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Abstract The Four Core Genotypes (FCG) is a mouse model system used to disentangle the function of sex chromosomes and hormones. We report that a copy of a 3.2 MB region of the X chromosome has translocated to the Y Sry- chromosome and thus increased the expression of X-linked genes including the single-stranded RNA sensor and autoimmune disease mediator Tlr7. This previously-unreported X-Y translocation complicates the interpretation of studies reliant on C57BL/6J FCG mice.

Abstract Background Whether or not to progress from a pilot study to a definitive trial is often guided by pre-specified quantitative progression criteria with three possible outcomes. Although the choice of these progression criteria will help to determine the statistical properties of the pilot trial, there is a lack of research examining how they, or the pilot sample size, should be determined. Methods We review three-outcome trial designs originally proposed in the phase II oncology setting and extend these to the case of external pilots, proposing a unified framework based on univariate hypothesis tests and the control of frequentist error rates. We apply this framework to an example and compare against a simple two-outcome alternative. Results We find that three-outcome designs can be used in the pilot setting, although they are not generally more efficient than simpler two-outcome alternatives. We show that three-outcome designs can help allow for other sources of information or other stakeholders to feed into progression decisions in the event of a borderline result, but this will come at the cost of a larger pilot sample size than the two-outcome case. We also show that three-outcome designs can be used to allow adjustments to be made to the intervention or trial design before commencing the definitive trial, providing the effect of the adjustment can be accurately predicted at the pilot design stage. An R package, tout, is provided to optimise progression criteria and pilot sample size. Conclusions The proposed three-outcome framework provides a way to optimise pilot trial progression criteria and sample size in a way that leads to desired operating characteristics. It can be applied whether or not an adjustment following the pilot trial is anticipated, but will generally lead to larger sample size requirements than simpler two-outcome alternatives.

Abstract Vincristine (VCR) is one of the most widely used chemotherapy agents in treating pediatric cancer. Nonetheless, it is known to cause dose-dependent neurotoxicity which can impact virtually every organ system. Despite its widespread use, the precise impact of VCR on the lower urinary tract (LUT) remains inadequately elucidated. Our initial clinical and translational investigations suggest a sex-specific influence of childhood VCR exposure on LUT function. Thus, the current study aimed to investigate the late effects of systemic VCR exposure on LUT physiology and the underlying mechanisms, focusing on dosage and male-sex, employing juvenile CD-1 mice as a model. Male mice subjected to VCR exhibited augmented functional bladder capacity accompanied by frequent non-void contractions during awake cystometry, alongside mast cell accumulation within the bladder, compared to the saline-treated control group. Noteworthy functional changes were observed in bladder strips from the VCR group, including decreased nerve-mediated contraction, heightened contractile responses to cholinergic and purinergic agonists, enhanced responsiveness to histamine—primarily via histamine receptor 1 (Hrh1)—and an augmented relaxation effect with compound 48/80 (a mast cell degranulator), relative to the control group. Significant changes in gene expression levels associated with neuroinflammation and nociception were observed in both the bladder and lumbosacral dorsal root ganglia (Ls-DRG) of the VCR group. These findings suggest that VCR exposure during childhood, particularly in males, triggers neuroimmune responses in the bladder and Ls-DRG, amplifying responsiveness to neurotransmitters in the bladder, thereby contributing to LUT dysfunction characterized by a mixed bladder phenotype as a late effect during survivorship.

Summary: The serine-/arginine-rich splicing factor 2 (SRSF2) plays pivotal roles in pre-mRNA processing and gene transcription. Recurrent mutations, particularly a proline-to-histidine substitution at position 95 (P95H), are common in neoplastic diseases. Here, we assess SRSF2’s diverse functions in squamous cell carcinoma. We show that SRSF2 deletion or homozygous P95H mutation both cause extensive DNA damage leading to cell-cycle arrest. Mechanistically, SRSF2 regulates efficient bi-directional transcription of DNA replication and repair genes, independent from its function in splicing. Further, SRSF2 haploinsufficiency induces DNA damage without halting the cell cycle. Exposing mouse skin to tumor-promoting carcinogens enhances the clonal expansion of heterozygous Srsf2 P95H epidermal cells but unexpectedly inhibits tumor formation. To survive carcinogen treatment, Srsf2 P95H+/− cells undergo substantial transcriptional rewiring and restore bi-directional gene expression. Thus, our study underscores SRSF2’s importance in regulating transcription to orchestrate the cell cycle and the DNA damage response.

