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Introduction: Recurrent uveitis (RU), an autoimmune disease, is a leading cause of ocular detriment in humans and horses. Equine and human RU share many similarities including spontaneous disease and aberrant cytokine signaling. Reduced levels of SOCS1, a critical regulator of cytokine signaling, is associated with several autoimmune diseases. Topical administration of SOCS1-KIR, a peptide mimic of SOCS1, was previously correlated to reduced ocular pathologies within ERU patients. Methods: To further assess the translational potential of a SOCS1 mimetic to treat RU, we assessed peptide-mediated modulation of immune functions in vitro , using equine peripheral blood mononuclear cells (PBMC), and in vivo through topical administration of SOCS1-KIR into the eyes of experimental (non-uveitic) horses. Equine PBMCs from non-uveitic control and ERU horses were cultured with or without SOCS1-KIR pretreatment, followed by 72 hours of mitogen stimulation. Proliferation was assessed using MTT, and cytokine production within cell supernatants was assessed by Luminex. SOCS1-KIR or carrier eye-drops were topically applied to experimental horse eyes twice daily for 21 days, followed by enucleation and isolation of ocular aqueous and vitreous humor. Histology was used to assess peptide treatment safety and localization within treated equine eyes. Cytokine secretion within aqueous humor and vitreous, isolated from experimental equine eyes, was measured by Luminex. Results: Following SOCS1-KIR pretreatment, cell proliferation significantly decreased in control, but not ERU-derived PBMCs. Despite differential regulation of cellular proliferation, SOCS1-KIR significantly reduced TNFα and IL-10 secretion in PHA-stimulated control and ERU equine PBMC. SOCS1-KIR increased PBMC secretion of IL-8. Topically administered SOCS1-KIR was well tolerated. Although SOCS1-KIR was undetectable within the eye, topically treated equine eyes had significant reductions in TNFα and IL-10. Interestingly, we found that while SOCS1-KIR treatment reduced TNFα and IL-10 production in healthy and ERU PBMC, SOCS1-KIR differentially modulated proliferation, IP-10 production, and RANTES within these two groups suggesting possible differences in cell types or activation status. Discussion: Topical administration of a SOCS1 peptide mimic is safe to the equine eye and reduces ERU associated cytokines IL-10 and TNFα serving as potential biomarkers of drug efficacy in a future clinical trial. [ABSTRACT FROM AUTHOR]

Introduction: Human metabolomics has made significant strides in understanding metabolic changes and their implications for human health, with promising applications in diagnostics and treatment, particularly regarding the gut microbiome. However, progress is hampered by issues with data comparability and reproducibility across studies, limiting the translation of these discoveries into practical applications. Objectives: This study aims to evaluate the fit-for-purpose of a suite of human stool samples as potential candidate reference materials (RMs) and assess the state of the field regarding harmonizing gut metabolomics measurements. Methods: An interlaboratory study was conducted with 18 participating institutions. The study allowed for the use of preferred analytical techniques, including liquid chromatography-mass spectrometry (LC–MS), gas chromatography-mass spectrometry (GC–MS), and nuclear magnetic resonance (NMR). Results: Different laboratories used various methods and analytical platforms to identify the metabolites present in human stool RM samples. The study found a 40% to 70% recurrence in the reported top 20 most abundant metabolites across the four materials. In the full annotation list, the percentage of metabolites reported multiple times after nomenclature standardization was 36% (LC–MS), 58% (GC–MS) and 76% (NMR). Out of 9,300 unique metabolites, only 37 were reported across all three measurement techniques. Conclusion: This collaborative exercise emphasized the broad chemical survey possible with multi-technique approaches. Community engagement is essential for the evaluation and characterization of common materials designed to facilitate comparability and ensure data quality underscoring the value of determining current practices, challenges, and progress of a field through interlaboratory studies. [ABSTRACT FROM AUTHOR]

The Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) requires applicants to visit local offices for in-person appointments. The face-to-face nature of WIC has been praised for fostering personal engagement between workers and participants, but some scholars argue that in-person requirements contribute to WIC's low take-up rate (51 percent in 2017). Because of public health concerns during COVID-19, the federal government allowed local WIC agencies to waive in-person requirements. Using linked primary waiver data and administrative WIC data, we implement a difference-in-differences design, comparing WIC caseloads in waiver and nonwaiver counties. We estimate that waivers increased WIC caseloads by about 11 percent, although we fail to detect effects for counties that did not offer modernized document submission options and did not allow applicants to self-attest eligibility or that only offered curbside—rather than fully remote—certification options. Results suggest that relaxing in-person requirements can improve WIC take-up. [ABSTRACT FROM AUTHOR]

