Arabidopsis thaliana RHAMNOSE 1 condensate formation drives UDP-rhamnose synthesis.

Rhamnose is an essential component of the plant cell wall and is synthesized from uridine diphosphate (UDP)-glucose by the RHAMNOSE1 (RHM1) enzyme. RHM1 localizes to biomolecular condensates in plants, but their identity, formation, and function remain elusive. Combining live imaging, genetics, and biochemical approaches in Arabidopsis and heterologous systems, we show that RHM1 alone is sufficient to form enzymatically active condensates, which we name rhamnosomes. Rhamnosome formation is required for UDP-rhamnose synthesis and organ development. Overall, our study demonstrates a novel role for biomolecular condensation in metabolism and organismal development, and provides further support for how organisms have harnessed this biophysical process to regulate small molecule metabolism.

Chloroplast phosphatases LPPγ and LPPε1 facilitate conversion of extraplastidic phospholipids to galactolipids.

Galactolipids comprise the majority of chloroplast membranes in plants, and their biosynthesis requires dephosphorylation of phosphatidic acid at the chloroplast envelope membranes. In Arabidopsis (Arabidopsis thaliana), the lipid phosphate phosphatases LPPγ, LPPε1, and LPPε2 have been previously implicated in chloroplast lipid assembly, with LPPγ being essential, as null mutants were reported to exhibit embryo lethality. Here, we show that lppγ mutants are in fact viable and that LPPγ, LPPε1, and LPPε2 do not appear to have central roles in the plastid pathway of membrane lipid biosynthesis. Redundant LPPγ and LPPε1 activity at the outer envelope membrane is important for plant development, and the respective lppγ lppε1 double mutant exhibits reduced flux through the ER pathway of galactolipid synthesis. While LPPε2 is imported and associated with interior chloroplast membranes, its role remains elusive and does not include basal nor phosphate limitation-induced biosynthesis of glycolipids. The specific physiological roles of LPPγ, LPPε1, and LPPε2 are yet to be uncovered, as does the identity of the phosphatidic acid phosphatase required for plastid galactolipid biosynthesis.

Integrated omics analysis unveils a DNA damage response to neurogenic injury.

Spinal cord injury (SCI) evokes profound bladder dysfunction. Current treatments are limited by a lack of molecular data to inform novel therapeutic avenues. Previously, we showed systemic inosine treatment improved bladder function following SCI in rats. Here, we applied multi-omics analysis to explore molecular alterations in the bladder and their sensitivity to inosine following SCI. Canonical pathways regulated by SCI included those associated with protein synthesis, neuroplasticity, wound healing, and neurotransmitter degradation. Upstream regulator analysis identified MYC as a key regulator, whereas causal network analysis predicted multiple regulators of DNA damage response signaling following injury, including PARP-1. Staining for both DNA damage (γH2AX) and PARP activity (poly-ADP-ribose) markers in the bladder was increased following SCI, and attenuated in inosine-treated tissues. Proteomics analysis suggested that SCI induced changes in protein synthesis-, neuroplasticity-, and oxidative stress-associated pathways, a subset of which were shown in transcriptomics data to be inosine-sensitive. These findings provide novel insights into the molecular landscape of the bladder following SCI, and highlight a potential role for PARP inhibition to treat neurogenic bladder dysfunction.

Redox regulation in chloroplast thylakoid lumen: The pmf changes everything, again.

Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force (pmf) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.

Understanding the needs of women undergoing breast ultrasound: Are male radiologists still needed?

INTRODUCTION: A trend towards less male radiologists specializing in breast ultrasound was observed. A common notion in the field of breast radiology is, that female patients feel more comfortable being treated by female radiologists. The aim of the study was to understand and report the needs of women undergoing breast ultrasound with regards to the sex of the radiologist performing the investigation. METHODS: Informed consent was obtained from all patients prior to inclusion in a prospective bi-center quality study. At center 1 (72 patients), the women were examined exclusively by female radiologists, at center 2 (100 patients) only by male radiologists. After the examination the patients were asked about their experiences and their wishes for the future. RESULTS: Overall, women made no distinction between female and male radiologists; 25% of them wanted a female radiologist and 1.2% wanted a male radiologist. The majority (74%) stated that it made no difference whether a female or male radiologist performed the examination. The majority of women in group 2, who were investigated exclusively by male radiologists, stated that they had no preferences with regard to the sex of the radiologist (93%); 5% of the women wished to be investigated solely by a female radiologist and 2% exclusively by a male radiologist. DISCUSSION: The majority of women undergoing breast ultrasound are unconcerned about the radiologist's sex. It would appear that women examined by male radiologists are less selective about the sex of the examining radiologist. TRIAL REGISTRATION: Written informed consent was obtained from all patients. All patient data were anonymized. The physicians had no access to any further personal data. National regulations did not require dedicated ethics approval with anonymized lists or retrospective questionnaires.

Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis.

ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

Arabidopsis stromal carbonic anhydrases exhibit non-overlapping roles in photosynthetic efficiency and development.

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO2 to HCO3 - . The Arabidopsis genome encodes members of the α-, ß- and γ-CA families, and it has been hypothesized that ßCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial ßCAs, ßCA1 and ßCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of ßCA5 induced the expression of ßCA1, supporting the existence of regulatory mechanisms to control the expression of stromal ßCAs. We also established that ßCA1 and ßCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that ßCA5 had a first-order rate constant ~10-fold lower than ßCA1, and that the loss of ßCA5 is detrimental to growth and could be rescued by high CO2 . Furthermore, we established that, while a ßCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of ßCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed ßCA1 does not compensate for the loss of a less active ßCA5, which in turn is involved in growth and photosynthesis at ambient CO2 levels. These results lend support to the hypothesis that, in Arabidopsis,ßCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal ßCA5 and a dispensable role for ßCA1.

Association between urinary biomarkers MMP-7/TIMP-2 and reduced renal function in children with ureteropelvic junction obstruction.

IMPORTANCE: Extracellular matrix proteins and enzymes involved in degradation have been found to be associated with tissue fibrosis and ureteropelvic junction obstruction (UPJO). In this study we developed a promising urinary biomarker model which can identify reduced renal function in UPJ obstruction patients. This can potentially serve as a non-invasive way to enhance surgical decision making for patients and urologists. OBJECTIVE: We sought to develop a predictive model to identify UPJO patients at risk for reduced renal function. DESIGN: Prospective cohort study. SETTING: Pre-operative urine samples were collected in a prospectively enrolled UPJO biomarker registry at our institution. Urinary MMP-2, MMP-7, TIMP-2, and NGAL were measured as well as clinical characteristics including hydronephrosis grade, differential renal function, t1/2, and UPJO etiology. PARTICIPANTS: Children who underwent pyeloplasty for UPJO. MAIN OUTCOME MEASUREMENT: Primary outcome was reduced renal function defined as MAG3 function <40%. Multivariable logistic regression was applied to identify the independent predictive biomarkers in the original Training cohort. Model validation and generalizability were evaluated in a new UPJO Testing cohort. RESULTS: We included 71 patients with UPJO in the original training cohort and 39 in the validation cohort. Median age was 3.3 years (70% male). By univariate analysis, reduced renal function was associated with higher MMP-2 (p = 0.064), MMP-7 (p = 0.047), NGAL (p = 0.001), and lower TIMP-2 (p = 0.033). Combining MMP-7 with TIMP-2, the multivariable logistic regression model predicted reduced renal function with good performance (AUC = 0.830; 95% CI: 0.722-0.938). The independent testing dataset validated the results with good predictive performance (AUC = 0.738). CONCLUSIONS AND RELEVANCE: Combination of urinary MMP-7 and TIMP-2 can identify reduced renal function in UPJO patients. With the high sensitivity cutoffs, patients can be categorized into high risk (aggressive management) versus lower risk (observation).

The Urinary Proteomic Profile Implicates Key Regulators for Urologic Chronic Pelvic Pain Syndrome (UCPPS): A MAPP Research Network Study.