Abstract Background DNA methylation is an important epigenetic modification which has numerous roles in modulating genome function. Its levels are spatially correlated across the genome, typically high in repressed regions but low in transcription factor (TF) binding sites and active regulatory regions. However, the mechanisms establishing genome-wide and TF binding site methylation patterns are still unclear. Results Here we use a comparative approach to investigate the association of DNA methylation to TF binding evolution in mammals. Specifically, we experimentally profile DNA methylation and combine this with published occupancy profiles of five distinct TFs (CTCF, CEBPA, HNF4A, ONECUT1, FOXA1) in the liver of five mammalian species (human, macaque, mouse, rat, dog). TF binding sites are lowly methylated, but they often also have intermediate methylation levels. Furthermore, biding sites are influenced by the methylation status of CpGs in their wider binding regions even when CpGs are absent from the core binding motif. Employing a classification and clustering approach, we extract distinct and species-conserved patterns of DNA methylation levels at TF binding regions. CEBPA, HNF4A, ONECUT1, and FOXA1 share the same methylation patterns, while CTCF's differ. These patterns characterize alternative functions and chromatin landscapes of TF-bound regions. Leveraging our phylogenetic framework, we find DNA methylation gain upon evolutionary loss of TF occupancy, indicating coordinated evolution. Furthermore, each methylation pattern has its own evolutionary trajectory reflecting its genomic contexts. Conclusions Our epigenomic analyses indicate a role for DNA methylation in TF binding changes across species including that specific DNA methylation profiles characterize TF binding and are associated with their regulatory activity, chromatin contexts, and evolutionary trajectories.

BackgroundFailure of Alzheimer's disease and related diseases (ADRD) research studies to include and engage Black participants is a major issue, which limits the impact and generalizability of research findings. Little is known about participation of Black adults in online ADRD-related research registries.ObjectivesAs part of the Community Engaged Digital Alzheimer's Research (CEDAR) Study, this study aims to increase our understanding of facilitators and barriers of Black adults to participating in ADRD-related online registries, as well as to understand their preferences for communication channels.Design, setting, participants, measurementsWe invited all Black participants enrolled in the Brain Health Registry (BHR) to complete a cross-sectional online survey. The survey consisted of rating scales and open-text questions asking about their attitudes towards brain health research, reasons for joining and continuing to participate in BHR, difficulties with participating, and preferences for modes of contact and website usage.ResultsOf all invited Black BHR participants (N=3,636), 198 (5.5%) completed the survey. The mean age was 58.4 (SD=11.3), mean years of education were 16.3 (SD=2.4), and 85.5% identified as female. Reported facilitators for joining and continuing to participate in BHR were personal interest (e.g., learning more about own brain health) and altruism (e.g., helping research). Among additional registry features which could encourage return, receiving feedback or scores about BHR tasks was rated the highest. Of those who found BHR participation difficult (21%), the most frequent reason was time burden. The most preferred way of receiving study information was via email. Participants reported that the websites that they used the most were YouTube and Facebook.DiscussionThe results of our study can inform the development of culturally-responsive registry features and engagement efforts to improve inclusion and participation of Black adults in online ADRD research. Providing participants with feedback about their registry performance and reducing the number of registry tasks are among the recommended strategies.

Rare-earth nickelates (chemical formula RNiO3, R being a rare-earth cation) display a temperature-dependent metal-to-insulator transition (MIT) together with a breathing distortion of the NiO6 octahedra units at a temperature ranging from 0 to 600 K depending on the size of the R cation. Their rich phase diagram is also characterized by a paramagnetic to antiferromagnetic transition that occurs at the same temperature as the MIT for R = Pr, Nd, while it arises at lower temperatures for all the other members of the series. In this work, we have investigated the order of the MIT in a portion of the phase diagram spanning from SmNiO3 to NdNiO3 by means of temperature dependent transport measurements and resonant elastic x-ray scattering performed on high quality epitaxial SmxNd1−xNiO3 solid solution thin films. Our results show that the order of the metal-to-insulator transition does not depend on whether or not the MIT is coupled with the magnetic transition.

