in parallel, rapid, and profound morphological changes and degeneration. Immunohistochemistry and electron microscopy of the outer retina and electroretinography of the CLC-2 KO mouse demonstrated normal morphology at postnatal day 2, followed by drastic changes in RPE and photoreceptor morphology and loss of vision during the first postnatal month. To investigate whether the RPE or the photoreceptors are the primary cause of the degeneration, we injected lentiviruses carrying HA-tagged CLC-2 with an RPE-specific promotor in the subretinal space of CLC-2-KO mice at the time of eye opening. As expected, CLC-2-HA was expressed exclusively in RPE; strikingly, this procedure rescued the degeneration of both RPE and photoreceptors. Light response in transduced eyes was also recovered. Only a fraction of RPE was transduced with the lentivirus; however, the entire RPE monolayer appears healthy, even the RPE cells not expressing the CLC-2-HA. Surprisingly, in contrast with previous physiological observations that postulate that CLC-2 has a basolateral localization in RPE, our immunofluorescence experiments demonstrated CLC-2 has an apical distribution, facing the subretinal space and the photoreceptor outer segments. Our findings suggest that CLC-2 does not play the postulated role in fluid transport at the basolateral membrane. Rather, they suggest that CLC-2 performs a critical homeostatic role in the subretinal compartment involving a chloride regulatory mechanism that is critical for the survival of both RPE and photoreceptors.