Urologic chronic pelvic pain syndrome (UCPPS) is a condition of unknown etiology characterized by pelvic pain and urinary frequency and/or urgency. As the proximal fluid of this syndrome, urine is an ideal candidate sample matrix for an unbiased study of UCPPS. In this study, a large, discovery-phase, TMT-based quantitative urinary proteomics analysis of 244 participants was performed. The participants included patients with UCPPS (n = 82), healthy controls (HC) (n = 94), and disparate chronic pain diseases, termed positive controls (PC) (n = 68). Using training and testing cohorts, we identified and validated a small and distinct set of proteins that distinguished UCPPS from HC (n = 9) and UCPPS from PC (n = 3). The validated UCPPS: HC proteins were predominantly extracellular matrix/extracellular matrix modifying or immunomodulatory/host defense in nature. Significantly varying proteins in the UCPPS: HC comparison were overrepresented by the members of several dysregulated biological processes including decreased immune cell migration, decreased development of epithelial tissue, and increased bleeding. Comparison with the PC cohort enabled the evaluation of UCPPS-specific upstream regulators, contrasting UCPPS with other conditions that cause chronic pain. Specific to UCPPS were alterations in the predicted signaling of several upstream regulators, including alpha-catenin, interleukin-6, epidermal growth factor, and transforming growth factor beta 1, among others. These findings advance our knowledge of the etiology of UCPPS and inform potential future clinical translation into a diagnostic panel for UCPPS.

Proteomic Discovery of Noninvasive Biomarkers Associated With Sport-Related Concussions.

BACKGROUND AND OBJECTIVES: Sport-related concussions affect millions of individuals across the United States each year, and current techniques to diagnose and monitor them rely largely on subjective measures. Our goal was to discover and validate objective, quantifiable noninvasive biomarkers with the potential to be used in sport-related concussion diagnosis. METHODS: Urine samples from a convenience series of healthy control collegiate athletes who had not sustained a concussion and athletes who sustained a concussion as diagnosed by a sports medicine physician within 7 days were collected prospectively and studied. Participants also completed an instrumented single-task gait analysis as a functional measure. Participants were recruited from a single collegiate athletic program and were ≥18 years of age and were excluded if they had a concomitant injury, active psychiatric conditions, or preexisting neurologic disorders. Using Tandem Mass Tags (TMT) mass spectroscopy and ELISA, we identified and validated urinary biomarkers of concussion. RESULTS: Forty-eight control and 47 age- and sex-matched athletes with concussion were included in the study (51.6% female, 48.4% male, average age 19.6 years). Participants represented both contact and noncontact sports. All but 1 of the postconcussion participants reported experiencing symptoms at the time of data collection. Insulin-like growth factor 1 (IGF-1) and IGF binding protein 5 (IGFBP5) were downregulated in the urine of athletes with concussions compared to healthy controls. Multivariable risk algorithms developed to predict the probability of sport-related concussion showed that IGF-1 multiplexed with single-task gait velocity predicts concussion risk across a range of postinjury time points (area under the curve [AUC] 0.786, 95% confidence interval [CI] 0.690-0.884). When IGF-1 and IGFBP5 are multiplexed with single-task gait velocity, they accurately distinguish between healthy controls and individuals with concussion at acute time points (AUC 0.835, 95% CI 0.701-0.968, p < 0.001). DISCUSSION: These noninvasive biomarkers, discovered in an objective and validated manner, may be useful in diagnosing and monitoring sport-related concussions in both acute phases of injury and several days after injury. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT02354469 (submitted February 2015, first patient enrolled August 2015). CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that urinary IGF-1 and IGFBP5 multiplexed with single-task gait velocity may be useful in diagnosing sport-related concussion.

Spectral Properties of Abdominal Tissues on Dual-energy Computed Tomography and the Effects of Contrast Agent.

BACKGROUND/AIM: Multiparametric dual energy comptuted tomography (CT) imaging allows for multidimensional tissue characterization beyond the measurement of Hounsfield units. The purpose of this study was to evaluate multiple imaging parameters for different abdominal organs in dual energy CT (DECT) and analyze the effects of the contrast agent on these different parameters and provide normal values for characterization of parenchymatous organs. PATIENTS AND METHODS: This retrospective analysis included a total of 484 standardized DECT scans of the abdomen. Hounsfield Units (HU), rho (electron density relative to water), Zeff (effective atomic number) and FF (fat fraction) were evaluated for liver, spleen, kidney, muscle, fat-tissue. Independent generalized estimation equation models were fitted. RESULTS: In DECT imaging there is only little difference in mean HUmixed for parenchymatous abdominal organs. Analysis including Zeff, rho and FF allows for better discrimination while a large overlap remains for liver, spleen and muscle. Including multidimensional analysis and the effects of contrast medium further enhances tissue characterization. Small differences remain for liver and spleen. CONCLUSION: Organ characterization using multiparametric dual energy CT analysis is possible. An increased number of parameters obtained from DECT improves organ characterization. To our knowledge this is the first attempt to provide normal values for characterization of parenchymatous organs.