The number of reference materials (RMs), including certified reference materials (CRMs) characterized for qualitative properties, has steadily increased in recent years. In response, the Technical Committee for Reference Materials of the International Organization for Standardization, ISO/TC 334, developed ISO 33406 to provide guidance to reference material producers (RMPs) on producing qualitative RMs. ISO 33406 builds on the general requirements in ISO 17034:2016 for RMP competence and offers specific guidance on value assignment, assessment of homogeneity and stability, statement of metrological traceability, and measurement uncertainty evaluation for RMs with qualitative property values. [ABSTRACT FROM AUTHOR]

Employment and earnings instability is common, particularly among low-income families, and can interfere with child-care arrangements. Child-care subsidies are designed to support parents' labor force participation and earnings, but the subsidy program's reach is limited, and enrollment may be hindered by changes in parents' employment or income; in turn, changes in child-care subsidy participation may lead to changes in parents' employment status, hours, or earnings. This study uses longitudinal administrative data from 2016 to 2019 on quarterly employment and earnings and child-care subsidy program participation from Virginia to examine associations between household subsidy receipt and parents' earnings and employment outcomes. Parental employment and earnings increased in the quarters following initial subsidy receipt, which then became stable. Among working parents, we find increased job and earnings stability in the quarters of subsidy receipt but increased multiple jobholding. Findings highlight the importance of child-care subsidies for the employment outcomes of low-income families. [ABSTRACT FROM AUTHOR]

As part of the southern Cascades, Mt. Hood is the tallest and most glaciated peak in Oregon, U.S.A. Despite alpine glaciers being one the clearest indicators of human-caused climate change, the 21st century behavior of glaciers on Mt. Hood has not been directly documented. Here we directly measure changes in Mt. Hood's glacier extents from 2003 to 2023 and find dramatic retreat of all glaciers, with one glacier disappearing, another two nearing this status, and a third retreating towards this status. The seven largest glaciers on the volcano lost ~2.8 km2, or ~40 % of their area in the 21st century. Comparison to historic records of glacier area back to 1907 shows that this 21st-century retreat is unprecedented with respect to the previous century and has outpaced modeled glacier changes. The rate of retreat over the last 23 years is more than double the fastest rate documented in the last century from 1907 to 1946. We demonstrate that this century-scale retreat strongly correlates with regional 30-year-average climate warming of ~1.1 ºC since the early 1900s, but not with regional changes in precipitation. We conclude that Mt. Hood's glaciers are retreating in response to a warming climate and that this recession has accelerated in the 21st century, with attendant consequences for water resources. [ABSTRACT FROM AUTHOR]

As part of the southern Cascades, Mt. Hood is the tallest and most glaciated peak in Oregon, U.S.A. Despite alpine glaciers being one the clearest indicators of human-caused climate change, the 21st century behavior of glaciers on Mt. Hood has not been directly documented. Here we directly measure changes in Mt. Hood's glacier extents from 2003 to 2023 and find dramatic retreat of all glaciers, with one glacier disappearing, another two nearing this status, and a third retreating towards this status. The seven largest glaciers on the volcano lost ~2.8 km², or ~40% of their area in the 21st century. Comparison to historic records of glacier area back to 1907 shows that this 21st-century retreat is unprecedented with respect to the previous century and has outpaced modeled glacier changes. The rate of retreat over the last 23 years is more than double the fastest rate documented in the last century from 1907 to 1946. We demonstrate that this century-scale retreat strongly correlates with regional 30-year-average climate warming of ~1.1°C since the early 1900s, but not with regional changes in precipitation. We conclude that Mt. Hood's glaciers are retreating in response to a warming climate and that this recession has accelerated in the 21st century, with attendant consequences for water resources. [ABSTRACT FROM AUTHOR]

