Analysis of cellularity in H&E-stained rat bone marrow tissue via deep learning.

Our objective was to develop an automated deep-learning-based method to evaluate cellularity in rat bone marrow hematoxylin and eosin whole slide images for preclinical safety assessment. We trained a shallow CNN for segmenting marrow, 2 Mask R-CNN models for segmenting megakaryocytes (MKCs), and small hematopoietic cells (SHCs), and a SegNet model for segmenting red blood cells. We incorporated the models into a pipeline that identifies and counts MKCs and SHCs in rat bone marrow. We compared cell segmentation and counts that our method generated to those that pathologists generated on 10 slides with a range of cell depletion levels from 10 studies. For SHCs, we compared cell counts that our method generated to counts generated by Cellpose and Stardist. The median Dice and object Dice scores for MKCs using our method vs pathologist consensus and the inter- and intra-pathologist variation were comparable, with overlapping first-third quartile ranges. For SHCs, the median scores were close, with first-third quartile ranges partially overlapping intra-pathologist variation. For SHCs, in comparison to Cellpose and Stardist, counts from our method were closer to pathologist counts, with a smaller 95% limits of agreement range. The performance of the bone marrow analysis pipeline supports its incorporation into routine use as an aid for hematotoxicity assessment by pathologists. The pipeline could help expedite hematotoxicity assessment in preclinical studies and consequently could expedite drug development. The method may enable meta-analysis of rat bone marrow characteristics from future and historical whole slide images and may generate new biological insights from cross-study comparisons.

Assessment of Color Reproducibility and Mitigation of Color Variation in Whole Slide Image Scanners for Toxicologic Pathology.

Digital pathology workflows in toxicologic pathology rely on whole slide images (WSIs) from histopathology slides. Inconsistent color reproduction by WSI scanners of different models and from different manufacturers can result in different color representations and inter-scanner color variation in the WSIs. Although pathologists can accommodate a range of color variation during their evaluation of WSIs, color variability can degrade the performance of computational applications in digital pathology. In particular, color variability can compromise the generalization of artificial intelligence applications to large volumes of data from diverse sources. To address these challenges, we developed a process that includes two modules: (1) assessing the color reproducibility of our scanners and the color variation among them and (2) applying color correction to WSIs to minimize the color deviation and variation. Our process ensures consistent color reproduction across WSI scanners and enhances color homogeneity in WSIs, and its flexibility enables easy integration as a post-processing step following scanning by WSI scanners of different models and from different manufacturers.

Toxicologic Pathology Forum: A Roadmap for Building State-of-the-Art Digital Image Data Resources for Toxicologic Pathology in the Pharmaceutical Industry.

Digitization of histologic slides brings with it the promise of enhanced toxicologic pathology practice through the increased application of computational methods. However, the development of these advanced methods requires access to substrate image data, that is, whole slide images (WSIs). Deep learning methods, in particular, rely on extensive training data to develop robust algorithms. As a result, pharmaceutical companies interested in leveraging computational methods in their digital pathology workflows must first invest in data infrastructure to enable data access for both data scientists and pathologists. The process of building robust image data resources is challenging and includes considerations of generation, curation, and storage of WSI files, and WSI access including via linked metadata. This opinion piece describes the collective experience of building resources for WSI data in the Roche group. We elaborate on the challenges encountered and solutions developed with the goal of providing examples of how to build a data resource for digital pathology analytics in the pharmaceutical industry.

Impact of color augmentation and tissue type in deep learning for hematoxylin and eosin image super resolution.

Single image super-resolution is an important computer vision task with applications including remote sensing, medical imaging, and surveillance. Modern work on super-resolution utilizes deep learning to synthesize high resolution (HR) images from low resolution images (LR). With the increased utilization of digitized whole slide images (WSI) in pathology workflows, digital pathology has emerged as a promising domain for super-resolution. Despite extensive existing research into super-resolution, there remain challenges specific to digital pathology. Here, we investigated image augmentation techniques for hematoxylin and eosin (H&E) WSI super-resolution and model generalizability across diverse tissue types. In addition, we investigated shortcomings with common quality metrics (peak signal-to-noise ratio (PSNR), structure similarity index (SSIM)) by conducting a perceptual quality survey for super-resolved pathology images. High performing deep super-resolution models were used to generate 20X HR images from LR images (5X or 10X equivalent) for 11 different tissues and 30 human evaluators were asked to score the quality of the generated versus the ground truth 20X HR images. The scores given by a human rater and the PSNR or the SSIM were compared to investigate the correlation between model training parameters. We found that models trained on multiple tissues generalized better than those trained on a single tissue type. We also found that PSNR correlated with perceptual quality (R = 0.26) less accurately than did SSIM (R = 0.64), suggesting that the SSIM quality metric is insufficient. The methods proposed in this study can be used to virtually magnify H&E images with better perceptual quality than interpolation methods (i.e., bicubic interpolation) commonly implemented in digital pathology software. The impact of deep SISR methods is more notable when scaling to 4X is needed, such as in the case of super-resolving a low magnification WSI from 10X to 40X.