Proteins associated with the Arabidopsis thaliana plastid rhomboid-like protein RBL10.

Rhomboid-like proteins are intramembrane proteases with a variety of regulatory roles in cells. Though many rhomboid-like proteins are predicted in plants, their detailed molecular mechanisms or cellular functions are not yet known. Of the 13 predicted rhomboids in Arabidopsis thaliana, one, RBL10, affects lipid metabolism in the chloroplast, because in the respective rbl10 mutant the transfer of phosphatidic acid through the inner envelope membrane is disrupted. Here we show that RBL10 is part of a high-molecular-weight complex of 250 kDa or greater in size. Nine likely components of this complex are identified by two independent methods and include Acyl Carrier Protein 4 (ACP4) and Carboxyltransferase Interactor1 (CTI1), which have known roles in chloroplast lipid metabolism. The acp4 mutant has decreased C16:3 fatty acid content of monogalactosyldiacylglycerol, similar to the rbl10 mutant, prompting us to offer a mechanistic model of how an interaction between ACP4 and RBL10 might affect chloroplast lipid assembly. We also demonstrate the presence of a seventh transmembrane domain in RBL10, refining the currently accepted topology of this protein. Taken together, the identity of possible RBL10 complex components as well as insights into RBL10 topology and distribution in the membrane provide a stepping-stone towards a deeper understanding of RBL10 function in Arabidopsis lipid metabolism.

Uromodulin Isolation and Its N-Glycosylation Analysis by NanoLC-MS/MS.

The glycoprotein uromodulin (UMOD) is the most abundant protein in urine, and N-glycans are critical for many biological functions of UMOD. Comprehensive glycan profiling of UMOD provides valuable information to understand the exact mechanisms of glycan-regulated functions. To perform comprehensive glycosylation analysis of UMOD from urine samples with limited volumes, we developed a streamlined workflow that included UMOD isolation from 5 mL of urine from 6 healthy adult donors (3 males and 3 females) and a glycosylation analysis using a highly sensitive and reproducible nanoLC-MS/MS based glycomics approach. In total, 212 N-glycan compositions were identified from the purified UMOD, and 17% were high-mannose glycans, 2% were afucosylated/asialylated, 3% were neutral fucosylated, 28% were sialylated (with no fucose), 46% were fucosylated and sialylated, and 4% were sulfated. We found that isolation of UMOD resulted in a significant decrease in the relative quantity of high-mannose and sulfated glycans with a significant increase of neutral fucosylated glycans in the UMOD-depleted urine relative to the undepleted urine, but depletion had little impact on the sialylated glycans. To our knowledge, this is the first study to perform comprehensive N-glycan profiling of UMOD using nanoLC-MS/MS. This analytical workflow would be very beneficial for studies with limited sample size, such as pediatric studies, and can be applied to larger patient cohorts not only for UMOD interrogation but also for global glycan analysis.

Modifying the RAPT Score to Reflect Discharge Destination in Current Practice.

