The Amount of Zeaxanthin Epoxidase But Not the Amount of Violaxanthin De-Epoxidase Is a Critical Determinant of Zeaxanthin Accumulation in Arabidopsis thaliana and Nicotiana tabacum.

The generation of violaxanthin (Vx) de-epoxidase (VDE), photosystem II subunit S (PsbS) and zeaxanthin (Zx) epoxidase (ZEP) (VPZ) lines, which simultaneously overexpress VDE, PsbS and ZEP, has been successfully used to accelerate the kinetics of the induction and relaxation of non-photochemical quenching (NPQ). Here, we studied the impact of the overexpression of VDE and ZEP on the conversion of the xanthophyll cycle pigments in VPZ lines of Arabidopsis thaliana and Nicotiana tabacum. The protein amount of both VDE and ZEP was determined to be increased to about 3- to 5-fold levels of wild-type (WT) plants for both species. Compared to WT plants, the conversion of Vx to Zx, and hence VDE activity, was only marginally accelerated in VPZ lines, whereas the conversion of Zx to Vx, and thus ZEP activity, was strongly increased in VPZ lines. This indicates that the amount of ZEP but not the amount of VDE is a critical determinant of the equilibrium of the de-epoxidation state of xanthophyll cycle pigments under saturating light conditions. Comparing the two steps of epoxidation, particularly the second step (antheraxanthin to Vx) was found to be accelerated in VPZ lines, implying that the intermediate Ax is released into the membrane during epoxidation by ZEP.

The relationship between clinical phenotype and kallikrein-kinin bioregulation in different forms of arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have shown increased levels of neutrophils generating kallikrein-kinin peptides in blood which are potent mediators of inflammation. This study investigated the association between the bioregulation of kinin-mediated inflammation with the clinical, quality of life, and imaging characteristics (e.g. ultrasonography) of different arthritides. METHODS: Patients with osteoarthritis (OA, n = 29), gout (n = 10) and RA (n = 8) were recruited and screened for clinical symptoms, quality of life, and ultrasonographical assessment of arthritis. Blood neutrophils were assessed for the expression of bradykinin receptors (B1R and B2R), kininogens and kallikreins by immunocytochemistry with visualization by bright field microscopy. Levels of plasma biomarkers were measured by ELISA and cytometric bead array. RESULTS: Quality of life (SF-36 domains and summary scores; including pain; and, HAQ) was similar across OA, gout and RA patients; with the exception of worse physical functioning scores between OA and gout patients. Synovial hypertrophy (on ultrasound) differed between groups (p = 0.001), and the dichotomised Power Doppler (PD) score of greater than or equal to 2 (PD-GE2) was marginally significant (p = 0.09). Plasma IL-8 were highest in patients with gout followed by RA and OA (both, P < 0.05). Patients with RA had higher plasma levels of sTNFR1, IL-1ß, IL-12p70, TNF and IL-6, compared to OA and gout patients (all, P < 0.05). Patients with OA had higher expression of K1B and KLK1 on blood neutrophils followed by RA and gout patients (both, P < 0.05). Bodily pain correlated with B1R expression on blood neutrophils (r = 0.334, p = 0.05), and inversely with plasma levels of CRP (r = -0.55), sTNFR1 (r = -0.352) and IL-6 (r = -0.422), all P < 0.05. Expression of B1R on blood neutrophils also correlated with Knee PD (r = 0.403) and PD-GE2 (r = 0.480), both P < 0.05. CONCLUSIONS: Pain levels and quality of life were similar between patients with OA, RA and gout with knee arthritis. Plasma inflammatory biomarkers and B1R expression on blood neutrophils correlated with pain. Targeting B1R to modulate the kinin-kallikrein system may pose as a new therapeutic target in the treatment of arthritis.

The actin bundling activity of ITPKA mainly accounts for its migration-promoting effect in lung cancer cells.