Abstract Cis-genetic effects are key determinants of transcriptional divergence in discrete tissues and cell types. However, how cis- and trans-effects act across continuous trajectories of cellular differentiation in vivo is poorly understood. Here, we quantify allele-specific expression during spermatogenic differentiation at single-cell resolution in an F1 hybrid mouse system, allowing for the comprehensive characterisation of cis- and trans-genetic effects, including their dynamics across cellular differentiation. Collectively, almost half of the genes subject to genetic regulation show evidence for dynamic cis-effects that vary during differentiation. Our system also allows us to robustly identify dynamic trans-effects, which are less pervasive than cis-effects. In aggregate, genetic effects were strongest in round spermatids, which parallels their increased transcriptional divergence we identified between species. Our approach provides a comprehensive quantification of the variability of genetic effects in vivo, and demonstrates a widely applicable strategy to dissect the impact of regulatory variants on gene regulation in dynamic systems.

Most perovskite oxides belong to the Pbnm space group, composed of an anisotropic unit cell, A-site antipolar displacements, and oxygen octahedral tilts. Mapping the orientation of the orthorhombic unit cell in epitaxial heterostructures that consist of at least one Pbnm compound is often needed for understanding and controlling the different degrees of coupling established at their coherent interfaces and, therefore, their resulting physical properties. However, retrieving this information from the strain maps generated with high-resolution scanning transmission electron microscopy can be challenging, because the three pseudocubic lattice parameters are very similar in these systems. Here, we present a novel methodology for mapping the crystallographic orientation in Pbnm systems. It makes use of the geometrical phase analysis algorithm, as applied to aberration-corrected scanning transition electron microscopy images, but in an unconventional way. The method is fast and robust, giving real-space maps of the lattice orientations in Pbnm systems, from both cross section and plan-view geometries, and across large fields of view. As an example, we apply our methodology to rare-earth nickelate heterostructures, in order to investigate how the crystallographic orientation of these films depends on various structural constraints that are imposed by the underlying single crystal substrates. We observe that the resulting domain distributions and associated defect landscapes mainly depend on a competition between the epitaxial compressive/tensile and shear strains, together with the matching of atomic displacements at the substrate/film interface. The results point toward strategies for controlling these characteristics by appropriate substrate choice.

Heterostructures consisting of SmNiO3 and NdNiO3 alternating layers with additional LaAlO3 spacer layers were grown and fully characterized by means of x-ray diffraction, atomic force microscopy, and scanning transmission electron microscopy. A change in the orientation of the orthorhombic long-axis of the nickelate layers is observed when a single unit cell of LaAlO3 is inserted between SmNiO3 and NdNiO3, in agreement with density functional theory calculations. At the same time, the structure of the ultra-thin rhombohedral LaAlO3 layers is affected by their proximity to orthorhombic nickelate layers, with both scanning transmission electron microscopy studies and density functional theory calculations revealing a weak antipolar motion of the La-cation in the LaAlO3 layers that is not present in the bulk rhombohedral structure of this compound.

Purpose: To investigate potential beneficial effects of tocotrienols which have been suggested to inhibit hypoxia-inducible factor (HIF) pathway, on partial bladder outlet obstruction (PBOO)-induced bladder pathology. Materials and Methods: PBOO was surgically created in juvenile male mice. Sham-operated mice were used as controls. Animals received daily oral administration of either tocotrienols (T3) or soybean oil (SBO, vehicle) from day 0 to 13 post-surgery. Bladder function was examined in vivo by void spot assay. At 2 weeks post-surgery, the bladders were subjected to physiological evaluation of detrusor contractility in vitro using bladder strips, histology by H&E staining and collagen imaging, and gene expression analyses by quantitative PCR. Results: A significant increase in the number of small voids was observed after 1 week of PBOO compared to the control groups. At 2 weeks post-surgery, PBOO+SBO mice showed a further increase in the number of small voids, which was not observed in PBOO+T3 group. PBOO-induced decrease in detrusor contractility was similar between two treatments. PBOO induced bladder hypertrophy to the same degree in both SBO and T3 treatment groups, however, fibrosis in the bladder was significantly less prominent in the T3 group than the SBO group following PBOO (1.8- vs. 3.0-fold increase in collagen content compared to the control). Enhanced levels of HIF target genes in the bladders were observed in PBOO+SBO group, but not in PBOO+T3 group compared to the control. Conclusions: Oral tocotrienol treatment reduced the progression of urinary frequency and bladder fibrosis by suppressing HIF pathways triggered by PBOO.

