Preservation of Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs) in Late Adult Mice: Implications as a Potential Biomarker for Early Onset Ocular Degenerative Diseases.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in non-image-forming visual functions. Given their significant loss observed in various ocular degenerative diseases at early stages, this study aimed to assess changes in both the morphology and associated behavioral functions of ipRGCs in mice between 6 (mature) and 12 (late adult) months old. The findings contribute to understanding the preservation of ipRGCs in late adults and their potential as a biomarker for early ocular degenerative diseases. Methods: Female and male C57BL/6J mice were used to assess the behavioral consequences of aging to mature and old adults, including pupillary light reflex, light aversion, visual acuity, and contrast sensitivity. Immunohistochemistry on retinal wholemounts from these mice was then conducted to evaluate ipRGC dendritic morphology in the ganglion cell layer (GCL) and inner nuclear layer (INL). Results: Morphological analysis showed that ipRGC dendritic field complexity was remarkably stable through 12 months old of age. Similarly, the pupillary light reflex, visual acuity, and contrast sensitivity were stable in mature and old adults. Although alterations were observed in ipRGC-independent light aversion distinct from the pupillary light reflex, aged wild-type mice continuously showed enhanced light aversion with dilation. No effect of sex was observed in any tests. Conclusions: The preservation of both ipRGC morphology and function highlights the potential of ipRGC-mediated function as a valuable biomarker for ocular diseases characterized by early ipRGC loss. The consistent stability of ipRGCs in mature and old adult mice suggests that detected changes in ipRGC-mediated functions could serve as early indicators or diagnostic tools for early-onset conditions such as Alzheimer's disease, Parkinson's disease, and diabetes, where ipRGC loss has been documented.

Organ-Chips Enhance the Maturation of Human iPSC-Derived Dopamine Neurons.

While cells in the human body function in an environment where the blood supply constantly delivers nutrients and removes waste, cells in conventional tissue culture well platforms are grown with a static pool of media above them and often lack maturity, limiting their utility to study cell biology in health and disease. In contrast, organ-chip microfluidic systems allow the growth of cells under constant flow, more akin to the in vivo situation. Here, we differentiated human induced pluripotent stem cells into dopamine neurons and assessed cellular properties in conventional multi-well cultures and organ-chips. We show that organ-chip cultures, compared to multi-well cultures, provide an overall greater proportion and homogeneity of dopaminergic neurons as well as increased levels of maturation markers. These organ-chips are an ideal platform to study mature dopamine neurons to better understand their biology in health and ultimately in neurological disorders.

Linked candidate genes of different functions for white mold resistance in common bean (Phaseolus vulgaris L) are identified by multiple QTL mapping approaches.

White mold (WM) is a major disease in common bean (Phaseolus vulgaris L.), and its complex quantitative genetic control limits the development of WM resistant cultivars. WM2.2, one of the nine meta-QTL with a major effect on WM tolerance, explains up to 35% of the phenotypic variation and was previously mapped to a large genomic interval on Pv02. Our objective was to narrow the interval of this QTL using combined approach of classic QTL mapping and QTL-based bulk segregant analysis (BSA), and confirming those results with Khufu de novo QTL-seq. The phenotypic and genotypic data from two RIL populations, 'Raven'/I9365-31 (R31) and 'AN-37'/PS02-029C-20 (Z0726-9), were used to select resistant and susceptible lines to generate subpopulations for bulk DNA sequencing. The QTL physical interval was determined by considering overlapping interval of the identified QTL or peak region in both populations by three independent QTL mapping analyses. Our findings revealed that meta-QTL WM2.2 consists of three regions, WM2.2a (4.27-5.76 Mb; euchromatic), WM 2.2b (12.19 to 17.61 Mb; heterochromatic), and WM2.2c (23.01-25.74 Mb; heterochromatic) found in both populations. Gene models encoding for gibberellin 2-oxidase 8, pentatricopeptide repeat, and heat-shock proteins are the likely candidate genes associated with WM2.2a resistance. A TIR-NBS-LRR class of disease resistance protein (Phvul.002G09200) and LRR domain containing family proteins are potential candidate genes associated with WM2.2b resistance. Nine gene models encoding disease resistance protein [pathogenesis-related thaumatin superfamily protein and disease resistance-responsive (dirigent-like protein) family protein etc] found within the WM2.2c QTL interval are putative candidate genes. WM2.2a region is most likely associated with avoidance mechanisms while WM2.2b and WM2.2c regions trigger physiological resistance based on putative candidate genes.