BACKGROUND: The Risk Assessment Prediction Tool (RAPT) is a validated 6-question survey designed to predict primary total joint arthroplasty (TJA) patients' discharge disposition. It is scored from 1 to 12 with patients stratified into high-, intermediate-, and low-risk groups. Given recent advancements in rapid-discharge protocols and increasing utilization of home services, the RAPT score may require modified scoring cutoffs. METHODS: A retrospective chart review of all patients undergoing primary TJA at a single academic center over 14 months was performed. The RAPT score was implemented during the sixth month. Patients undergoing revision TJA, complex TJA, and TJA after resection of malignancy were excluded. Outcomes before and after RAPT implementation were analyzed with additional subanalysis investigating of post-RAPT data. RESULTS: A total of 1264 patients (624 Pre-RAPT and 640 Post-RAPT) were evaluated. The post-RAPT group (245 total hip arthroplasty and 395 total knee arthroplasty) experienced significant decreases in mean hospital length of stay (2.22 days pre-RAPT to 1.82 days post-RAPT, P < .001) and the proportion of patients discharged to facility (21.8% pre-RAPT to 15.2% post-RAPT, P = .002). The modified system demonstrated the highest overall predictive accuracy at 92% and was found to be predictive of hospital length of stay. CONCLUSION: Owing to the recent trends favoring in-home services over rehab facility after discharge, previously published RAPT scoring cutoffs are inaccurate for modern practice. Using mRAPT cutoffs maximizes the number of patients for whom a discharge prediction can be made, while maintaining excellent predictive accuracy.

Applicant Ranking Criteria in Adult Reconstruction Fellowship: Your Interview Counts When Applying for Fellowship.

Background The criteria for successful ranking in the fellowship match remains unclear. Although some data are available regarding the qualities sought after in medical students for the residency match, little information is available regarding the fellowship match. In this study, for arthroplasty applications to our institution, the interview was hypothesized to be the most important factor for ranking, with little impact from other commonly assessed variables. Methodology All 40 applicants who applied for fellowship were selected to interview for the 2017 Brown University Comprehensive Adult Reconstruction Fellowship and were evaluated on the interview, United States Medical Licensing Examination (USMLE) scores, letters of recommendation, personal statement, extracurricular activities, research, and caliber of undergraduate institution, medical school, and residency program. The interview score was based on a combined score of individual components of personality, program fit, and research. Results Of the 40 applicants who were interviewed, eight did not match. The interview score was the only statistically significant variable and had the highest correlation with ranking (r = 0.92). Moreover, extracurricular activities correlated with a higher ranking whereas USMLE Step 1/Step 2 scores had a relatively low correlation (r = 0.32 and 0.29, respectively). Recommendation letters and caliber of medical school, undergraduate education, and residency demonstrated low correlations. The personal statement and research components had the lowest correlations. Conclusions The combined interview score, in particular the personality and program fit components, was the most important determinant of successful ranking at our institution. Because all 40 applicants who applied for an arthroplasty fellowship at our institution were selected for an interview, there was no pre-interview selection bias that would confound these results. However, the ranking does not correlate with an applicant's success in fellowship, and further research is required to determine the qualifications of a successful surgeon.

Association of Longitudinal Changes in Symptoms and Urinary Biomarkers in Patients with Urological Chronic Pelvic Pain Syndrome: A MAPP Research Network Study.

PURPOSE: We analyzed a series of novel noninvasive urinary biomarkers for their ability to objectively monitor the longitudinal clinical status of patients with urological chronic pelvic pain syndrome. MATERIALS AND METHODS: Baseline, 6 and 12-month urine samples were collected (216) and used to quantify vascular endothelial growth factor, vascular endothelial growth factor (VEGF) receptor 1 (R1), neutrophil gelatinase associated lipocalin (NGAL), matrix metalloproteinase-2, matrix metalloproteinase (MMP)-9, and MMP-9/NGAL complex by enzyme-linked immunosorbent assays. Patient symptom changes were classified as improved, stable or worse using a functional clustering algorithm. Proportional odds models were used to evaluate the association between symptom change and urinary biomarkers. RESULTS: Across all sampled participants, longitudinal decreases in normalized VEGF concentration (pg/µg) were associated with pain severity improvement, and decreases in MMP-9, NGAL and VEGF-R1 concentration (pg/ml) as well as NGAL normalized concentration were associated with improved urinary symptoms. Longitudinal decreases in normalized VEGF-R1 were associated with pain improvement in patients with moderate widespreadness, no bladder symptoms and no painful filling. Lower baseline normalized VEGF-R1 concentration was associated with pain improvement in patients with pelvic pain only. Higher baseline MMP-9/NGAL levels were associated with pain and urinary improvement across all participants. Moreover, longitudinal increases in MMP-2 concentration was associated with improved pain in men and patients with painful filling. CONCLUSIONS: Our results suggest these urinary biomarkers may be useful in monitoring urological chronic pelvic pain syndrome symptom changes with respect to both urinary severity and pain severity. With further testing, they may represent objective biological measures of urological chronic pelvic pain syndrome progression and/or resolution while also providing insight into the pathophysiology of urological chronic pelvic pain syndrome.

