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Abstract Squamous or epidermoid cancer arises in stratified epithelia but also is frequent in the non-epidermoid epithelium of the lung by unclear mechanisms. A poorly studied mitotic checkpoint drives epithelial cells bearing irreparable genetic damage into epidermoid differentiation. We performed an RNA-sequencing gene search to target unknown regulators of this response and selected the SUMO regulatory protein SENP2. Alterations of SENP2 expression have been associated with some types of cancer. We found the protein to be strongly localised to mitotic spindles of freshly isolated human epidermal cells. Primary cells rapidly differentiated after silencing SENP2 with specific shRNAs. Loss of SENP2 produced in synchronised epithelial cells delays in mitotic entry and exit and defects in chromosomal alignment. The results altogether strongly argue for an essential role of SENP2 in the mitotic spindle and hence in controlling differentiation. In addition, the expression of SENP2 displayed an inverse correlation with the immuno-checkpoint biomarker PD-L1 in a pilot collection of aggressive lung carcinomas. Consistently, metastatic head and neck cancer cells that do not respond to the mitosis-differentiation checkpoint were resistant to depletion of SENP2. Our results identify SENP2 as a novel regulator of the epithelial mitosis-differentiation checkpoint and a potential biomarker in epithelial cancer.

Sex differences are widespread during neurodevelopment and play a role in neuropsychiatric conditions such as autism, which is more prevalent in males than females. In humans, males have been shown to have larger brain volumes than females with development of the hippocampus and amygdala showing prominent sex differences. Mechanistically, sex steroids and sex chromosomes drive these differences in brain development, which seem to peak during prenatal and pubertal stages. Animal models have played a crucial role in understanding sex differences, but the study of human sex differences requires an experimental model that can recapitulate complex genetic traits. To fill this gap, human induced pluripotent stem cell–derived brain organoids are now being used to study how complex genetic traits influence prenatal brain development. For example, brain organoids from individuals with autism and individuals with X chromosome–linked Rett syndrome and fragile X syndrome have revealed prenatal differences in cell proliferation, a measure of brain volume differences, and excitatory-inhibitory imbalances. Brain organoids have also revealed increased neurogenesis of excitatory neurons due to androgens. However, despite growing interest in using brain organoids, several key challenges remain that affect its validity as a model system. In this review, we discuss how sex steroids and the sex chromosomes each contribute to sex differences in brain development. Then, we examine the role of X chromosome inactivation as a factor that drives sex differences. Finally, we discuss the combined challenges of modeling X chromosome inactivation and limitations of brain organoids that need to be taken into consideration when studying sex differences.

Abstract The extent to which PSA screening is related to prostate cancer mortality reduction in the United States (US) is controversial. US Surveillance, Epidemiology, and End Results Program (SEER) data from 1980 to 2016 were examined to assess the relationship between prostate cancer mortality and cumulative excess incidence (CEI) in the PSA screening era and to clarify the impact of race on this relationship. CEI was considered as a surrogate for the intensity of prostate cancer screening with PSA testing and subsequent biopsy as appropriate. Data from 163,982,733 person-years diagnosed with 544,058 prostate cancers (9 registries, 9% of US population) were examined. Strong inverse linear relationships were noted between CEI and prostate cancer mortality, and 317,356 prostate cancer deaths were avoided. Eight regions of the US demonstrated prostate cancer mortality reduction of 46.0–63.7%. On a per population basis, the lives of more black men than white men were saved in three of four registries with sufficient black populations for comparison. Factor(s) independent of CEI (potential effects of treatment advances) explained 14.6% of the mortality benefit (p-value = 0.3357) while there was a significant main effect of CEI (effect = −0.0064; CI: [−0.0088, −0.0040]; p-value

