A local subset of mesenchymal cells expressing the transcription factor Osr1 orchestrates lymph node initiation.

During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.

Clinical and radiological outcome following trauma-related reverse shoulder arthroplasty.

BACKGROUND: Reverse shoulder arthroplasty (RSA) is a frequently used therapy for complex proximal humeral fractures and posttraumatic disorders. The present study's purpose was to assess the clinical and radiological outcome of primary and secondary RSA, and to analyze the impact of refixation of the greater tuberosity (GT). PATIENTS AND METHODS: 28 patients with primary fracture RSA and 18 patients with RSA due to posttraumatic disorders were examined with a mean clinical follow-up of 2.5 ± 1.73 years. Operative details and radiographs were retrospectively reviewed. Additional analyses were performed for healed and non-healed GT in primary RSA. RESULTS: Patients with fracture RSA had higher Constant-Murley score (CMS) than secondary RSA without reaching significance (p = 0.104). No significant difference was present for the quality of life measured by the Short Form 36 (SF 36) and the range of motion. In primary RSA, 78.6% GT healed anatomically. Compared to non-healed GT, patients with healed GT had a significantly higher CMS (p = 0.011), external rotation (p = 0.026) and forward flexion (p = 0.083), whereas DASH score was lower without a significant difference (p = 0.268). SF 36 showed no significant difference. Patients with healed GT had a more neutral glenoid version (p = 0.009). CONCLUSION: Superior range of motion and clinical outcome scores were present for anatomically healed GT. Therefore, refixation of the tuberosities is recommended. Secondary RSA can result in inferior results compared to primary RSA, so patients need to be adequately informed.

Are there associations between hip geometry and bone quality? An analysis on 3074 adults from a general population.

INTRODUCTION: Patients with reduced bone mineral density and altered hip geometry are susceptible for hip pathologies. Knowledge on associations between bone properties and hip geometric parameters might facilitate identification of patients at risk for hip pathologies. The aim of the present study was to identify associations of bone properties assessed by quantitative ultrasound (QUS) at the heel and hip geometric parameters like center-edge angle (CE), neck-shaft angle (NSA) and alpha angle. MATERIALS AND METHODS: Hip geometric parameters (CE, NSA and alpha angle) of 3074 participants from the population-based Study of Health in Pomerania were assessed on magnetic resonance imaging. QUS was performed on both calcanei providing broadband ultrasound attenuation (BUA), speed of sound (SOS) and stiffness-index. Based on the stiffness-index the individual osteoporotic fracture risk (low, moderate or high) was determined. Associations between QUS-based and hip geometric parameters were calculated in linear regression models adjusted for age, sex, body height and weight. Interactions of QUS markers with age and sex on hip geometric parameters were tested. RESULTS: Significant inverse associations between BUA (ß = - 0.068), SOS (ß = - 0.024) as well as stiffness-index (ß = - 0.056) and CE were present, while fracture risk was positively associated with CE (ß for high = 1.28 and moderate = 2.54 vs. low fracture risk). Interactions between BUA and sex as well as between SOS and age were detected in the models for CE. Furthermore, there was an inverse relation between fracture risk and NSA that was restricted to the moderate risk (ß for moderate vs. low fracture risk = - 0.60). There were no significant associations between QUS parameters and alpha angle. CONCLUSIONS: In the general population, several associations between QUS-based bone properties or fracture risk and hip geometry are present. Less dysplastic hips had a lower stiffness-index and a higher fracture risk, whereas more valgus hips had a lower fracture risk.

Nonproductive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses.

Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

Platelet-derived factors dysregulate placental sphingosine-1-phosphate receptor 2 in human trophoblasts.