Expression of Ins(1,4,5)P3-kinase-A (ITPKA), the neuronal isoform of Ins(1,4,5)P3-kinases, is up-regulated in many tumor types. In particular, in lung cancer cells this up-regulation is associated with bad prognosis and it has been shown that a high level of ITPKA increases migration and invasion of lung cancer cell lines. However, since ITPKA exhibits actin bundling and Ins(1,4,5)P3-kinase activity, it was not clear which of these activities account for ITPKA-promoted migration and invasion of cancer cells. To address this issue, we inhibited endogenous actin bundling activity of ITPKA in lung cancer H1299 cells by overexpressing the dominant negative mutant ITPKAL34P. Analysis of actin dynamics in filopodia as well as wound-healing migration revealed that ITPKAL34P inhibited both processes. Moreover, the formation of invasive protrusions into collagen I was strongly blocked in cells overexpressing ITPKAL34P. Furthermore, we found that ATP stimulation slightly but significantly (by 13%) increased migration of cells overexpressing ITPKA while under basal conditions up-regulation of ITPKA had no effect. In accordance with these results, overexpression of a catalytic inactive ITPKA mutant did not affect migration, and the Ins(1,4,5)P3-kinase-inhibitor GNF362 reversed the stimulating effect of ITPKA overexpression on migration. In summary, we demonstrate that under basal conditions the actin bundling activity controls ITPKA-facilitated migration and invasion and in presence of ATP the Ins(1,4,5)P3-kinase activity slightly enhances this effect.

Overexpression of Lin28A in neural progenitor cells in vivo does not lead to brain tumor formation but results in reduced spine density.

LIN28A overexpression has been identified in malignant brain tumors called embryonal tumors with multilayered rosettes (ETMR) but its specific role during brain development remains largely unknown. Radial glia cells of the ventricular zone (VZ) are proposed as a cell of origin for ETMR. We asked whether an overexpression of LIN28A in such cells might affect brain development or result in the formation of brain tumors.Constitutive overexpression of LIN28A in hGFAP-cre::lsl-Lin28A (GL) mice led to a transient increase of proliferation in the cortical VZ at embryonic stages but no postnatal brain tumor formation. Postnatally, GL mice displayed a pyramidal cell layer dispersion of the hippocampus and altered spine and dendrite morphology, including reduced dendritic spine densities in the hippocampus and cortex. GL mice displayed hyperkinetic activity and differential quantitative MS-based proteomics revealed altered time dependent molecular functions regarding mRNA processing and spine morphogenesis. Phosphoproteomic analyses indicated a downregulation of mTOR pathway modulated proteins such as Map1b being involved in microtubule dynamics.In conclusion, we show that Lin28A overexpression transiently increases proliferation of neural precursor cells but it is not sufficient to drive brain tumors in vivo. In contrast, Lin28A impacts on protein abundancy patterns related to spine morphogenesis and phosphorylation levels of proteins involved in microtubule dynamics, resulting in decreased spine densities of neurons in the hippocampus and cortex as well as in altered behavior. Our work provides new insights into the role of LIN28A for neuronal morphogenesis and development and may reveal future targets for treatment of ETMR patients.

The kneeling test is a valid method of assessing kneeling tolerance.

PURPOSE: No quantitative test exists to reliably assess kneeling tolerance before and after surgery. The aim of this study was to validate a kneeling test, designed to quantify kneeling tolerance. METHOD: A total of 179 participants (324 knees) were enrolled into the study, including 124 asymptomatic knees, 98 with osteoarthritis (OA), 85 following total knee arthroplasty (TKA) and 17 following anterior cruciate ligament reconstruction (ACLR). Patients were asked to kneel on a custom-built platform on a soft, firm and hard surface, at both 90° and 110° of knee flexion. A kneeling score of 0-100 was established for 90° and 110° with 100 being a complete absence of pain or discomfort. A linear mixed model with random intercept was used to estimate differences between conditions (healthy, OA, ACLR and TKA). Pearson's correlation coefficient was used to test the strength of the association between the kneeling test and the forgotten joint score (FJS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score. Test-retest reliability was estimated by the intraclass correlation coefficient (ICC). RESULTS: The kneeling test demonstrated good discriminative validity with differences at 110° between asymptomatic knees and knees with OA (difference = - 42, p < 0.001), following ACLR (- 12, p = 0.013) and TKA (- 26, p < 0.001). Similar differences were observed at 90°. The kneeling test demonstrated construct validity, with a moderately strong correlation observed between the kneeling test and the FJS and the WOMAC at 90° (FJS 0.474 [95%CI: 0.357, 0.577], WOMAC - 0.503 [- 0.389, - 0.602]) and 110° (FJS 0.579 [95% CI: 0.479, 0.665], WOMAC - 0.648 [- 0.560, - 0.722]). The ICC for the kneeling test at 90° and 110° was 0.843 (95% CI: 0.745, 0.905) and 0.926 (95% CI: 0.877, 0.956), respectively. CONCLUSIONS: The kneeling test is a valid technique to quantitatively determine kneeling tolerance. It can aid in the assessment and modification of current surgical techniques to improve patient outcomes. LEVEL OF EVIDENCE: III.