The National Institute of Standards and Technology (NIST) has prepared four seafood reference materials (RMs) for use in food safety and nutrition studies: wild-caught and aquacultured salmon (RM 8256 and RM 8257) and wild-caught and aquacultured shrimp (RM 8258 and RM 8259). These materials were characterized using genetic, metabolomic (1H-NMR, nuclear magnetic resonance and LC-HRMS/MS, liquid chromatography high-resolution tandem mass spectrometry), lipidomic, and proteomic methods to explore their use as matrix-matched, multi-omic differential materials for method development towards identifying product source and/or as quality control in untargeted omics studies. The results from experimental replicates were reproducible for each reference material and analytical method, with the most abundant features reported. Additionally, differences between the materials could be detected, where wild-caught and aquacultured seafood could be distinguished using untargeted metabolite, lipid, and protein analyses. Further processing of the fresh-frozen RMs by freeze-drying revealed the freeze-dried seafoods could still be reliably discerned. These results demonstrate the usefulness of these reference materials as tools for omics instrument validation and measurement harmonization in seafood-related studies. Furthermore, their use as differential quality control (QC) materials, regardless of preparation method, may also provide a tool for laboratories to demonstrate proficiency at discriminating between products based on source/species. [ABSTRACT FROM AUTHOR]

The last decade has witnessed dramatic improvements in whole-genome sequencing capabilities coupled to drastically decreased costs, leading to an inundation of high-quality de novo genomes. For this reason, the continued development of genome quality metrics is imperative. Using the 2016 Atlantic bottlenose dolphin NCBI RefSeq annotation and mass spectrometry-based proteomic analysis of six tissues, we confirmed 10,402 proteins from 4711 protein groups, constituting nearly one-third of the possible predicted proteins. Since the identification of larger proteins with more identified peptides implies reduced database fragmentation and improved gene annotation accuracy, we propose the metric NP10, which attempts to capture this quality improvement. The NP10 metric is calculated by first stratifying proteomic results by identifying the top decile (or 10th 10-quantile) of identified proteins based on the number of peptides per protein and then returns the median molecular weight of the resulting proteins. When using the 2016 versus 2012 Tursiops truncatus genome annotation to search this proteomic data set, there was a 21% improvement in NP10. This metric was further demonstrated by using a publicly available proteomic data set to compare human genome annotations from 2004, 2013 and 2016, which showed a 33% improvement in NP10. These results demonstrate that proteomics may be a useful metrological tool to benchmark genome accuracy, though there is a need for reference proteomic datasets across species to facilitate the evaluation of new de novo and existing genome. [ABSTRACT FROM AUTHOR]