RESEARCH QUESTION: Sphingosine-1-phosphate (S1P) is an essential and bioactive sphingolipid with various functions, which acts through five different G-protein-coupled receptors (S1PR1-5). What is the localization of S1PR1-S1PR3 in the human placenta and what is the effect of different flow rates, various oxygen concentrations and platelet-derived factors on the expression profile of S1PR in trophoblasts? DESIGN: Expression dynamics of placental S1PR1-S1PR3 were determined in human first trimester (n = 10), pre-term (n = 9) and term (n = 10) cases. Furthermore, the study investigated the expression of these receptors in different primary cell types isolated from human placenta, verified the findings with publicly available single-cell RNA-Seq data from first trimester and immunostaining of human first trimester and term placentas. The study also tested whether the placental S1PR subtypes are dysregulated in differentiated BeWo cells under different flow rates, different oxygen concentrations or in the presence of platelet-derived factors. RESULTS: Quantitative polymerase chain reaction revealed that S1PR2 is the predominant placental S1PR in the first trimester and reduces towards term (P < 0.0001). S1PR1 and S1PR3 increased from first trimester towards term (P < 0.0001). S1PR1 was localized in endothelial cells, whereas S1PR2 and S1PR3 were predominantly found in villous trophoblasts. Furthermore, S1PR2 was found to be significantly down-regulated in BeWo cells when co-incubated with platelet-derived factors (P = 0.0055). CONCLUSION: This study suggests that the placental S1PR repertoire is differentially expressed across gestation. S1PR2 expression in villous trophoblasts is negatively influenced by platelet-derived factors, which could contribute to down-regulation of placental S1PR2 over time of gestation as platelet presence and activation in the intervillous space increases from the middle of the first trimester onwards.

In closed wedge distal femur osteotomies for correction of valgus malalignment overcorrection of mLDFA should be avoided.

PURPOSE: The purpose of the study was to investigate the influence of the mLDFA (mechanical lateral distal femur angle) as a parameter in varus realignment osteotomies for valgus deformities of the knee. We hypothesized that joint line obliquity with mLDFA > 90° after distal femur osteotomy (DFO) is associated with inferior clinical outcome. METHODS: In a retrospective study, a total of 52 patients with isolated femoral valgus deformities were included in the study. The mean postoperative follow-up was 70.5 (SD 33.3) months (standard deviation SD±33.3). In all patients, a distal femur osteotomy was performed. A clinical examination and survey of questionnaires was conducted with the HSS (Hospital for Special Surgery), LG (Lysholm-Gilquist), and KOOS (Knee Injury and Osteoarthritis Outcome Score) scores. Several radiological parameters were assessed on long-standing x-rays: mechanical tibio-femoral angle (mTFA), mLDFA, mechanical medial proximal tibia angle (mMPTA), joint-line convergence angle (JLCA). The t test was used for normally distributed data. The Mann-Whitney U test was performed in non-normally distributed data. RESULTS: The mLDFA was 84.9° (SD±2.3) preop and changed to 91.9° (SD±3, 22.9) postop. The mTFA (mechanical tibio-femoral angle) was 5.2° (SD±2.9°) preop and - 1.8° (SD±2.9) postop demonstrating a difference of 6.7°. For analysis, the data was divided into two groups based on postop mLDFA. Group 1: mLDFA ≤ 90°; Group 2: > 90°. Postoperatively, a mean mLDFA of 88.6° (SD±1.4°) was measured in group 1 and 93.9° (SD±2.1) in group 2. The change in mLDFA was 4.7° (SD±1.6) in group 1 and 8.4° (SD±2.8) in group 2. Preoperatively, the mTFA was 4.8° (SD±1.9) in group 1 and 5.5° (SD±3.3) in group 2. Postoperatively, the mTFA decreased in group 1 by 4.8° (SD±2.3) to - 0.1° (SD±2.1). In group 2, the mTFA decreased by 8.2° (SD±3.8) to - 2.8° (SD±2.9). Regarding the HSS, group 1 showed a 10.4 points better score than group 2 (p<0.01). Also, regarding the Lysholm, a significant difference of 16.9 points was found (p<0.01). CONCLUSION: Correction of valgus knees using closed wedge DFO leads to good clinical results. A postoperative mLDFA of 85-90° results in superior clinical outcome compared to mLDFA > 90°. Joint-line obliquity should be avoided using double level osteotomy, if needed. LEVEL OF EVIDENCE: III.

Reference values and influencing factors of the glenohumeral subluxation index: a study on 3004 participants.

BACKGROUND: The primary objective of this study was to examine the glenohumeral subluxation index (GHSI) in a large general population cohort and to define reference values. Glenohumeral subluxation is important in the development and prediction of pathological states of the shoulder joint and in total shoulder arthroplasty. Therefore, another objective was to examine the influence of age, sex, body mass index, and body height and weight on GHSI. METHODS: GHSI according to Walch was measured on bilateral magnetic resonance imaging of 3004 participants of the Study of Health in Pomerania (SHIP, aged 21-90 years). SHIP drew a sample of the adult general population of Pomerania (Northeastern Germany). Reference values for GHSI were assessed by quantile regression models. Associations of sex, age, and anthropometric markers with the GHSI were calculated by linear regression models. RESULTS: A reference range between 42% and 55% for men with a mean of 49% ± 4% was defined, while the upper reference limit for women was 1% higher (mean, 50% ± 4%). Age was inversely associated with the GHSI in males (P < 0.001), while no significant association in females was observed (P = .625). Body weight and body mass index were positively associated (P < .001) without effect modification by sex. Heavy mechanical oscillations on the upper extremity showed no significant association with GHSI (P = .268). CONCLUSION: The reference values for GHSI were expanded to a range of 42%-57% on magnetic resonance imaging. Several associations between GHSI and anthropometric properties are present. According to these associations, adjusted formulas are provided to enable individual, patient-specific diagnostics and therapy. Nevertheless, the clinical picture cannot be neglected.