Multiscale generative model using regularized skip-connections and perceptual loss for anomaly detection in toxicologic histopathology.

Background: Automated anomaly detection is an important tool that has been developed for many real-world applications, including security systems, industrial inspection, and medical diagnostics. Despite extensive use of machine learning for anomaly detection in these varied contexts, it is challenging to generalize and apply these methods to complex tasks such as toxicologic histopathology (TOXPATH) assessment (i.e.,finding abnormalities in organ tissues). In this work, we introduce an anomaly detection method using deep learning that greatly improves model generalizability to TOXPATH data. Methods: We evaluated a one-class classification approach that leverages novel regularization and perceptual techniques within generative adversarial network (GAN) and autoencoder architectures to accurately detect anomalous histopathological findings of varying degrees of complexity. We also utilized multiscale contextual data and conducted a thorough ablation study to demonstrate the efficacy of our method. We trained our models on data from normal whole slide images (WSIs) of rat liver sections and validated on WSIs from three anomalous classes. Anomaly scores are collated into heatmaps to localize anomalies within WSIs and provide human-interpretable results. Results: Our method achieves 0.953 area under the receiver operating characteristic on a real-worldTOXPATH dataset. The model also shows good performance at detecting a wide variety of anomalies demonstrating our method's ability to generalize to TOXPATH data. Conclusion: Anomalies in both TOXPATH histological and non-histological datasets were accurately identified with our method, which was only trained with normal data.

Shape Memory Nitinol Based Minimally Invasive Spinal Cord Stimulation Device Concept for Improved Pain Management.

BACKGROUND: Spinal cord stimulation (SCS) is a common treatment for neuropathic pain. There are 2 main categories of SCS leads: paddle leads and cylindrical leads. Paddle leads have reduced long-term complications and provide better coverage of target dermatomes when compared to cylindrical leads. However, insertion of a paddle lead requires invasive surgery that comes with significantly higher costs and more short-term complications, such as postoperative pain and infection. In contrast, cylindrical leads can be inserted minimally invasively using percutaneous techniques but provide less coverage of targeted dermatomes and have a higher tendency to migrate from intended neuronal targets. OBJECTIVES: Our objective is to develop a novel improved cylindrical spinal cord stimulation device that can convert into an optimal geometry once exposed to the body's environment after minimally invasive surgery. Such a device would be able to reduce long-term complications, lead migration, and better cover targeted dermatomes. STUDY DESIGN: Biomaterial selection, medical intervention device design with an in-vitro lab-scale test, and cadaveric experimental study. METHODS: A shape memory alloy nitinol-based cylindrical lead was designed, and its nitinol core material was processed and geometrically programmed for percutaneous insertion into the epidural space and morphing into an optimal geometry once exposed to the body's environment. Deployment of the nitinol component of the design was tested in the lab and human cadaveric models of the epidural space. RESULTS: Deployment of the nitinol component of the proposed cylindrical lead was successfully demonstrated in both a lab model of the epidural space and in the epidural space of a human cadaver in a minimally invasive fashion, indicating that a similar component could be used clinically in a full SCS electrode manufactured in a custom final geometry. LIMITATIONS: The focus of this study was to test the deployment of a novel minimally invasive lead that provides optimal coverage of intended dermatomes using in-vitro methods. Our study does not include in vivo trials. We do not test the electrical components of the design proposed since our design does not make changes to the electrical components of current commercially used cylindrical leads. CONCLUSION: The unique shape memory property of nitinol shows promise in allowing cylindrical spinal cord stimulation leads to expand into a more optimal geometry within the epidural space. By having a body temperature-dependent geometry change, nitinol-based cylindrical leads could reduce lead migration, increase dermatomal coverage, and increase electrode density while maintaining the advantages of minimally invasive insertion.