The purpose of the current study was to investigate the feasibility of simultaneously estimating the cellular water efflux rate (kie), intracellular longitudinal relaxation rate (R10i), and intracellular volume fraction (vi) of a cell suspension using multiple samples with different gadolinium concentrations. Numerical simulation studies were conducted to assess the uncertainty in the estimation of kie, R10i, and vi from saturation recovery data using single (SC) or multiple concentrations (MC) of gadolinium‐based contrast agent (GBCA). In vitro experiments with 4 T1 murine breast cancer and SCCVII squamous cell cancer models were conducted at 11 T to compare parameter estimation using the SC protocol with that using the MC protocol. The cell lines were challenged with a Na+/K+‐ATPase inhibitor, digoxin, to assess the treatment response in terms of kie, R10i, and vi. Data analysis was conducted using the two‐compartment exchange model for parameter estimation. The simulation study data demonstrate that the MC method, compared with the SC method, reduces the uncertainty of the estimated kie by decreasing the interquartile ranges from 27.3% ± 3.7% to 18.8% ± 5.1% and the median differences from ground truth from 15.0% ± 6.3% to 7.2% ± 4.2%, while estimating R10i and vi simultaneously. In the cell studies, the MC method demonstrated reduced uncertainty in overall parameter estimation compared with the SC approach. MC method‐measured parameter changes in cells treated with digoxin increased R10i by 11.7% (p = 0.218) and kie by 5.9% (p = 0.234) for 4 T1 cells, respectively, and decreased R10i by 28.8% (p = 0.226) and kie by 1.6% (p = 0.751) for SCCVII cells, respectively. vi did not change noticeably by the treatment. The results of this study substantiate the feasibility of using saturation recovery data of multiple samples with different GBCA concentrations for simultaneous measurement of the cellular water efflux rate, intracellular volume fraction, and intracellular longitudinal relaxation rate in cancer cells. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) is a debilitating mental disorder that can be treated with a number of different antidepressant therapies, each with its own unique prescribing considerations. Complicating the selection of an appropriate antidepressant for adults with MDD is the heterogeneity of clinical profiles and depression subtypes. Additionally, patient comorbidities, preferences, and likelihood of adhering to treatment must all be considered when selecting an appropriate therapy. With the majority of prescriptions being written by primary care practitioners, it is appropriate to review the unique characteristics of all available antidepressants, including safety considerations. Prior to initiating antidepressant treatment and when patients do not respond adequately to initial therapy and/or exhibit any hypomanic or manic symptoms, bipolar disorder must be ruled out, and evaluation for psychiatric comorbidities must be considered as well. Patients with an inadequate response may then require a treatment switch to another drug with a different mechanism of action, combination, or augmentation strategy. In this narrative review, we propose that careful selection of the most appropriate antidepressant for adult patients with MDD based on their clinical profile and comorbidities is vital for initial treatment selection. Strategies must be considered for addressing partial and inadequate responses as well to help patients achieve full remission and sustained functional recovery. This review also highlights data for MDD clinical outcomes for which gaps in the literature have been identified, including the effects of antidepressants on functional outcomes, sleep disturbances, emotional and cognitive blunting, anxiety, and residual symptoms of depression. Major depressive disorder (MDD) is a leading cause of disability worldwide and can affect each patient differently. Antidepressants play a critical role in treatment; however, with multiple antidepressant options available, it is important that providers select the best fit for each patient. Rather than use a "one size fits all" approach, it is important to consider each patient's symptoms, medical and psychiatric comorbidities, as well as their treatment preferences. A clear summary of each antidepressant's distinctive characteristics is essential for providers to select antidepressants to best match each patient's needs. This narrative review aims to discuss the latest information on available antidepressants, including their risks and benefits and how they impact symptoms of MDD such as sleep disturbances, anxiety, emotional blunting, and changes in cognition, as well as different treatment goals, such as the ability to function in everyday life. This information can guide clinical practice recommendations and further enable shared decision-making between the provider and patient, incorporating individual treatment needs and preferences. In addition, many patients do not reach their treatment goals with the first antidepressant or may continue to have symptoms of depression after treatment. This review discusses strategies to increase the likelihood of symptom improvement and creates awareness of patient-specific considerations. Overall, careful, personalized selection of antidepressant treatment is critical for finding the right balance of maximized antidepressant effect with minimized side effects, leading to the best possibility for patients to tolerate the medication and ultimately helping patients reach their treatment goals. [ABSTRACT FROM AUTHOR]

Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples. [ABSTRACT FROM AUTHOR]

Glaucoma is the leading cause of irreversible blindness worldwide. Therapies for glaucoma are directed toward reducing intraocular pressure (IOP), the leading risk factor and only reliable therapeutic target via topical medications or with procedural intervention including laser or surgery. Though topical therapeutics are typically first line, less than 50% of patients take drops as prescribed. Sustained release technologies that decrease IOP for extended periods of time are being examined for clinical use. We recently identified Stanniocalcin-1, a naturally occurring hormone, as an IOP-lowering agent. Here, we show that a single injection into the anterior chamber of mice with an adeno-associated viral vector containing the transgene of stanniocalcin-1 results in diffuse and sustained expression of the protein and produces IOP reduction for up to 6 months. As the treatment effect begins to wane, IOP-lowering can be rescued with a repeat injection. Aqueous humor dynamic studies revealed an increase in outflow facility as the mechanism of action. This first-in-class therapeutic approach has the potential to improve care and reduce the rates of vision loss in the 80 million people worldwide currently affected by glaucoma. [ABSTRACT FROM AUTHOR]

Primary care clinicians have a vital role to play in the diagnosis and management of patients with major depressive disorder (MDD). This includes screening for MDD as well as identifying other possible psychiatric disorders including bipolar disorder and/or other comorbidities. Once MDD is confirmed, partnering with patients in the shared decision-making process while considering different treatment options and best management of MDD over the course of their illness is recommended. Vortioxetine has been approved for the treatment of adults with MDD since 2013, and subsequent US label updates indicate that vortioxetine may be particularly beneficial for specific populations of patients with MDD, including those with treatment-emergent sexual dysfunction and patients experiencing certain cognitive symptoms. Given these recent label updates, this prescribing guide for vortioxetine aims to provide clear and practical guidance for primary care clinicians on the safe and effective use of vortioxetine for the treatment of MDD, including how to identify appropriate patients for treatment. [ABSTRACT FROM AUTHOR]