Single-nucleus RNA sequencing of plant tissues using a nanowell-based system.

Single-cell genomics provides unprecedented potential for research on plant development and environmental responses. Here, we introduce a generic procedure for plant nucleus isolation combined with nanowell-based library preparation. Our method enables the transcriptome analysis of thousands of individual plant nuclei. It serves as an alternative to the use of protoplast isolation, which is currently a standard methodology for plant single-cell genomics, although it can be challenging for some plant tissues. We show the applicability of our nucleus isolation method by using different plant materials from different species. The potential of our single-nucleus RNA sequencing method is shown through the characterization of transcriptomes of seedlings and developing flowers from Arabidopsis thaliana. We evaluated the transcriptome dynamics during the early stages of anther development, identified stage-specific activities of transcription factors regulating this process, and predicted potential target genes of these transcription factors. Our nucleus isolation procedure can be applied in different plant species and tissues, thus expanding the toolkit for plant single-cell genomics experiments.

Overweight patients benefit from high tibial osteotomy to the same extent as patients with normal weights but show inferior mid-term results.

PURPOSE: The purpose of this prospective study was to analyze the impact of obesity on the clinical and radiological outcomes 6 years after open-wedge high tibial osteotomy (HTO). METHODS: A total of 120 prospectively recorded patients with medial compartment osteoarthritis underwent open-wedge HTO between 2008 and 2011. The study cohort was frequently examined over a minimum of a 6-year follow-up. The cohort was divided into three groups according to body mass index (BMI): normal weight patients (BMI < 25 kg/m2), pre-obese patients (BMI 25-30 kg/m2) and obese patients (BMI > 30 kg/m2). Clinical and functional outcomes (Oxford Knee Score, Hospital for Special Surgery Score, Lequesne Score, Tegner Activity Scale), subjective health-related quality of life (SF-36), change in mechanical limb alignment (mTFA) as well as conversion to unicompartmental or total knee arthroplasty (TKA) were evaluated. To compare clinical scoring between the groups, univariate variance analysis was applied. Changes in outcome variables over time were analyzed with dependent t tests. RESULTS: From 120 patients, 85 were followed-up over a 6.7-year period on average (6-11.8 years) after HTO. The mean BMI was 28.6 ± 4.6 kg/m2. Each group showed a significant pre- to postoperative increase in all recorded scores (p < 0.05). In absolute terms, both mental and clinical scores of overweight patients did not reach the peak values of the normal weighted population during the period of observation. There was a conversion to TKA in 10.5% after an average of 50.1 ± 25.0 months following surgery. A total of five complications occurred without significant differences (BMI < 25: n = 1, BMI 25-30: n = 2, BMI > 30: n = 2; n.s.). There was a mean pre- to postoperative (six weeks after surgery) correction difference of 6.9° ± 3.2° (mTFA) with higher loss of correction over time in overweight patients. CONCLUSION: In terms of clinical outcome and health-related quality of life, overweight patients may receive a benefit from open-wedge HTO to the same extent as patients with normal weights and show similar complication rates. However, they have inferior preoperative clinical and functional results and mid-term results after open-wedge HTO compared to patients with normal weights. LEVEL OF EVIDENCE: Level III.

Os acromiale: prevalence and associated patient-related factors-a population-based study of three thousand and fifty participants.