Protective effects of dietary grape against atopic dermatitis-like skin lesions in NC/NgaTndCrlj mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with significant health/economic burdens. Existing therapies are not fully effective, necessitating development of new approaches for AD management. Here, we report that dietary grape powder (GP) mitigates AD-like symptoms in 2,4-dinitrofluorobenzene (DNFB)-induced AD in NC/NgaTndCrlj mice. Using prevention and intervention protocols, we tested the efficacy of 3% and 5% GP-fortified diet in a 13-weeks study. We found that GP feeding markedly inhibited development and progression of AD-like skin lesions, and caused reduction in i) epidermal thickness, mast cell infiltration, ulceration, excoriation and acanthosis in dorsal skin, ii) spleen weight, extramedullary hematopoiesis and lymph nodes sizes, and iii) ear weight and IgE levels. We also found significant modulations in 15 AD-associated serum cytokines/chemokines. Next, using quantitative global proteomics, we identified 714 proteins. Of these, 68 (normal control) and 21 (5% GP-prevention) were significantly modulated (≥2-fold) vs AD control (DNFB-treated) group, with many GP-modulated proteins reverting to normal levels. Ingenuity pathway analysis of GP-modulated proteins followed by validation using ProteinSimple identified changes in acute phase response signaling (FGA, FGB, FGG, HP, HPX, LRG1). Overall, GP supplementation inhibited DNFB-induced AD in NC/NgaTndCrlj mice in both prevention and intervention trials, and should be explored further.

Prolactin synergizes with canonical Wnt signals to drive development of ER+ mammary tumors via activation of the Notch pathway.

Prolactin (PRL) cooperates with other factors to orchestrate mammary development and lactation, and is epidemiologically linked to higher risk for breast cancer. However, how PRL collaborates with oncogenes to foster tumorigenesis and influence breast cancer phenotype is not well understood. To understand its interactions with canonical Wnt signals, which elevate mammary stem cell activity, we crossed heterozygous NRL-PRL mice with ApcMin/+ mice and treated pubertal females with a single dose of mutagen. PRL in the context of ApcMin/+ fueled a dramatic increase in tumor incidence in nulliparous mice, compared to ApcMin/+ alone. Although carcinomas in both NRL-PRL/ApcMin/+ and ApcMin/+ females acquired a mutation in the remaining wildtype Apc allele and expressed abundant ß-catenin, PRL-promoted tumors displayed higher levels of Notch-driven target genes and Notch-dependent cancer stem cell activity, compared to ß-catenin-driven activity in ApcMin/+ tumors. This PRL-induced shift to dominant Notch signals was evident in preneoplastic epithelial hyperplasias at 120 days of age. In NRL-PRL/ApcMin/+ females, rapidly proliferating hyperplasias, characterized by ß-catenin at cell junctions and high NOTCH1 expression, contrasted with slower growing lesions with nuclear ß-catenin in ApcMin/+ females. These studies demonstrate that PRL can powerfully modulate the incidence and phenotype of mammary tumors, shedding light on mechanisms whereby PRL elevates risk of breast cancer.

p53 Is Not Required for High CIN to Induce Tumor Suppression.

Chromosomal instability (CIN) is a hallmark of cancer. While low levels of CIN can be tumor promoting, high levels of CIN cause cell death and tumor suppression. The widely used chemotherapeutic, paclitaxel (Taxol), exerts its anticancer effects by increasing CIN above a maximally tolerated threshold. One significant outstanding question is whether the p53 tumor suppressor is required for the cell death and tumor suppression caused by high CIN. Both p53 loss and reduction of the mitotic kinesin, centromere-associated protein-E, cause low CIN. Combining both genetic insults in the same cell leads to high CIN. Here, we test whether high CIN causes cell death and tumor suppression even in the absence p53. Despite a surprising sex-specific difference in tumor spectrum and latency in p53 heterozygous animals, these studies demonstrate that p53 is not required for high CIN to induce tumor suppression. Pharmacologic induction of high CIN results in equivalent levels of cell death due to loss of essential chromosomes in p53+/+ and p53-/- cells, further demonstrating that high CIN elicits cell death independently of p53 function. IMPLICATIONS: These results provide support for the efficacy of anticancer therapies that induce high CIN, even in tumors that lack functional p53.

Zika virus in rhesus macaque semen and reproductive tract tissues: a pilot study of acute infection†.