Introduction: Bevacizumab and ranibizumab, which are anti-vascular endothelial growth factor (VEGF) medications, are used frequently in the treatment for retinopathy of prematurity (ROP) in infants. Aflibercept, or VEGF Trap, has been used anecdotally, but translation and clinical studies are lacking. Objective: This study investigates the efficacy of aflibercept at reducing areas of non-perfused retina and studies its effect on normal angiogenesis in the oxygen-induced retinopathy mouse model of ROP. Methods: C57BL/6 J mice were assigned to room air control (n = 21 eyes) or hyperoxia with 75% oxygen (n = 84 eyes). The hyperoxic mice were assigned to 1 of 3 groups: 0 ng (n = 14 eyes), 100 ng (n = 35 eyes), or 1,000 ng (n = 35 eyes) of intravitreal aflibercept administered on postnatal day 14. Eyes were enucleated at PN17 and PN25 postinjection. Retinas were stained with anti-collagen IV antibody and photographed with microscopy. Areas of perfused and non-perfused retina were quantified using ImageJ software. Statistical comparisons were made using ANOVA with Tukey post hoc comparisons. Results: At PN17, there was no significant difference in the area of non-perfused retina between the hyperoxic control and the 100 and 1,000 ng aflibercept groups. At PN25, the 100 ng (p < 0.05) and 1,000 ng (p = 0.008) treatment groups displayed less non-perfusion compared to hyperoxic controls. At the 1,000 ng dose, there was increased non-perfusion compared to the 100 ng dose (p = 0.02). There was reduced non-perfusion by PN25 compared to PN17 for the 100 ng group (p < 0.05), with no difference in the 1,000 ng group. Conclusions: The study shows that the area of non-perfused retina decreases effectively with aflibercept at PN25 with 100 ng dosage. With the 1,000 ng dosage, there is an inhibition of the physiologic angiogenesis with a higher area of non-perfused retina. [ABSTRACT FROM AUTHOR]

Biological reference materials (RMs) are essential for quality assurance, traceability of measurement results and for method validation. When addressing new measurement questions or emerging regulatory issues, rigorous large-scale CRM production may not be time efficient or economically practical using current production methods. By amending a relatively small matrix batch with a compound(s) of interest at the homogenization step, the National Institute of Standards and Technology (NIST) can create a custom material on an "as-needed" basis and circumvent the time delay inherent in large-batch production, thereby generating a fit-for-purpose, rapid-response RM. Here, Coho salmon (Oncorhynchus kisutch) was cryohomogenized and spiked with an aquaculture antibiotic and antibiotic metabolite. The resultant material was analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) to determine the effectiveness of the amendment technique in a fresh-frozen matrix by assessing homogeneity and accuracy to the target concentration (e.g. mass fraction). Target mass fractions were achieved for both spike components, with RSDs below 5% in replicate measurements of each compound (n = 8). The stability of the spiked compounds was assessed one year post-production and mass fractions were stable, within 1–6% of the initial measurement results, indicating minimal change to the amended analyte concentrations over time. The results support this method as a promising new technique for custom, small-batch RM generation. [ABSTRACT FROM AUTHOR]