Abstract Background Structural differences exist in the brains of autistic individuals. To date only a few studies have explored the relationship between fetal brain growth and later infant autistic traits, and some have used fetal head circumference (HC) as a proxy for brain development. These findings have been inconsistent. Here we investigate whether fetal subregional brain measurements correlate with autistic traits in toddlers. Methods A total of 219 singleton pregnancies (104 males and 115 females) were recruited at the Rosie Hospital, Cambridge, UK. 2D ultrasound was performed at 12-, 20- and between 26 and 30 weeks of pregnancy, measuring head circumference (HC), ventricular atrium (VA) and transcerebellar diameter (TCD). A total of 179 infants were followed up at 18–20 months of age and completed the quantitative checklist for autism in toddlers (Q-CHAT) to measure autistic traits. Results Q-CHAT scores at 18–20 months of age were positively associated with TCD size at 20 weeks and with HC at 28 weeks, in univariate analyses, and in multiple regression models which controlled for sex, maternal age and birth weight. Limitations Due to the nature and location of the study, ascertainment bias could also have contributed to the recruitment of volunteer mothers with a higher than typical range of autistic traits and/or with a significant interest in the neurodevelopment of their children. Conclusion Prenatal brain growth is associated with toddler autistic traits and this can be ascertained via ultrasound starting at 20 weeks gestation.

Abstract The ratio of index to ring finger (2D:4D) has been hypothesised to indicate prenatal androgen exposure, yet evidence for its validity is lacking. We report the first pre-registered study to investigate mothers’ early pregnancy sex hormone concentrations in relation to their children’s digit ratios measured at 18–22-month follow-up. Although the testosterone (T) to estradiol (E) ratio correlated negatively with right hand digit ratio (R2D:4D) and directional asymmetry (right-minus-left) in digit ratio (D[R−L]), neither effect remained statistically significant once demographic and obstetric covariates were controlled for. Nevertheless, the multivariate level of analysis did reveal that T correlated positively with left hand digit ratio (L2D:4D) and negatively with D[R−L]. However, the first of these effects is in the opposite direction to that predicted by theory. Taken together, the results of our study suggest research with larger samples is required to determine whether digit ratios are valid proxies for maternal sex hormone exposure.

BackgroundAutism likelihood is a largely heritable trait. Autism prevalence has a skewed sex ratio, with males being diagnosed more often than females. Steroid hormones play a mediating role in this, as indicated by studies of both prenatal biology and postnatal medical conditions in autistic men and women. It is currently unclear if the genetics of steroid regulation or production interact with the genetic liability for autism.MethodsTo address this, two studies were conducted using publicly available datasets, which focused respectively on rare genetic variants linked to autism and neurodevelopmental conditions (study 1) and common genetic variants (study 2) for autism. In Study 1 an enrichment analysis was conducted, between autism-related genes (SFARI database) and genes that are differentially expressed (FDR

BackgroundLevels of steroid hormones in the first three months of life, a period referred to as ‘mini-puberty’, are one of the earliest physiological differences between typical males and females postnatally. Autistic traits also show consistent typical sex differences in later infancy, after the 18th month of life. Previous studies have shown prenatal testosterone is associated with later levels of autistic traits. Studies testing if postnatal testosterone levels are associated with autistic traits have reported null results. No studies to date have investigated mini-puberty longitudinally or tested for interactions with baseline sex differences or familial likelihood of autism.MethodsThe ‘Cambridge Human Imaging and Longitudinal Development Study’ (CHILD) is a prospective enriched cohort study in Cambridge, UK. It includes physiological measurements in early infancy, as well as neurodevelopmental follow-ups over the first two years of life. A subset of the cohort also includes children with a family history of autism (a diagnosed parent or sibling). Salivary testosterone levels were assessed at two time-points, just after the 2nd and 6th month of life. Autistic traits were measured using the Quantitative Checklist of Autism in Toddlers (Q-CHAT) when the children were 18 months of age.ResultsSalivary testosterone levels were significantly higher during ‘mini-puberty’ in the 2nd and 3rd month of life, compared to after the 6th month of life, in both males and females. There was no significant sex difference at either time-point. Log-transformed testosterone levels were not associated with autistic traits (Q-CHAT). There was no interaction effect with infant sex, autism family history or baseline testosterone levels after mini-puberty (at >6 months of age).ConclusionBoth male and female infants have elevated levels of salivary testosterone during mini-puberty but in this relatively small sample this was not associated with their later autistic traits at 18 months or their family history of autism. This suggests that prenatal rather than postnatal testosterone levels are more relevant for understanding the causes of autism. Future studies should test these relationships in larger samples.