Although sexual transmission of Zika virus (ZIKV) is well-documented, the viral reservoir(s) in the male reproductive tract remains uncertain in humans and immune-intact animal models. We evaluated the presence of ZIKV in a rhesus macaque pilot study to determine persistence in semen, assess the impact of infection on sperm functional characteristics, and define the viral reservoir in the male reproductive tract. Five adult male rhesus monkeys were inoculated with 105 PFU of Asian-lineage ZIKV isolate PRVABC59, and two males were inoculated with the same dose of African-lineage ZIKV DAKAR41524. Viremia and viral RNA (vRNA) shedding in semen were monitored, and a cohort of animals were necropsied for tissue collection to assess tissue vRNA burden and histopathology. All animals exhibited viremia for limited periods (1-11 days); duration of shedding did not differ significantly between viral isolates. There were sporadic low levels of vRNA in the semen from some, but not all animals. Viral RNA levels in reproductive tract tissues were also modest and present in the epididymis in three of five cases, one case in the vas deferens, but not detected in testis, seminal vesicles or prostate. ZIKV infection did not impact semen motility parameters as assessed by computer-assisted sperm analysis. Despite some evidence of prolonged ZIKV RNA shedding in human semen and high tropism of ZIKV for male reproductive tract tissues in mice deficient in Type 1 interferon signaling, in the rhesus macaques assessed in this pilot study, we did not consistently find ZIKV RNA in the male reproductive tract.

Protective Effects of Dietary Grape on UVB-Mediated Cutaneous Damages and Skin Tumorigenesis in SKH-1 Mice.

Non-melanoma skin cancers (NMSCs) are the most diagnosed cancers in the US and occur more frequently in males. We previously demonstrated chemoprotective effects of dietary grape powder (GP) against UVB-mediated skin tumorigenesis in female SKH-1 mice. To expand on this, here, we determined the effects of GP in a short-term UVB exposure protocol (0 or 5% GP, followed by UVB every other day for 2 weeks) in male and female SKH-1 mice, as well as explored any sex-related differences in UVB carcinogenesis via male SKH-1 mice (0, 3, or 5% GP; UVB twice weekly for 28 weeks). In the short-term study, we found that GP protects against early-stage epithelial hyperplasia and mast cell infiltration in both sexes. In the long term, GP markedly reduced tumor counts and malignant conversion, along with significant decreases in mast cell infiltration, serum IgE and Eotaxin. We also found inhibition of P38 phosphorylation and reduced PCNA, Ki67 and BCL2 levels, suggesting that the anti-inflammatory effects of GP inhibits P38, acting as an upstream regulator to inhibit proliferation and reduce tumor cell survival. Together, GP appears to protect against UVB-mediated skin damage and carcinogenesis in SKH-1 mice and should be explored further as a supplement for NMSC prevention.

The microRNA miR-7a-5p ameliorates ischemic brain damage by repressing α-synuclein.

Ischemic stroke, which is caused by a clot that blocks blood flow to the brain, can be severely disabling and sometimes fatal. We previously showed that transient focal ischemia in a rat model induces extensive temporal changes in the expression of cerebral microRNAs, with a sustained decrease in the abundance of miR-7a-5p (miR-7). Here, we evaluated the therapeutic efficacy of a miR-7 mimic oligonucleotide after cerebral ischemia in rodents according to the Stroke Treatment Academic Industry Roundtable (STAIR) criteria. Rodents were injected locally or systemically with miR-7 mimic before or after transient middle cerebral artery occlusion. Decreased miR-7 expression was observed in both young and aged rats of both sexes after cerebral ischemia. Pre- or postischemic treatment with miR-7 mimic decreased the lesion volume in both sexes and ages studied. Furthermore, systemic injection of miR-7 mimic into mice at 30 min (but not 2 hours) after cerebral ischemia substantially decreased the lesion volume and improved motor and cognitive functional recovery with minimal peripheral toxicity. The miR-7 mimic treatment substantially reduced the postischemic induction of α-synuclein (α-Syn), a protein that induces mitochondrial fragmentation, oxidative stress, and autophagy that promote neuronal cell death. Deletion of the gene encoding α-Syn abolished miR-7 mimic-dependent neuroprotection and functional recovery in young male mice. Further analysis confirmed that the transcript encoding α-Syn was bound and repressed by miR-7. Our findings suggest that miR-7 mimics may therapeutically minimize stroke-induced brain damage and disability.

