Joint sequence & chromatin neural networks characterize the differential abilities of Forkhead transcription factors to engage inaccessible chromatin.

The DNA-binding activities of transcription factors (TFs) are influenced by both intrinsic sequence preferences and extrinsic interactions with cell-specific chromatin landscapes and other regulatory proteins. Disentangling the roles of these binding determinants remains challenging. For example, the FoxA subfamily of Forkhead domain (Fox) TFs are known pioneer factors that can bind to relatively inaccessible sites during development. Yet FoxA TF binding also varies across cell types, pointing to a combination of intrinsic and extrinsic forces guiding their binding. While other Forkhead domain TFs are often assumed to have pioneering abilities, how sequence and chromatin features influence the binding of related Fox TFs has not been systematically characterized. Here, we present a principled approach to compare the relative contributions of intrinsic DNA sequence preference and cell-specific chromatin environments to a TF's DNA-binding activities. We apply our approach to investigate how a selection of Fox TFs (FoxA1, FoxC1, FoxG1, FoxL2, and FoxP3) vary in their binding specificity. We over-express the selected Fox TFs in mouse embryonic stem cells, which offer a platform to contrast each TF's binding activity within the same preexisting chromatin background. By applying a convolutional neural network to interpret the Fox TF binding patterns, we evaluate how sequence and preexisting chromatin features jointly contribute to induced TF binding. We demonstrate that Fox TFs bind different DNA targets, and drive differential gene expression patterns, even when induced in identical chromatin settings. Despite the association between Forkhead domains and pioneering activities, the selected Fox TFs display a wide range of affinities for preexiting chromatin states. Using sequence and chromatin feature attribution techniques to interpret the neural network predictions, we show that differential sequence preferences combined with differential abilities to engage relatively inaccessible chromatin together explain Fox TF binding patterns at individual sites and genome-wide.

Vaporized Hydrogen Peroxide Sterilization in the Production of Protein Therapeutics: Uptake and Effects on Product Quality.

The aseptic filling of drug products is carried out in pharmaceutical isolators that have been sterilized. A commonly used method for achieving a high level of sterility assurance is vaporized hydrogen peroxide (VHP) sterilization, which is favorable to other methods, such as ethylene oxide sterilization, due to its low cycle times and nontoxic residuals. While VHP cycles are often employed to create a sterile environment within an isolator, they can leave residual levels of hydrogen peroxide behind that can enter the product during fill-finish operations. Due to the oxidizing potential of hydrogen peroxide and the multiple possible sources of uptake along filling lines, the extent of the potential impact on product quality needs to be understood during pharmaceutical development. Herein, different factors affecting hydrogen peroxide uptake, points of entry along the filling line, and possible impacts on product quality are reviewed.

Allo: Accurate allocation of multi-mapped reads enables regulatory element analysis at repeats.

Transposable elements (TEs) and other repetitive regions have been shown to contain gene regulatory elements, including transcription factor binding sites. Unfortunately, regulatory elements harbored by repeats have proven difficult to characterize using short-read sequencing assays such as ChIP-seq or ATAC-seq. Most regulatory genomics analysis pipelines discard "multi-mapped" reads that align equally well to multiple genomic locations. Since multi-mapped reads arise predominantly from repeats, current analysis pipelines fail to detect a substantial portion of regulatory events that occur in repetitive regions. To address this shortcoming, we developed Allo, a new approach to allocate multi-mapped reads in an efficient, accurate, and user-friendly manner. Allo combines probabilistic mapping of multi-mapped reads with a convolutional neural network that recognizes the read distribution features of potential peaks, offering enhanced accuracy in multi-mapping read assignment. Allo also provides read-level output in the form of a corrected alignment file, making it compatible with existing regulatory genomics analysis pipelines and downstream peak-finders. In a demonstration application on CTCF ChIP-seq data, we show that Allo results in the discovery of thousands of new CTCF peaks. Many of these peaks contain the expected cognate motif and/or serve as TAD boundaries. We additionally apply Allo to a diverse collection of ENCODE ChIP-seq datasets, resulting in multiple previously unidentified interactions between transcription factors and repetitive element families. Finally, we show that Allo may be particularly effective in identifying ChIP-seq peaks in younger TEs, which hold evolutionary significance due to their emergence during human evolution from primates.

The vaginal and uterine blood flow changes during the ovsynch program and its impact on the pregnancy rates in Holstein dairy cows.

AIM: OvSynch is a hormonal protocol for synchronization of estrus and use of artificial insemination (AI) at an optimal time without adverse effects on the ovaries or uterus. This study investigated the use of noninvasive color Doppler ultrasound to assess changes in uterine and vaginal blood flow during the Ovsynch program for synchronization of estrus and its relation to the pregnancy rates in Holstein cows. MATERIALS AND METHODS: The experimental cows received an intramuscular dose of 10 µg of a GnRH analogue (G1), followed 7 days later with an intramuscular injection of synthetic prostaglandin F2α (P: PGF2α) analogue (500 µg cloprostenol sodium), and given a 10 µg, injection of the GnRH analogue (G2) i.m. 48 h after the PGF2α treatment, and the cows were bred 14-16 h after. Uterine and vaginal perfusion were investigated by performing transrectal Doppler ultrasonography of both the uterine and vaginal arteries in Holstein cows at different time points during the Ovsynch program to determine: peak systolic velocity (PSV), time-averaged maximum velocity (TAMV), the volume of blood flow (BFV), pulsatility index (PI), resistance index (RI), resistance impedance (S/D) and diameters of uterine (UA) and vaginal (VA) arteries. Steroid hormones were also assayed. Transrectal ultrasonography (TUS) was performed at 32 and 60 days to confirm the pregnancy per artificial insemination (P/AI). RESULTS: The uterine PSV, TAMV, and PV were greater at the time of the cloprostenol sodium and second GnRH injections (p<0.05) than at the time of the first GnRH injection. The vaginal PSV, PV were greater at the time of the cloprostenol sodium than at the time of the first and second GnRH injections (p<0.05). The receiver operating characteristic curve (ROC curve) indicated a high correlation between the uterine and vaginal blood flow and the rate of the pregnancy (p<0.05). The area under the ROC curve was 0.920 and 0.87 (p<0.05) for vaginal and uterine arteries respectively at time of G2. The serum levels of progesterone, estrogen and cortisol were correlated with the P/AI (p<0.05). The P/AI significantly decreased from 43.9 % at 32 d to 35.37 % at 60 d. CONCLUSION: These results indicate that noninvasive Doppler ultrasonography is a valid method to evaluate changes in the characteristics of uterine and vaginal blood flow in cows during the Ovsynch protocol. Furthermore, vaginal and uterine blood flow are two determinant factors for the higher conception rates in Holstein dairy cows.

A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.