Integrating de novo QTL-seq and linkage mapping to identify quantitative trait loci conditioning physiological resistance and avoidance to white mold disease in dry bean.

White mold (WM), caused by the ubiquitous fungus Sclerotinia sclerotiorum, is a devastating disease that limits production and quality of dry bean globally. In the present study, classic linkage mapping combined with QTL-seq were employed in two recombinant inbred line (RIL) populations, "Montrose"/I9365-25 (M25) and "Raven"/I9365-31 (R31), with the initial goal of fine-mapping QTL WM5.4 and WM7.5 that condition WM resistance. The RILs were phenotyped for WM reactions under greenhouse (straw test) and field environments. The general region of WM5.4 and WM7.5 were reconfirmed with both mapping strategies within each population. Combining the results from both mapping strategies, WM5.4 was delimited to a 22.60-36.25 Mb interval in the heterochromatic regions on Pv05, while WM7.5 was narrowed to a 0.83 Mb (3.99-4.82 Mb) region on the Pv07 chromosome. Furthermore, additional QTL WM2.2a (3.81-7.24 Mb), WM2.2b (11.18-17.37 Mb, heterochromatic region), and WM2.2c (23.33-25.94 Mb) were mapped to a narrowed genomic interval on Pv02 and WM4.2 in a 0.89 Mb physical interval at the distal end of Pv04 chromosome. Gene models encoding gibberellin 2-oxidase proteins regulating plant architecture are likely candidate genes associated with WM2.2a resistance. Nine gene models encoding a disease resistance protein (quinone reductase family protein and ATWRKY69) found within the WM5.4 QTL interval are putative candidate genes. Clusters of 13 and 5 copies of gene models encoding cysteine-rich receptor-like kinase and receptor-like protein kinase-related family proteins, respectively, are potential candidate genes associated with WM7.5 resistance and most likely trigger physiological resistance to WM. Acquired knowledge of the narrowed major QTL intervals, flanking markers, and candidate genes provides promising opportunities to develop functional molecular markers to implement marker-assisted selection for WM resistant dry bean cultivars.

Conserved and variable heat stress responses of the Heat Shock Factor transcription factor family in maize and Setaria viridis.

The Heat Shock Factor (HSF) transcription factor family is a central and required component of plant heat stress responses and acquired thermotolerance. The HSF family has dramatically expanded in plant lineages, often including a repertoire of 20 or more genes. Here we assess and compare the composition, heat responsiveness, and chromatin profiles of the HSF families in maize and Setaria viridis (Setaria), two model C4 panicoid grasses. Both species encode a similar number of HSFs, and examples of both conserved and variable expression responses to a heat stress event were observed between the two species. Chromatin accessibility and genome-wide DNA-binding profiles were generated to assess the chromatin of HSF family members with distinct responses to heat stress. We observed significant variability for both chromatin accessibility and promoter occupancy within similarly regulated sets of HSFs between Setaria and maize, as well as between syntenic pairs of maize HSFs retained following its most recent genome duplication event. Additionally, we observed the widespread presence of TF binding at HSF promoters in control conditions, even at HSFs that are only expressed in response to heat stress. TF-binding peaks were typically near putative HSF-binding sites in HSFs upregulated in response to heat stress, but not in stable or not expressed HSFs. These observations collectively support a complex scenario of expansion and subfunctionalization within this transcription factor family and suggest that within-family HSF transcriptional regulation is a conserved, defining feature of the family.

Mapping responsive genomic elements to heat stress in a maize diversity panel.

BACKGROUND: Many plant species exhibit genetic variation for coping with environmental stress. However, there are still limited approaches to effectively uncover the genomic region that regulates distinct responsive patterns of the gene across multiple varieties within the same species under abiotic stress. RESULTS: By analyzing the transcriptomes of more than 100 maize inbreds, we reveal many cis- and trans-acting eQTLs that influence the expression response to heat stress. The cis-acting eQTLs in response to heat stress are identified in genes with differential responses to heat stress between genotypes as well as genes that are only expressed under heat stress. The cis-acting variants for heat stress-responsive expression likely result from distinct promoter activities, and the differential heat responses of the alleles are confirmed for selected genes using transient expression assays. Global footprinting of transcription factor binding is performed in control and heat stress conditions to document regions with heat-enriched transcription factor binding occupancies. CONCLUSIONS: Footprints enriched near proximal regions of characterized heat-responsive genes in a large association panel can be utilized for prioritizing functional genomic regions that regulate genotype-specific responses under heat stress.