Increased transport of acetyl-CoA into the endoplasmic reticulum causes a progeria-like phenotype.

The membrane transporter AT-1/SLC33A1 translocates cytosolic acetyl-CoA into the lumen of the endoplasmic reticulum (ER), participating in quality control mechanisms within the secretory pathway. Mutations and duplication events in AT-1/SLC33A1 are highly pleiotropic and have been linked to diseases such as spastic paraplegia, developmental delay, autism spectrum disorder, intellectual disability, propensity to seizures, and dysmorphism. Despite these known associations, the biology of this key transporter is only beginning to be uncovered. Here, we show that systemic overexpression of AT-1 in the mouse leads to a segmental form of progeria with dysmorphism and metabolic alterations. The phenotype includes delayed growth, short lifespan, alopecia, skin lesions, rectal prolapse, osteoporosis, cardiomegaly, muscle atrophy, reduced fertility, and anemia. In terms of homeostasis, the AT-1 overexpressing mouse displays hypocholesterolemia, altered glycemia, and increased indices of systemic inflammation. Mechanistically, the phenotype is caused by a block in Atg9a-Fam134b-LC3ß and Atg9a-Sec62-LC3ß interactions, and defective reticulophagy, the autophagic recycling of the ER. Inhibition of ATase1/ATase2 acetyltransferase enzymes downstream of AT-1 restores reticulophagy and rescues the phenotype of the animals. These data suggest that inappropriately elevated acetyl-CoA flux into the ER directly induces defects in autophagy and recycling of subcellular structures and that this diversion of acetyl-CoA from cytosol to ER is causal in the progeria phenotype. Collectively, these data establish the cytosol-to-ER flux of acetyl-CoA as a novel event that dictates the pace of aging phenotypes and identify intracellular acetyl-CoA-dependent homeostatic mechanisms linked to metabolism and inflammation.

Common basis for orofacial clefting and cortical interneuronopathy.

Orofacial clefts (OFCs) of the lip and/or palate are among the most common human birth defects. Current treatment strategies focus on functional and cosmetic repair but even when this care is available, individuals born with OFCs are at high risk for persistent neurobehavioral problems. In addition to learning disabilities and reduced academic achievement, recent evidence associates OFCs with elevated risk for a constellation of psychiatric outcomes including anxiety disorders, autism spectrum disorder, and schizophrenia. The relationship between these outcomes and OFCs is poorly understood and controversial. Recent neuroimaging studies in humans and mice demonstrate subtle morphological brain abnormalities that co-occur with OFCs but specific molecular and cellular mechanisms have not been investigated. Here, we provide the first evidence directly linking OFC pathogenesis to abnormal development of GABAergic cortical interneurons (cINs). Lineage tracing revealed that the structures that form the upper lip and palate develop in molecular synchrony and spatiotemporal proximity to cINs, suggesting these populations may have shared sensitivity to genetic and/or teratogenic insult. Examination of cIN development in a mouse model of nonsyndromic OFCs revealed significant disruptions in cIN proliferation and migration, culminating in misspecification of the somatostatin-expressing subgroup. These findings reveal a unified developmental basis for orofacial clefting and disrupted cIN development, and may explain the significant overlap in neurobehavioral and psychiatric outcomes associated with OFCs and cIN dysfunction. This emerging mechanistic understanding for increased prevalence of adverse neurobehavioral outcomes in OFC patients is the entry-point for developing evidence-based therapies to improve patient outcomes.

Development and evaluation of the Andhra Pradesh Children and Parent Study Physical Activity Questionnaire (APCAPS-PAQ): a cross-sectional study.

BACKGROUND: There is limited availability of context-specific physical activity questionnaires in low and middle income countries. The aim of this study was to develop and examine the validity of a new Indian physical activity questionnaire, the Andhra Pradesh Children and Parent Study Physical Activity Questionnaire (APCAPS-PAQ). METHODS: The current study was conducted with the cohort from the Hyderabad DXA Study (n = 2321), recruited in 2009-2010. Criterion validity (n = 245) was examined by comparing the APCAPS-PAQ to a combined heart rate and motion sensor worn for 8 days. Construct validity (n = 2321) was assessed with linear regression, comparing APCAPS-PAQ against BMI, percent body fat, and pulse rate. RESULTS: The APCAPS-PAQ criterion validity was variable depending on the PA intensity groups (ρ = 0.26, 0.07, 0.39; к = 0.14, 0.04, 0.16 for sedentary, light, moderate/vigorous physical activity (MVPA) respectively). Sedentary and light intensity activities from the questionnaire were underestimated when compared to the criterion data while MVPA in APCAPS-PAQ was overestimated. Higher time spent in sedentary activity in APCAPS-PAQ was associated with higher BMI and percent body fat, suggesting construct validity. CONCLUSIONS: The APCAPS-PAQ validity is comparable to other physical activity questionnaires. This tool is able to assess sedentary behavior, moderate/vigorous activity and physical activity energy expenditure on a group level with reasonable validity. This new questionnaire may be used for ranking individuals according to their sedentary time and physical activity in southern India.