PURPOSE: The presence of os acromiale is of clinical relevance before performing shoulder surgery but ethnic differences and little information regarding associated factors seem to be present. Population-based studies to clarify these topics are essential so the purpose of this study was to assess the prevalence, anatomy, and associations of os acromiale in a general adult population. METHODS: Both shoulders of 3050 participants from the population-based Study of Health in Pomerania (SHIP) were assessed on magnetic resonance imaging (MRI). Associations with the os acromiale were calculated for sex, age, body height, body weight, and heavy mechanical oscillations on the upper extremity. RESULTS: In total, 1.9% (58/3050) had an os acromiale, while 21 were unilateral left, 23 were unilateral right, and 14 were bilateral. Sixty-eight meso-acromions, three pre-acromions, and one meta-acromion were detected. Os acromiale were more frequent in men (right side: p = 0.037, left side: p = 0.005). Overall, no differences in sides (p = 0.808), to participants' age (right: p = 0.993, left: p = 0.499), body height (right side: p = 0.241, left side: p = 0.154), and the exposure to heavy mechanical oscillations on the upper extremity (right: p = 0.054, left: p = 0.117) were detected. CONCLUSION: Our results support the genetic theory for the aetiology of the os acromiale due to the lower prevalence of the os acromiale in north-eastern Germany compared to the worldwide prevalence (1.9 to 7%) and the lacking association to lifestyle, age, gender, or sides. Additionally, it is important to be aware of possible os acromiale before surgery.

Candesartan Does Not Activate PPARγ and Its Target Genes in Early Gestation Trophoblasts.

Angiotensin II receptor 1 blockers are commonly used to treat hypertension in women of childbearing age. While the fetotoxic effects of these drugs in the second and third trimesters of pregnancy are well documented, their possible impacts on placenta development in early gestation are unknown. Candesartan, a member of this group, also acts as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, a key regulator shown to be important for placental development. We have previously shown that trophoblasts do not express the candesartan target-receptor angiotensin II type 1 receptor AGTR1. This study investigated the possible role of candesartan on trophoblastic PPARγ and its hallmark target genes in early gestation. Candesartan did not affect the PPARγ protein expression or nuclear translocation of PPARγ. To mimic extravillous trophoblasts (EVTs) and cytotrophoblast/syncytiotrophoblast (CTB/SCT) responses to candesartan, we used trophoblast cell models BeWo (for CTB/SCT) and SGHPL-4 (EVT) cells as well as placental explants. In vitro, the RT-qPCR analysis showed no effect of candesartan treatment on PPARγ target genes in BeWo or SGHPL-4 cells. Treatment with positive control rosiglitazone, another PPARγ agonist, led to decreased expressions of LEP and PPARG1 in BeWo cells and an increased expression of PPARG1 in SGHPL-4 cells. Our previous data showed early gestation-placental AGTR1 expression in fetal myofibroblasts only. In a CAM assay, AGTR1 was stimulated with angiotensin II and showed increased on-plant vessel outgrowth. These results suggest candesartan does not negatively affect PPARγ or its target genes in human trophoblasts. More likely, candesartan from maternal serum may first act on fetal-placental AGTR1 and influence angiogenesis in the placenta, warranting further research.

Heterogeneous Sorption of Radionuclides Predicted by Crystal Surface Nanoroughness.

Reactive transport modeling (RTM) is an essential tool for the prediction of contaminants' behavior in the bio- and geosphere. However, RTM of sorption reactions is constrained by the reactive surface site assessment. The reactive site density variability of the crystal surface nanotopography provides an "energetic landscape", responsible for heterogeneous sorption efficiency, not covered in current RTM approaches. Here, we study the spatially heterogeneous sorption behavior of Eu(III), as an analogue to trivalent actinides, on a polycrystalline nanotopographic calcite surface and quantify the sorption efficiency as a function of surface nanoroughness. Based on experimental data from micro-focus time-resolved laser-induced luminescence spectroscopy (µTRLFS), vertical scanning interferometry, and electron back-scattering diffraction (EBSD), we parameterize a surface complexation model (SCM) using surface nanotopography data. The validation of the quantitatively predicted spatial sorption heterogeneity suggests that retention reactions can be considerably influenced by nanotopographic surface features. Our study presents a way to implement heterogeneous surface reactivity into a SCM for enhanced prediction of radionuclide retention.

Pulsating dissolution of crystalline matter.

Fluid-solid reactions result in material flux from or to the solid surface. The prediction of the flux, its variations, and changes with time are of interest to a wide array of disciplines, ranging from the material and earth sciences to pharmaceutical sciences. Reaction rate maps that are derived from sequences of topography maps illustrate the spatial distribution of reaction rates across the crystal surface. Here, we present highly spatially resolved rate maps that reveal the existence of rhythmic pulses of the material flux from the crystal surface. This observation leads to a change in our understanding of the way crystalline matter dissolves. Rhythmic fluctuations of the reactive surface site density and potentially concomitant oscillations in the fluid saturation imply spatial and temporal variability in surface reaction rates. Knowledge of such variability could aid attempts to upscale microscopic rates and predict reactive transport through changing porous media.