Introduction: Here we report an infant with a 46,XY karyotype and novel de novo PPP1R12A stop-loss variant with a concomitant anorectal malformation (ARM) and difference in sex development (DSD). Case report: This is an infant born with ambiguous genitalia and rectoperineal fistula. Genetic testing confirmed a 46,XY chromosomal complement and whole exome sequencing demonstrated a novel de novo heterozygous PPP1R12A stop-loss variant. The infant underwent tapering jejunoplasty, proximal jejunostomy and mucous fistula creation for jejunal atresia and posterior sagittal anorectoplasty for the ARM. The gonad will be addressed with a biopsy and orchiopexy when the infant reaches six months of age. Conclusion: Although Müllerian anomalies and hypospadias may be common with ARM, a true DSD of this nature is exceedingly rare. Multidisciplinary care with urology, endocrinology, genetics, and colorectal surgery is paramount to providing optimal care for this patient.

We present the development of a new algorithm which combines state-of-the-art energy-dispersive x-ray (EDX) spectroscopy theory and a suitable machine learning formulation for the hyperspectral unmixing of scanning transmission electron microscope EDX spectrum images. The algorithm is based on non-negative matrix factorization (NMF) incorporating a physics-guided factorization model. It optimizes a Poisson likelihood, under additional simplex constraint together with user-chosen sparsity-inducing and smoothing regularizations, and is based on iterative multiplicative updates. The fluorescence of x-rays is fully modeled thanks to state-of-the-art theoretical work. It is shown that the output of the algorithm can be used for a direct chemical quantification. With this approach, it is straightforward to include a priori knowledge on the specimen such as the presence or absence of certain chemical elements in some of its phases. This work is implemented within two open-source Python packages, espm and emtables , which are used here for data simulation, data analysis and quantification. Using simulated data, we demonstrate that incorporating physical modeling in the decomposition helps retrieve meaningful components from spatially and spectrally mixed phases, even when the data are very noisy. For synthetic data with a higher signal, the regularizations yield a tenfold increase in the quality of the reconstructed abundance maps compared to standard NMF. Our approach is further validated on experimental data with a known ground truth, where state-of-the art results are achieved by using prior knowledge about the sample. Our model can be generalized to any other scanning spectroscopy techniques where underlying physical modeling can be linearized.

Abstract The discovery of superconductivity in doped infinite‐layer nickelate thin films has brought increased attention to the behavior of the doped perovskite phase. Despite this interest, the majority of existing studies pertain to hole‐doped perovskite rare‐earth nickelate thin films, while most electron‐doping studies have been performed on bulk materials so far. To tackle this imbalance, a detailed study that addresses doping of NdNiO3 thin films using A‐site substitution is presented, using Pb as a dopant and taking advantage of its valence‐skipping nature. Through a combination of complementary techniques including X‐ray diffraction, transport measurements, X‐ray absorption spectroscopy, electron energy‐loss spectroscopy and scanning transmission electron microscopy, the valence of Pb in the Nd1−xPbxNiO3 structure is confirmed to be 4+, and the behavior of the doped thin films is found to be controlled by a competition between carrier injection and structural distortions, which respectively reduce and increase the metal‐to‐insulator transition temperature. This work provides a systematic study of electron doping in NdNiO3, demonstrating that A‐site substitution with Pb is an appropriate method for such doping in perovskite rare‐earth nickelate systems.