Can Prophylactic Cables Stop Crack Propagation in Revision Arthroplasty: A Biomechanical Study.

BACKGROUND: Intraoperative femur fractures are a common complication of revision hip arthroplasty. This study examined the use of a prophylactic cable in stopping a crack from propagating beyond the cable. METHODS: Seventy sheep femora were prepared. A 5-mm vertical incision was performed. Using a force-controlled materials testing machine, a Wagner shaft was advanced until a crack occurred. Cracks were visualized with green ink. In the first part, the control group without any cable (n = 10) was compared with polyethylene (n = 15) and single CoCr cable (n = 15) groups. The cables were positioned 15 mm distal to the osteotomy. In the second part, three different CoCr configurations were compared, single-wrapped (n = 15), double-wrapped (n = 125), and two separate cables at 10 and 15 mm distal to the osteotomy (n = 15). RESULTS: The polyethylene cable stopped only 3 of 15 cracks (20%), whereas the CoCr cable stopped 11 of 15 cracks (73%) (P = .009). The force needed to initiate the crack between the different groups was not significant. Twelve (80%) of 15 cracks were stopped at the level of the cable with two separate CoCr cables and 15 (100%) of 15 cracks with a double-wrapped cable (P = .11). CONCLUSION: This study demonstrated that an elastic cable is not suitable for preventive cabling. The force required to form a crack is not improved with the use of a prophylactic cable placed 10-15 mm below the osteotomy. While the results on the different configurations were not conclusive, the double-wrapped cable was able to stop all cracks from progressing distally.

A systematic review of psychological interventions in total hip and knee arthroplasty.

BACKGROUND: The current practice in elective orthopaedics does not routinely include psychological interventions despite evidence that psychological factors such as personality, anxiety, depression and negative thinking styles can influence outcomes and recovery from surgery. The objective of this paper was to review the effectiveness of psychological interventions used in conjunction with total hip (THA) and knee arthroplasty (TKA), in improving patient reported joint outcomes. METHODS: An extensive literature search was conducted according to Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. Search terms included psychology, interventions, and orthopaedics. Articles were included if they were randomised controlled trials (RCTs) of psychological interventions involving active patient participation measured with patient reported joint outcomes in patients undergoing hip or knee arthroplasty. RESULTS: A total of 19,489 titles were screened. Seven studies met the inclusion criteria and were included. Five of seven studies did not show improvements in patient reported outcomes after surgery. Specifically, psycho-education alone was not effective at improving patient reported joint outcomes in two out of two studies. CONCLUSION: The current literature does not support routine psychological interventions for TKA and THA. However, it should be noted that the literature for psychological interventions in conjunction with TKA and THA is still in its infancy. This gap in the literature is surprising, considering the importance of the role of psychological factors in recovery. Further RCTs with long term follow ups, multidisciplinary involvement, and more comprehensive and focused interventions that go beyond educating patients are needed. Future studies should account for the demand effect, include measures of psychological variables to determine whether psychological interventions are more beneficial for some patients compared to others, and compare the different modes of delivery and timing of interventions to determine the optimal nature and duration of psychological interventions for TKA and THA.

Pseudoexon activation increases phenotype severity in a Becker muscular dystrophy patient.

We report a dystrophinopathy patient with an in-frame deletion of DMD exons 45-47, and therefore a genetic diagnosis of Becker muscular dystrophy, who presented with a more severe than expected phenotype. Analysis of the patient DMD mRNA revealed an 82 bp pseudoexon, derived from intron 44, that disrupts the reading frame and is expected to yield a nonfunctional dystrophin. Since the sequence of the pseudoexon and canonical splice sites does not differ from the reference sequence, we concluded that the genomic rearrangement promoted recognition of the pseudoexon, causing a severe dystrophic phenotype. We characterized the deletion breakpoints and identified motifs that might influence selection of the pseudoexon. We concluded that the donor splice site was strengthened by juxtaposition of intron 47, and loss of intron 44 silencer elements, normally located downstream of the pseudoexon donor splice site, further enhanced pseudoexon selection and inclusion in the DMD transcript in this patient.