The neck-shaft angle: an update on reference values and associated factors.

Background and purpose - The neck-shaft angle (NSA) is valuable for diagnostics and therapy of the hip, but current reference values derive mostly from studies on anatomic specimens, small cohorts, or are hospital-based. Moreover, associated factors such as age, sex, or anthropometric data have rarely been considered. Therefore, we determined associated factors for NSA and reassessed the historical reference values in a general adult population.Methods - NSAs on both sides of 3,226 volunteers from the population-based Study of Health in Pomerania (SHIP) were measured with MRI. SHIP drew a representative sample of the population of Pomerania (northeastern Germany). NSAs were compared with sex, age, and anthropometric data by bivariable linear regression models. Reference values were assessed by quantile regressions for 2.5th and 97.5th percentiles.Results - The mean NSA was 127° (SD 7), while men had a lower NSA than women (95% confidence interval [CI] 0.4°-1.4°). The reference range was 114°-140°. Age was inversely associated with NSA (CI -0.2 to -0.1). Body height was positively associated with the NSA, while BMI and waist circumference showed a negative association. There was no association between body weight and NSA.Interpretation - The historical lower limit of 120° might be too high, so the radiological prevalence of hip pathology might have been overestimated. The previously reported influence of age, sex, and body height on the NSA has been confirmed.

What Are the Reference Values and Associated Factors for Center-edge Angle and Alpha Angle? A Population-based Study.

BACKGROUND: The available evidence regarding normal ranges for the center-edge angle and the alpha angle derives from a few small studies, and associated factors such as sex and anthropometric factors have not been well evaluated. Knowing more about normal values for these parameters is critical, because this can inform decisions about when to perform elective hip preservation surgery. Population-level studies would provide considerable clarity on these issues, but to our knowledge, no such studies are available. QUESTIONS/PURPOSES: The purposes of this study were (1) to use MRI in patients of a population-based study to establish normal values for the alpha and center-edge angles in the normal adult hip; and (2) to determine whether age, sex, or anthropometric variables were associated with differences in these values. METHODS: We used MRI images (1.5 T) of 3226 participants of the Study of Health in Pomerania (SHIP). SHIP is a population-based study that started with 4308 participants in 1997. Participants were recruited randomly from official inhabitant data files as a stratified cluster sample of the population from a defined region in northeastern Germany. To ensure a representative epidemiologic cohort, stratification variables were sex, age, and city of residence. Between 2008 and 2012, 1094 of these participants underwent whole-body MRI with pelvic sequences in the second followup. In parallel, the next cohort, SHIP-Trend, started with the same protocol in which 2132 participants underwent MRI examination. Reference values were calculated by quantile regressions for the 2.5 and 97.5 percentiles. Associations with the demographic features sex, age, weight, height, body mass index (BMI), and waist circumference were analyzed by bivariate linear regression models. RESULTS: The mean center-edge angle was 31° (± 7°) with a corresponding calculated normal range of 17° to 45°. The mean alpha angle was 55° (± 8°) with a corresponding calculated normal range between 39° and 71°. Men (30° ± 7°) had a lower center-edge angle than women (32° ± 8°) (p < 0.001, ß = 1.4°; 95% confidence interval [CI], 0.9°-1.9°) and a higher alpha angle (57° ± 8° versus 52° ± 7°, p < 0.001, ß = 5.7°; 95% CI, 5.2°-6.3°). Moreover, a higher center-edge angle for the left side was found (33° ± 8° versus 30° ± 8°, p < 0.001, ß =3.2°; 95% CI, 3.0°-3.4°). In addition to sex, we found that age, height, waist circumference, and BMI affected both center-edge angle and alpha angle. Weight was associated with the alpha angle as well. Because of these associations, age- and sex-adjusted reference values with belonging formulas were calculated. CONCLUSIONS: The range of normal center-edge and alpha angles is quite wide. Therefore, only markedly abnormal angles may be associated with pathology. Moreover, center-edge angle and alpha angle are associated with age, sex, and anthropometric factors, which have to be taken into account for better interpretation. CLINICAL RELEVANCE: The association of abnormal radiographic values with true clinical hip pathology is tenuous at best. Assuming that a patient with an abnormal radiograph requires treatment is unwise. The clinical picture has to be substantial for therapeutic decisions.

Variability of Zinc Oxide Dissolution Rates.