A randomized complete block design experiment with 32 yearling crossbred steers (average body weight [BW] = 442 ± 17.0 kg) fed a steam-flaked corn-based diet was used to evaluate the effects of dietary Zn (KemTRACE Zn propionate 27; Kemin Industries, Inc., Des Moines, IA) supplementation on live growth performance, skeletal muscle fiber, and beta-adrenergic receptor (β-AR) characteristics during the finishing phase. Steers were blocked by BW (n = 4 blocks; 8 steers/block), assigned to pens (n = 4 steers/pen), and randomly assigned to the following treatments: control (CON; 0.0 g/[head (hd) · d] of additional Zn) or additional dietary Zn (ZnP; 1.0 g/[hd · d] additional Zn). The basal diet contained Zn (60 ppm dry matter basis) from ZnSO4; additional Zn was top-dressed at feeding. Ractopamine hydrochloride (RH; Optaflexx: Elanco Animal Health, Greenfield, IN) was included at 300 mg/(hd · d) for the final 28 d of the 111-d feeding period. Longissimus muscle biopsy samples, BW, and blood were obtained on days 0, 42, 79, and 107. Final BW was collected prior to shipping on day 111. Biopsy samples were used for immunohistochemical (IHC), mRNA, and protein analysis. Serum urea nitrogen (SUN) and nonesterified fatty acid (NEFA) concentrations were measured. Steers fed ZnP had a greater average daily gain (P = 0.02) and gain to feed ratio (G:F; P = 0.03) during the RH feeding period compared with CON. There were no differences (P > 0.05) in other growth performance variables, carcass traits, mRNA abundance, or relative protein concentration for fiber type and β-AR. Fiber types I and IIA had no differences in the cross-sectional area; however, the IIX area was greater for CON (P < 0.04) compared with ZnP and increased (P < 0.02) over time. There were no differences between treatments for the β1-AR density (P > 0.05) in skeletal muscle tissue throughout the study. A treatment × day interaction was observed in β2-AR density (P = 0.02) and β3-AR density (P = 0.02) during the RH feeding period, where the abundance of the receptors increased with ZnP but did not change in CON. Compared with CON, ZnP had greater (P < 0.01) mean NEFA concentrations. Mean SUN concentrations did increase by day (P < 0.01). Additional dietary Zn, supplied as Zn propionate, upregulates β2-AR and β3-AR and improves growth performance in feedlot steers during the RH feeding period, likely through a shift of resource utilization from lipogenesis to muscle maintenance and hypertrophy. [ABSTRACT FROM AUTHOR]

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALK F1174L ) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALK F1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK -mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN , but not FN1 , delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling. [Display omitted] • ALK cooperates with MYCN to accelerate tumorigenesis in human neural crest cells • ALK tumors activate focal adhesion signaling via extracellular matrix proteins • POSTN and WNT signaling are involved in ALK-mediated tumor growth Huang et al. used a human stem cell model to elucidate the mechanism for cooperation between MYCN and ALK. ALK contributes to tumor growth by upregulating the extracellular matrix protein periostin and activating WNT signaling. Periostin and WNT signal through a feedforward loop. [ABSTRACT FROM AUTHOR]

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin‐1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT‐PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single‐cell RNA‐sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock‐in mouse with a hemagglutinin (HA) epitope‐tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1‐specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

The National Institute of Standards and Technology (NIST) is creating new, economical, qualitative reference materials and data for proteomics comparisons, benchmarking and harmonization. Here we describe a large dataset from shotgun proteomic analysis of RM 8461 Human Liver for Proteomics, a reference material being developed. Consensus identifications using multiple search engines and sample preparations demonstrate a homogeneous and fit-for-purpose material that can be incorporated into automated or manual sample preparation workflows, with the resulting data used to directly assess complete sample-to-data workflows and provide harmonization and benchmarking between laboratories and techniques. Data are available via PRIDE with identifier PXD013608. Measurement(s) peptide sequence-level identification attribute • protein expression profiling Technology Type(s) liquid chromatography-tandem mass spectrometry Factor Type(s) mass of liver sample Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11310485 [ABSTRACT FROM AUTHOR]

Purpose: Radiation dose calculated on tumors for radiopharmaceutical therapy varies significantly from tumor to tumor and from patient to patient. Accurate estimation of radiation dose requires multiple time point measurements using radionuclide imaging modalities such as SPECT or PET. In this report, we show our technical development of reducing the number of scans needed for reasonable estimation of tumor and normal organ dose in our pretherapy imaging and dosimetry platform of 124I‐metaiodobenzylguanidine (MIBG) positron emission tomography/computed tomography (PET/CT) for 131I‐MIBG therapy of neuroblastoma. Methods: We analyzed the simplest kinetic data, areas of two‐time point data for five patients with neuroblastoma who underwent 3 or 4 times of 124I‐MIBG PET/CT scan prior to 131I‐MIBG therapy. The data for which we derived areas were percent of injected activity (%IA) and standardized uptake value of tumors. These areas were correlated with time‐integrated activity coefficients (TIACs) from full data (3 or 4 time points). TIACs are direct correlates with radiation dose as long as the volume and the radionuclide are known. Results: The areas of %IAs between data obtained from all the two‐time points with time points 1 and 2 (day 0 and day 1), time points 2 and 3 (day 1 and day 2), and time points 1 and 3 (day 0 and day 2) showed reasonable correlation (Pearson's correlation coefficient |r| > 0.5) with not only tumor and organ TIACs but also tumor and organ absorbed doses. The tumor and organ doses calculated using %IA areas of time point 1 and time point 2 were our best fits at about 20% individual percent difference compared to doses calculated using 3 or 4 time points. Conclusions: We could achieve reasonable accuracy of estimating tumor doses for subsequent radiopharmaceutical therapy using only the two‐time point imaging sessions. Images obtained from these time points (within the 48‐h after administration of radiopharmaceutical) were also viewed as useful for diagnostic reading. Although our analysis was specific to 124I‐MIBG PET/CT pretherapy imaging data for 131I‐MIBG therapy of neuroblastoma and the number of imaging datasets was not large, this feasible methodology would generally be applicable to other imaging and therapeutic radionuclides with an appropriate data analysis similar to our analysis to other imaging and therapeutic radiopharmaceuticals. [ABSTRACT FROM AUTHOR]