Prediction of conserved and variable heat and cold stress response in maize using cis-regulatory information.

Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes.

Calbindin expression in adult vestibular epithelia.

The mammalian vestibular epithelia exhibit a remarkably stereotyped organization featuring cellular characteristics under planar cell polarity (PCP) control. PCP mechanisms are responsible for the organization of hair cell morphologic polarization vectors, and are thought to be responsible for the postsynaptic expression of the calcium-binding protein calretinin that defines the utricular striola and cristae central zone. However, recent analyses revealed that subtle differences in the topographic expression of oncomodulin, another calcium-binding protein, reflects heterogeneous factors driving the subtle variations in expression. Calbindin represents a third calcium-binding protein that has been previously described to be expressed in both hair cells and afferent calyces in proximity to the utricular striola and crista central zone. The objective of the present investigation was to determine calbindin's topographic pattern of expression to further elucidate the extent to which PCP mechanisms might exert control over the organization of vestibular neuroepithelia. The findings revealed that calbindin exhibited an expression pattern strikingly similar to oncomodulin. However, within calyces of the central zone calbindin was colocalized with calretinin. These results indicate that organizational features of vestibular epithelia are governed by a suite of factors that include PCP mechanisms as well others yet to be defined.

New developments on the Encyclopedia of DNA Elements (ENCODE) data portal.

The Encyclopedia of DNA Elements (ENCODE) is an ongoing collaborative research project aimed at identifying all the functional elements in the human and mouse genomes. Data generated by the ENCODE consortium are freely accessible at the ENCODE portal (https://www.encodeproject.org/), which is developed and maintained by the ENCODE Data Coordinating Center (DCC). Since the initial portal release in 2013, the ENCODE DCC has updated the portal to make ENCODE data more findable, accessible, interoperable and reusable. Here, we report on recent updates, including new ENCODE data and assays, ENCODE uniform data processing pipelines, new visualization tools, a dataset cart feature, unrestricted public access to ENCODE data on the cloud (Amazon Web Services open data registry, https://registry.opendata.aws/encode-project/) and more comprehensive tutorials and documentation.

The ENCODE Portal as an Epigenomics Resource.

The Encyclopedia of DNA Elements (ENCODE) web portal hosts genomic data generated by the ENCODE Consortium, Genomics of Gene Regulation, The NIH Roadmap Epigenomics Consortium, and the modENCODE and modERN projects. The goal of the ENCODE project is to build a comprehensive map of the functional elements of the human and mouse genomes. Currently, the portal database stores over 500 TB of raw and processed data from over 15,000 experiments spanning assays that measure gene expression, DNA accessibility, DNA and RNA binding, DNA methylation, and 3D chromatin structure across numerous cell lines, tissue types, and differentiation states with selected genetic and molecular perturbations. The ENCODE portal provides unrestricted access to the aforementioned data and relevant metadata as a service to the scientific community. The metadata model captures the details of the experiments, raw and processed data files, and processing pipelines in human and machine-readable form and enables the user to search for specific data either using a web browser or programmatically via REST API. Furthermore, ENCODE data can be freely visualized or downloaded for additional analyses. © 2019 The Authors. Basic Protocol: Query the portal Support Protocol 1: Batch downloading Support Protocol 2: Using the cart to download files Support Protocol 3: Visualize data Alternate Protocol: Query building and programmatic access.

NUCLEAR FACTOR-Y: still complex after all these years?

The NUCLEAR FACTOR-Y (NF-Y) families of transcription factors are important regulators of plant development and physiology. Though NF-Y regulatory roles have recently been suggested for numerous aspects of plant biology, their roles in flowering time, early seedling development, stress responses, hormone signaling, and nodulation are the best characterized. The past few years have also seen significant advances in our understanding of the mechanistic function of the NF-Y, and as such, increasingly complex and interesting questions are now more approachable. This review will primarily focus on these developmental, physiological, and mechanistic roles of the NF-Y in recent research.

Evaluation of a pharmacy-driven methicillin-resistant Staphylococcus aureus surveillance protocol in pneumonia.