A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model of Mycobacterium smegmatis Infection.

Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid-loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 µg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models.

Associations between diet, physical activity and body fat distribution: a cross sectional study in an Indian population.

BACKGROUND: Obesity is a growing health problem in India and worldwide, due to changes in lifestyle. This study aimed to explore the independent associations between dietary and physical activity exposure variables and total body fat and distribution in an Indian setting. METHODS: Individuals who had participated in the Indian Migration Study (IMS) or the Andhra Pradesh Children And Parents' Study (APCAPS), were invited to participate in the Hyderabad DXA Study. Total and abdominal body fat of study participants was measured using DXA scans. Diet and physical activity (PA) levels were measured using questionnaires. RESULTS: Data on 2208 participants was available for analysis; mean age was 49 yrs in IMS, 21 yrs in APCAPS. Total energy intake was positively associated with total body fat in the APCAPS sample: a 100 kcal higher energy intake was associated with 45 g higher body fat (95% CI 22, 68). In the IMS sample no association was found with total energy intake, but there was a positive association with percent protein intake (1% higher proportion of energy from protein associated with 509 g (95% CI 138,880) higher total body fat). Broadly the same pattern of associations was found with proportion of fat in the abdominal region as the outcome. PA was inversely associated with total body fat in both populations (in APCAPS, one MET-hour higher activity was associated with 46 g (95% CI 12, 81) less body fat; in the IMS it was associated with 145 g less body fat (95% CI 73, 218)). An inverse association was observed between PA and percentage abdominal fat in the IMS but no association was seen in the APCAPS population. CONCLUSIONS: In this Indian population, there was an inverse association between PA and body fat. Associations between body fat and dietary variables differed between the younger APCAPS population and older IMS population. Further longitudinal research is needed to elucidate causality and directions of these associations across the life course.

Genetic etiology of renal agenesis: fine mapping of Renag1 and identification of Kit as the candidate functional gene.

Congenital anomalies of the kidney and urogenital tract (CAKUT) occur in approximately 0.5% of live births and represent the most frequent cause of end-stage renal disease in neonates and children. The genetic basis of CAKUT is not well defined. To understand more fully the genetic basis of one type of CAKUT, unilateral renal agenesis (URA), we are studying inbred ACI rats, which spontaneously exhibit URA and associated urogenital anomalies at an incidence of approximately 10%. URA is inherited as an incompletely dominant trait with incomplete penetrance in crosses between ACI and Brown Norway (BN) rats and a single responsible genetic locus, designated Renag1, was previously mapped to rat chromosome 14 (RNO14). The goals of this study were to fine map Renag1, identify the causal genetic variant responsible for URA, confirm that the Renag1 variant is the sole determinant of URA in the ACI rat, and define the embryologic basis of URA in this rat model. Data presented herein localize Renag1 to a 379 kilobase (kb) interval that contains a single protein coding gene, Kit (v-kit Hardy-Zukerman 4 feline sarcoma viral oncogene homolog); identify an endogenous retrovirus-derived long terminal repeat located within Kit intron 1 as the probable causal variant; demonstrate aberrant development of the nephric duct in the anticipated number of ACI rat embryos; and demonstrate expression of Kit and Kit ligand (Kitlg) in the nephric duct. Congenic rats that harbor ACI alleles at Renag1 on the BN genetic background exhibit the same spectrum of urogenital anomalies as ACI rats, indicating that Renag1 is necessary and sufficient to elicit URA and associated urogenital anomalies. These data reveal the first genetic link between Kit and URA and illustrate the value of the ACI rat as a model for defining the mechanisms and cell types in which Kit functions during urogenital development.

Loss of BMAL1 in ovarian steroidogenic cells results in implantation failure in female mice.

The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.