This study involved 108 subjects who were randomly assigned to individual (contingent on role performance) and systems (contingent on participation) reward conditions. Analysis of variance found that satisfaction was lower in the individual reward conditions even though it was correlated positively to performance. Although those who did not want to participate again were dissatisfied, they were not, on the average lower performers. [ABSTRACT FROM AUTHOR]

A Standard Reference Material (SRM) of seaweed, SRM 3232 Kelp Powder ( Thallus laminariae) has been developed to support food and dietary supplement measurements in compliance with the Food Safety Modernization Act (FSMA) and the Dietary Supplement Health and Education Act of 1994 (DSHEA). The material was characterized for nutritional minerals, arsenic species, isomers of vitamin K, proximates, and toxic elements. Kelp is a rich source of vitamins and minerals, and it is an excellent source of dietary iodine. Kelp also contains a large amount of arsenic, which is toxic as inorganic species but much less so as organic species. To capture the dietary profile of kelp, certified values were issued for As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Mo, Na, Pb, and Zn. Reference values for proximates were assigned. For the first time, a certified value for iodine, reference values for isomers of vitamin K, and reference values for arsenic species including arsenosugars were assigned in a seaweed. SRM 3232 fills a gap in Certified Reference Materials (CRMs) needed for quality assurance and method validation in the compositional measurements of kelp and similar seaweeds used as food and as dietary supplements. [ABSTRACT FROM AUTHOR]

The APOBEC3 family of cytidine deaminases cause lethal hypermutation of retroviruses via deamination of newly reverse-transcribed viral DNA. Their ability to bind RNA is essential for virion infiltration and antiviral activity, yet the mechanisms of viral RNA recognition are unknown. By screening naturally occurring, polymorphic, non-human primate APOBEC3H variants for biological and crystallization properties, we obtained a 2.24-Å crystal structure of pig-tailed macaque APOBEC3H with bound RNA. Here, we report that APOBEC3H forms a dimer around a short RNA duplex and, despite the bound RNA, has potent cytidine deaminase activity. The structure reveals an unusual RNA-binding mode in which two APOBEC3H molecules at opposite ends of a seven-base-pair duplex interact extensively with both RNA strands, but form no protein-protein contacts. CLIP-seq analysis revealed that APOBEC3H preferentially binds to sequences in the viral genome predicted to contain duplexes, a property that may facilitate both virion incorporation and catalytic activity. [ABSTRACT FROM AUTHOR]

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF) after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin) decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules. [ABSTRACT FROM AUTHOR]

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder. [ABSTRACT FROM AUTHOR]

Purpose: Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5) protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina. Methods: Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins. Results: PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold) less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium of photoreceptors, where BBS5 is also localized. Like BBS5, the binding of BBS5L to arrestin1 can be modulated by phosphorylation through protein kinase C. Conclusions: In this study we have identified a novel splice variant of BBS5 that appears to be expressed only in the retina. The BBS5 splice variant is expressed at approximately 10% of full-length BBS5 level. No unique functional or localization properties could be identified for the splice variant compared to BBS5. [ABSTRACT FROM AUTHOR]

Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1-/- neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. [ABSTRACT FROM AUTHOR]