Zinc oxide (ZnO) is of widespread use for numerous applications, including many in the cosmetic industry. Thus, ZnO particles are quite likely to enter the environment. ZnO may be harmful because of the release of cytotoxic Zn2+ ions during dissolution reactions. Here, we analyze the dissolution kinetics of the polar zinc-terminated (000-1) and nonpolar (10-10) crystal surfaces in ultrapure water to examine the impact of the crystal defects on dissolution. By using a complementary approach of atomic force microscopy and vertical scanning interferometry, we quantify the difference in reaction rate between the crystal faces, the overall range of rate variability, and the rate components that combine to an overall rate. The mean dissolution rate of the (000-1) crystal surface is more than 4 times that of the (10-10) surface. By using the rate spectrum analysis, we observed an overall dissolution rate variability of more than 1 order of magnitude. The rate components and the range of dissolution rate are important input parameters in reactive transport models for the prediction of potential release of Zn2+ into the environment.

Efficient Application of De Novo RNA Assemblers for Proteomics Informed by Transcriptomics.

RNA sequencing is a powerful method to build reference transcriptome assemblies and eventually sample-specific protein databases for mass-spectrometry-based analyses. This novel proteomics informed by transcriptomics (PIT) workflow improves proteome characterization of dynamic and especially nonmodel organism proteomes and moreover helps to identify novel gene products. With increasing popularity of such proteogenomics applications a growing number of researchers demand qualitative but resource-friendly and easy to use analysis strategies. Most PIT applications so far rely on the initially introduced Trinity de novo assembly tool. To aid potential users to start off with PIT, we compared main performance criteria of Trinity and other alternative RNA assembly tools known from the transcriptomics field including Oases, SOAPdenovo-Trans, and Trans-ABySS. Using exemplary data sets and software-specific default parameters, Trinity and alternative assemblers produced comparable and high-quality reference data for vertebrate transcriptomes/proteomes of varying complexity. However, Trinity required large computational resources and time. We found that alternative de novo assemblers, in particular, SOAPdenovo-Trans but also Oases and Trans-ABySS, rapidly produced protein databases with far lower computational requirements. By making choice among various RNA assembly tools, proteomics researchers new to transcriptome assembly and with future projects with high sample numbers can benefit from alternative approaches to efficiently apply PIT.

Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria.

A known (1) and a structurally related new natural product (2), both belonging to the amorfrutin benzoic acid class, were isolated from the roots of Glycyrrhiza foetida. Compound 1 (amorfrutin B) is an efficient agonist of the nuclear peroxisome proliferator activated receptor (PPAR) gamma and of other PPAR subtypes. Compound 2 (amorfrutin C) showed comparably lower PPAR activation potential. Amorfrutin C exhibited striking antiproliferative effects for human colorectal cancer cells (HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC50 values ranging from 8 to 16 µM in these cancer cell lines). Notably, amorfrutin C (2) showed less potent antiproliferative effects in primary colon cells. For HT-29 cells, compound 2 induced G0/G1 cell cycle arrest and modulated protein expression of key cell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studies revealed that 2 disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC50 0.6 µM) and permanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumption and extracellular acidification. Structure-activity-relationship experiments revealed the carboxylic acid and the hydroxy group residues of 2 as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstrated stimulation of the death receptor signaling pathway. Taken together, amorfrutin C (2) represents a promising lead for the development of anticancer drugs.

PHOXTRACK-a tool for interpreting comprehensive datasets of post-translational modifications of proteins.

UNLABELLED: We introduce PHOXTRACK (PHOsphosite-X-TRacing Analysis of Causal Kinases), a user-friendly freely available software tool for analyzing large datasets of post-translational modifications of proteins, such as phosphorylation, which are commonly gained by mass spectrometry detection. In contrast to other currently applied data analysis approaches, PHOXTRACK uses full sets of quantitative proteomics data and applies non-parametric statistics to calculate whether defined kinase-specific sets of phosphosite sequences indicate statistically significant concordant differences between various biological conditions. PHOXTRACK is an efficient tool for extracting post-translational information of comprehensive proteomics datasets to decipher key regulatory proteins and to infer biologically relevant molecular pathways. AVAILABILITY: PHOXTRACK will be maintained over the next years and is freely available as an online tool for non-commercial use at http://phoxtrack.molgen.mpg.de. Users will also find a tutorial at this Web site and can additionally give feedback at https://groups.google.com/d/forum/phoxtrack-discuss.

Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.