Background: Restrictive cardiomyopathy (RCM) places patients at high risk for adverse events. In this study, we aim to evaluate the association between left atrial function and time to adverse events such as all-cause mortality and cardiovascular hospitalizations related to RCM. Material and Methods: In this single-center study, ninety-eight patients with a clinical diagnosis of RCM were recruited from our registry: 30 women (31%); age (mean ± standard deviation) 61 ± 13 years. These patients underwent cardiac magnetic resonance (CMR) imaging from May 2007 to September 2015. Left atrial (LA) function (reservoir, contractile, and conduit strain), LA diameter and area, and left ventricular function (global longitudinal strain, ejection fraction), and volume were quantified, and the presence of late gadolinium enhancement was visually assessed. The cutoff value of the LA reservoir strain was selected based on tertile. An adjusted Cox proportional regression analysis was used to assess time to adverse outcomes with a median follow up of 49 months. Results: In our cohort, all-cause mortality was 36% (35/98). Composite events (all-cause mortality and cardiovascular hospitalizations) occurred in 56% of patients (55/98). All-cause mortality and composite events were significantly associated with a decreased LA reservoir strain (adjusted hazard ratio (aHR) = 0.957, p = 0.002 and aHR = 0.969, p = 0.008) using a stepwise elimination of imaging variables, demographics, and comorbidities. All-cause mortality and composite events were six and almost four times higher, respectively, in patients with the LA reservoir strain p = 0.005, and HR = 4.252, p = 0.001) compared to patients with the LA reservoir strain >34%. Survival was significantly reduced in patients with an LA reservoir strain p = 0.008). Conclusions: The decreased LA reservoir strain is independently associated with time to adverse events in patients with RCM.

OBJECTIVES/SPECIFIC AIMS: The study aims to understand if pro inflammatory epicardial white adipose phenotype is positively associated with coronary atherosclerosis, while the brown adipose phenotype is negatively associated. Primary outcome is association between epicardial fat fraction and coronary atherosclerosis and cardiac function. Secondary outcome is transcriptomic and lipidomic profiling between epicardial, extra pericardial, and subcutaneous depots and how these profiles correlate with fat fraction. METHODS/STUDY POPULATION: Recruited patients undergoing open-heart surgery provided informed consent at their second visit and underwent laboratory testing and imaging (cardiac magnetic resonance including water-fat imaging and coronary calcium computed tomography) prior to their surgery. Cardiac function such as cardiac chamber volume, mass, function, and strain, and depo-specific fat fraction were calculated from cardiac MR and Agatston calcium score and epicardial adipose volume from CT images. At the time of surgery, a tissue specimens from the epicardial, extrapericardial, and subcutaneous depots were obtained for transcriptomic and lipidomic analysis. Linear and logistic regression analyses adjusted for other variables were performed to evaluate significance level between variables. RESULTS/ANTICIPATED RESULTS: 37 subjects were enrolled in the study, 13 (35%) of which were women. Cardiac function and fat fraction was quantified in all patients, whereas tissue analyses were performed in 22 patients. Epicardial and extrapericardial fat fraction were independently associated with coronary atherosclerosis (p-value 0.01 and 0.04 respectively) Only epicardial fat fraction was negatively associated with global circumferential shortening of the left ventricle (0.03), while neither the extrapericardial fat fraction nor epicardial adipose volume were not (p =0.33 and 0.97 respectively) All three adipose depots have unique gene signatures with differentially expressed genes and pathways. RNA sequencing of epicardial, extrapericardial, and subcutaneous depots demonstrated tight clustering of epicardial and subcutaneous signatures based on PCA analysis (Figure 2). 19 lipid classes and 59 lipids showed differential expression between at least 2 of the fat depots (Figure 3). Hierarchal clustering of the lipids showed that epicardial and extrapericardial depots were more closely related than subcutaneous adipose. Plasmenyl-phosphatidylcholines, with an ether-linked fatty acid at the sn-1 position of the lipid, were higher in subcutaneous fat while most other lipids were higher in epicardial fat per tissue weight, such as ceramides (p=0.002). DISCUSSION/SIGNIFICANCE OF IMPACT: Epicardial, extrapericardial, and subcutaneous adipose depots express different lipidome and transcriptome signatures and different pathways. GSEA analysis demonstrated enrichment of genes related to antigen presentation and B cell immunity in epicardial compared to subcutaneous adipose tissue. Epicardial fat fraction is associated with coronary atherosclerosis and decreased left ventricular global circumferential shortening as an early predictor of decreased left ventricular stroke volume. Epicardial fat fraction is also associated with cermides which may play role in the development of coronary atherosclerosis and decreased cardiac function.