Human osteoarthritis cartilage-derived stromal cells activate joint degeneration through TGF-beta lateral signaling.

Human osteoarthritis cartilage contains chondrocytes (OAC) and mesenchymal stromal cells (OA-MSC). Here, we found that TGF-ß had different effects on OA-MSC and OAC, and revealed its lateral signaling mechanism in OA. RNAseq analysis indicated that OA-MSC expressed the same level of Bone Morphogenetic Protein (BMP) Receptor-1A as OAC but only 1/12 of Transforming Growth Factor beta (TGF-ß) Receptor-1. While TGF-ß specifically activated SMAD2 in OAC, it also activated BMP signaling-associated SMAD1 in OA-MSC. While TGF-ß stimulated chondrogenesis in OAC, it induced hypertrophy, mineralization, and MMP-13 in OA-MSC. Inhibiting TGF-ßR1 suppressed MMP-13 in OA-MSC but stimulated it in OAC. In contrast, by specifically targeting BMPR1A/ACVR1 in both cell types, LDN193189 inhibits cartilage degeneration through suppressing hypertrophy and MMP-13 in a mouse osteoarthritis model. Thus, LDN193189, a drug under development to inhibit constitutive BMP signaling during heterotopic ossification, may be re-purposed for OA treatment.

An in-depth Comparison of the Pediatric and Adult Urinary N-glycomes.

We performed an in-depth characterization and comparison of the pediatric and adult urinary glycomes using a nanoLC-MS/MS based glycomics method, which included normal healthy pediatric (1-10 years, n = 21) and adult (21-50 years, n = 22) individuals. A total of 116 N-glycan compositions were identified, and 46 of them could be reproducibly quantified. We performed quantitative comparisons of the 46 glycan compositions between different age and sex groups. The results showed significant quantitative changes between the pediatric and adult cohorts. The pediatric urinary N-glycome was found to contain a higher level of high-mannose (HM), asialylated/afucosylated glycans (excluding HM), neutral fucosylated and agalactosylated glycans, and a lower level of trisialylated glycans compared with the adult. We further analyzed gender-associated glycan changes in the pediatric and adult group, respectively. In the pediatric group, there was almost no difference of glycan levels between males and females. In adult, the majority of glycans were more abundant in males than females, except the high-mannose and tetrasialylated glycans. These findings highlight the importance to consider age-matching and adult sex-matching for urinary glycan studies. The identified normal pediatric and adult urinary glycomes can serve as a baseline reference for comparisons to other disease states affected by glycosylation.

Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome.

Recurrent urinary tract infections (UTIs) pose a significant burden on the health care system. Underlying mechanisms predisposing children to UTIs and associated changes in the urinary proteome are not well understood. We aimed to investigate the urinary proteome of a subset of children who have vesicoureteral reflux (VUR) and recurrent UTIs because of their risk of developing infection-related renal damage. Improving diagnostic modalities to identify UTI risk factors would significantly alter the clinical management of children with VUR. We profiled the urinary proteomes of 22 VUR patients with low grade VUR (1-3 out of 5), a history of recurrent UTIs, and renal scarring, comparing them to those obtained from 22 age-matched controls. Urinary proteins were analyzed by mass spectrometry followed by protein quantitation based on spectral counting. Of the 2,551 proteins identified across both cohorts, 964 were robustly quantified, as defined by meeting criteria with spectral count (SC) ≥2 in at least 7 patients in either VUR or control cohort. Eighty proteins had differential expression between the two cohorts, with 44 proteins significantly up-regulated and 36 downregulated (q <0.075, FC ≥1.2). Urinary proteins involved in inflammation, acute phase response (APR), modulation of extracellular matrix (ECM), and carbohydrate metabolism were altered among the study cohort.

PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4.

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid-associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild-type levels of 16:1t. The FAD4-PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co-production of PRXQ with FAD4 was required to produce Δ3-desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site-directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.