Glial cells play a critical role in shaping neuronal development, structure, and function. In a screen for Caenorhabditis elegans mutants that display dopamine (DA)-dependent, Swimming-Induced Paralysis (Swip), we identified a novel gene, swip-10, the expression of which in glia is required to support normal swimming behavior. swip-10 mutants display reduced locomotion rates on plates, consistent with our findings of elevated rates of presynapticDAvesicle fusion using fluorescence recovery after photobleaching. In addition, swip-10 mutants exhibit elevated DA neuron excitability upon contact with food, as detected by in vivo Ca2+ monitoring, that can be rescued by glial expression of swip-10. Mammalian glia exert powerful control of neuronal excitability via transporter-dependent buffering of extracellular glutamate (Glu). Consistent with this idea, swip-10 paralysis was blunted in mutants deficient in either vesicular Glu release or Glu receptor expression and could be phenocopied by mutations that disrupt the function of plasma membrane Glu transporters, most noticeably glt-1, the ortholog of mammalian astrocytic GLT1 (EAAT2). swip-10 encodes a protein containing a highly conserved metallo- β-lactamase domain, within which our swip-10 mutations are located and where engineered mutations disrupt Swip rescue. Sequence alignments identify the CNS-expressed gene MBLAC1 as a putative mammalian ortholog. Together, our studies provide evidence of a novel pathway in glial cells regulated by swip-10 that limits DA neuron excitability, DA secretion, and DA-dependent behaviors through modulation of Glu signaling. [ABSTRACT FROM AUTHOR]

The MYCN proto-oncogene is associated with poor outcome across a broad range of pediatric tumors. While amplification of MYCN drives subsets of high-risk neuroblastoma and medulloblastoma, dysregulation of MYCN in medulloblastoma (in the absence of amplification) also contributes to pathogenesis. Since PI3K stabilizes MYCN, we have used inhibitors of PI3K to drive degradation. In this study, we show PI3K inhibitors by themselves induce cell cycle arrest, with modest induction of apoptosis. In screening inhibitors of PI3K against MYCN, we identified PIK-75 and its derivative, PW-12, inhibitors of both PI3K and of protein kinases, to be highly effective in destabilizing MYCN. To determine the effects of PW-12 treatment in vivo, we analyzed a genetically engineered mouse model for MYCN-driven neuroblastoma and a model of MYCN-driven medulloblastoma. PW-12 showed significant activity in both models, inducing vascular collapse and regression of medulloblastoma with prominent apoptosis in both models. These results demonstrate that inhibitors of lipid and protein kinases can drive apoptosis in MYCN-driven cancers and support the importance of MYCN as a therapeutic target. [ABSTRACT FROM AUTHOR]

Summary The development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy. [ABSTRACT FROM AUTHOR]

Background: The simple and well-described structure of the C. elegans nervous system offers an unprecedented opportunity to identify the genetic programs that define the connectivity and function of individual neurons and their circuits. A correspondingly precise gene expression map of C. elegans neurons would facilitate the application of genetic methods toward this goal. Here we describe a powerful new approach, SeqCeL (RNA-Seq of C. elegans cells) for producing gene expression profiles of specific larval C. elegans neurons. Methods and Results: We have exploited available GFP reporter lines for FACS isolation of specific larval C. elegans neurons for RNA-Seq analysis. Our analysis showed that diverse classes of neurons are accessible to this approach. To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the C. elegans pharynx. These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes. Significance: This work offers a simple and robust protocol for expression profiling studies of post-embryonic C. elegans neurons and thus provides an important new method for identifying candidate genes for key roles in neuron-specific development and function. [ABSTRACT FROM AUTHOR]

An emergent challenge in macromolecular crystallography is the identification of the substructure from native anomalous scatterers in crystals that diffract to low to moderate resolution. Increasing the multiplicity of data sets has been shown to make previously intractable phasing problems solvable and to increase the useful resolution in model refinement. For the West Nile virus nonstructural protein 1 (NS1), a protein of novel fold, the utility of exceptionally high multiplicity data is demonstrated both in solving the crystal structure from the anomalous scattering of the native S atoms and in extending the useful limits of resolution during refinement. A high-multiplicity data set from 18 crystals had sufficient anomalous signal to identify sulfur sites using data to 5.2 Å resolution. Phases calculated to 4.5 Å resolution and extended to 3.0 Å resolution were of sufficient quality for automated building of three-quarters of the final structure. Crystallographic refinement to 2.9 Å resolution proceeded smoothly, justifying the increase in resolution that was made possible by combining multiple data sets. The identification and exclusion of data from outlier crystals is shown to result in more robust substructure determination. [ABSTRACT FROM AUTHOR]