OBJECTIVES/SPECIFIC AIMS: The goal is to understand the underlying mechanism of epicaridial fat biology and its response to cardiometabolic disease by using quantitative multi-echo Dixon (mDixon) of water and lipid sequence, T2* blood-oxygen-level-dependent (BOLD) sequence of iron content, and data analysis methods to determine the quantity of brown Versus white fat. To accomplish this goal, we propose to define the histological, genetic, and metabolite state of epicardial fat and to confirm the relationship between fat phenotype and magnetic resonance (MR) characteristics. We will then investigate whether MR is more effective in identifying patients with lower cardiovascular disease risk than computed tomography (CT). METHODS/STUDY POPULATION: We will recruit 100 patients undergoing open-heart surgery and will quantify mDixon (proton density fat fraction), BOLD (T2*), and T2/T1 maps of epicardial, extrapericardial, and subcutaneous fat before their surgery. We will then (a) validate MR findings by direct depot-specific tissue analysis for lipid content, histological, and genetic markers of inflammation and brown and white fat, (b) develop plasma and fat depot specific metabolite profiling of cardiovascular disease risk and correlate with imaging characteristics. We will categorize cardiovascular risk score (Cardiovascular Health Status) of our 100 patients on quartiles. We will then build models where the categorized cardiovascular risk score are regressed on MR measures (epicardial fat fraction, T2*, and T2/T1 maps) and CT measures (epicardial fat volume and coronary calcium score). RESULTS/ANTICIPATED RESULTS: We anticipate to learn about epicardial fat biology and the role of inflammation in cardiometabolic disease. We will validate proton density fat fraction, T2* and T2 map against histology of epicardial fat for lipid content, established markers of brown and white fat and inflammation, respectively, to help us translate imaging technique to clinical practice. In respect to our second aim we anticipate that MR identifies patients at lower cardiovascular risk quartile than CT. DISCUSSION/SIGNIFICANCE OF IMPACT: Interest in epicardial fat as a visceral fat of the heart and coronary arteries is rapidly growing as the scientific based evidence indicates that the anatomic specificity is an important contributor to the cardiovascular diseases. The transformation of epicardial fat from a cardioprotective phenotype to a pro-inflammatory, atherosclerosis-promoting state triggers inflammation that is coincident with the expansion of epicardial fat volume detected by anatomic imaging. This study will impact the management of patients at risk for cardiovascular disease because it will demonstrate that quantification of epicardial fat status by MR identifies fat tissue changes validated by histology at lower cardiovascular disease risk quartile than CT.