Background Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of pneumonia and clinicians must determine when empiric antimicrobial therapy directed toward MRSA is needed. Objective To evaluate the effect of a pharmacy-driven protocol utilizing the nasal swab MRSA polymerase chain reaction (PCR) test to discontinue vancomycin on duration of vancomycin therapy and clinical outcomes in patients with suspected community-acquired pneumonia (CAP) or healthcare-associated pneumonia (HCAP). Setting A teaching hospital in Huntington, WV, USA. Methods This retrospective study included adult patients who received at least one dose of vancomycin for suspected CAP or HCAP. The pre-intervention group consisted of patients prior to the addition of the nasal swab MRSA PCR test to the CAP/HCAP order set. The post-intervention group consisted of patients after the addition of the nasal swab MRSA PCR test to the CAP/HCAP order set. Main outcome measure The primary outcome was vancomycin hours of therapy. Results Of the 196 patients included in the study, 121 patients were in the pre-intervention group and 75 patients were in the post-intervention group. The median duration of vancomycin therapy was significantly shorter in the post-intervention group than the pre-intervention group (49 vs. 18 h, p < 0.001). There were no statistically significant differences in the secondary outcomes including hospital length of stay, 30-day readmission rate, and in-hospital all-cause mortality. Conclusion The addition of a pharmacy-driven protocol utilizing the nasal swab MRSA PCR test was associated with shorter duration of empiric vancomycin therapy by approximately 31 h per patient without increasing adverse clinical outcomes.

The multifaceted roles of NUCLEAR FACTOR-Y in Arabidopsis thaliana development and stress responses.

NUCLEAR FACTOR-Y (NF-Y) is a heterotrimeric transcription factor (TF) consisting of evolutionarily distinct NF-YA, NF-YB and NF-YC subunits. The functional NF-Y heterotrimer binds to CCAAT elements in eukaryotic gene promoters and influences their expression. The genome of the model organism Arabidopsis thaliana encodes 10 distinct NF-YA, NF-YB, and NF-YC proteins, allowing for enormous combinatorial and functional diversity. Two decades of research have elucidated the importance of NF-Ys in plant growth, development and stress responses; however, the molecular mechanisms of action remain largely unexplored. Intriguingly, recent evidence suggests that NF-Ys are frequently associated with other groups of TFs, expanding the potential NF-Y combinatorial complexity. Further, information regarding the regulation of individual NF-Y subunits at the transcriptional and post-transcriptional level is beginning to emerge. In this review, we will identify developing trends within the NF-Y field and discuss recent progress towards a better understanding of NF-Y function, molecular action, and regulation in the context of Arabidopsis. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

NUCLEAR FACTOR Y, Subunit A (NF-YA) Proteins Positively Regulate Flowering and Act Through FLOWERING LOCUS T.

Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal "CORE" elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.

NUCLEAR FACTOR Y, Subunit C (NF-YC) Transcription Factors Are Positive Regulators of Photomorphogenesis in Arabidopsis thaliana.

Recent reports suggest that NF-Y transcription factors are positive regulators of skotomorphogenesis in Arabidopsis thaliana. Three NF-YC genes (NF-YC3, NF-YC4, and NF-YC9) are known to have overlapping functions in photoperiod dependent flowering and previous studies demonstrated that they interact with basic leucine zipper (bZIP) transcription factors. This included ELONGATED HYPOCOTYL 5 (HY5), which has well-demonstrated roles in photomorphogenesis. Similar to hy5 mutants, we report that nf-yc3 nf-yc4 nf-yc9 triple mutants failed to inhibit hypocotyl elongation in all tested light wavelengths. Surprisingly, nf-yc3 nf-yc4 nf-yc9 hy5 mutants had synergistic defects in light perception, suggesting that NF-Ys represent a parallel light signaling pathway. As with other photomorphogenic transcription factors, nf-yc3 nf-yc4 nf-yc9 triple mutants also partially suppressed the short hypocotyl and dwarf rosette phenotypes of CONSTITUTIVE PHOTOMORPHOGENIC 1 (cop1) mutants. Thus, our data strongly suggest that NF-Y transcription factors have important roles as positive regulators of photomorphogenesis, and in conjunction with other recent reports, implies that the NF-Y are multifaceted regulators of early seedling development.

Physician ePortfolio: the missing piece for linking performance with improvement.

The movement toward improvement in healthcare quality and patient safety has led to greater emphasis on practice performance measurement and physician accountability. Health information technology provides clinical data for quality measurement but hasn't provided the link to practice-based learning and improvement. An electronic portfolio for practice-based learning and improvement (ePortfolio) that combines practice data for identification of competency and performance gaps along with learning and process interventions offers true practice-based learning and performance improvement. Automated reporting can assist in the ever-increasing burden of documentation for maintenance of licensure, maintenance of specialty board certification, credentialing, payer recognition programs, and other physician accountability requirements.