Identifying risk factors for poor multidimensional recovery after major surgery: A systematic review.

Traditional risk factors used for predicting poor postoperative recovery have focused on postoperative complications, adverse symptoms (nausea, pain), length of hospital stay, and patient quality of life. Despite these being traditional performance indicators of patient postoperative "status," they may not fully define the multidimensional nature of patient recovery. The definition of postoperative recovery is thus evolving to include patient-reported outcomes that are important to the patient. Previous reviews have focused on risk factors for the above traditional outcomes after major surgery. Yet, there remains a need for further study of risk factors predicting multidimensional patient-focused recovery, and investigation beyond the immediate postoperative period after patients are discharged from the hospital. This review aimed to appraise the current literature identifying risk factors for multidimensional patient recovery. METHODS: A systematic review without meta-analysis was performed to qualitatively summarize preoperative risk factors for multidimensional recovery 4-6 weeks after major surgery (PROSPERO, CRD42022321626). We reviewed three electronic databases between January 2012 and April 2022. The primary outcome was risk factors for multidimensional recovery at 4-6 weeks. A GRADE quality appraisal and a risk of bias assessment were completed. RESULTS: In total, 5150 studies were identified, after which 1506 duplicates were removed. After the primary and secondary screening, nine articles were included in the final review. Interrater agreements between the two assessors for the primary and secondary screening process were 86% (k = 0.47) and 94% (k = 0.70), respectively. Factors associated with poor recovery were found to include ASA grade, recovery tool baseline score, physical function, number of co-morbidities, previous surgery, and psychological well-being. Mixed results were reported for age, BMI, and preoperative pain. Due to the observational nature, heterogeneity, multiple definitions of recovery, and moderate risk of bias of the primary studies, the quality of evidence was rated from very low to low. CONCLUSION: Our review found that there were few studies assessing preoperative risk factors as predictors for poor postoperative multidimensional recovery. This confirms the need for higher quality studies assessing risk for poor recovery, ideally with a consistent and multi-dimensional definition of recovery.

Characteristics of sarcopenia subjects in arterial pulse spectrum analysis.

Aims: Sarcopenia is significantly associated with the number of cardiovascular and metabolic diseases, however, the underlying pathophysiological processes are largely unknown. This study performed harmonic index of finger photoplethysmography (PPG) waveforms with the aims of distinguishing different arterial pulse waveform signals between sarcopenia, presarcopenia, dynapenia, and healthy subjects. Methods: Sixty-eight subjects were enrolled and obtained 1-min PPG signals, then were assigned to four age-matched groups: control, dynapenia, presarcopenia, and sarcopenia which definition according to Asian Working Group for Sarcopenia (AWGS): 2019 Consensus Update on Sarcopenia Diagnosis and Treatment. Harmonics 1-10 of the PPG waveform were obtained and calculated each of the amplitude proportions (C n ), standard deviations (SD n ), coefficients of variations (CV n ), and vascular elasticity index (VEI) for to evaluating the blood-pressure harmonic variability. Results: The prevalence of sarcopenia in women gender (8 out of 9, 88.9%, p = 0.046) and osteoporosis in dynapenia (7 out of 16, 43.8%, p = 0.005) were significant higher. Among the four groups, compared with control, dynapenia, and presarcopenia, sarcopenia had largest SD n -values for harmonics 1, 2, 3, and 5 (ratio 1, 2, 3, 5 = 0.354, 0.209, 0.137, 0.074); whereas sarcopenia had largest coefficients of variations (CV n ) values for harmonics 1, 2, 3 and 10 (ratio 1, 2, 3, 10 = 0.263, 0.310, 0.402, 0.791). Besides, the Δ odds ratio of ratio 3, 4,and 6 tertile values were significantly increased in sarcopenia and possible sarcopenia group compared with control group. Subjects with sarcopenia had significantly higher VEI in mean, SD, and CV of the PPG waveform (mean = 2.332, SD = 1.479, CV = 0.634, p = 0.007) among the groups and the results of binary logistic regression analysis in the tertiles met statistical significance between the sarcopenia and non-sarcopenia groups whether adjusted or unadjusted (adjusted odds ratio 6.956, p = 0.030, unadjusted odds ratio 3.937, p = 0.039). Conclusions: The elasticity of vessels among sarcopenia groups in lower-frequency components of harmonic ratio in which we defined as VEI showed a significantly highest VEI mean, SD, and CV in sarcopenia indicates the poorer elasticity of the arteries. The present findings showed finger PPG waveform measurements may be useful for early detection of vascular diseases with patients with sarcopenia in a non-invasive and easy-to-perform technique which may expand the clinical applicability in the future.

Prognostic and Theranostic Biomarkers in Ovarian Clear Cell Carcinoma.

In this study, we aimed to test whether prognostic biomarkers can achieve a clinically relevant stratification of patients with stage I ovarian clear cell carcinoma (OCCC) and to survey the expression of 10 selected actionable targets (theranostic biomarkers) in stage II to IV cases. From the population-based Alberta Ovarian Tumor Type study, 160 samples of OCCC were evaluated by immunohistochemistry and/or silver-enhanced in situ hybridization for the status of 5 prognostic (p53, p16, IGF2BP3, CCNE1, FOLR1) and 10 theranostic biomarkers (ALK, BRAF V600E, ERBB2, ER, MET, MMR, PR, ROS1, NTRK1-3, VEGFR2). Kaplan-Meier survival analyses were performed. Cases with abnormal p53 or combined p16/IFG2BP3 abnormal expression identified a small subset of patients (6/54 cases) with stage I OCCC with an aggressive course (5-yr ovarian cancer-specific survival of 33.3%, compared with 91.5% in the other stage I cases). Among theranostic targets, ERBB2 amplification was present in 11/158 (7%) of OCCC, while MET was ubiquitously expressed in OCCC similar to a variety of normal control tissues. ER/PR showed a low prevalence of expression. No abnormal expression was detected for any of the other targets. We propose a combination of 3 biomarkers (p53, p16, IGF2BP3) to predict prognosis and the potential need for adjuvant therapy for patients with stage I OCCC. This finding requires replication in larger cohorts. In addition, OCCC could be tested for ERBB2 amplification for inclusion in gynecological basket trials targeting this alteration.

Combined CCNE1 high-level amplification and overexpression is associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma.

CCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high-level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut-off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high-level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut-off for IHC to predict CCNE1 high-level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high-level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high-level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38-2.26, p < 0.0001). CCNE1 high-level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high-level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors.

Histological and molecular diversity and heterogeneity of precancerous lesions associated with inflammatory bowel diseases.

AIMS: Inflammatory bowel disease (IBD)-associated precancerous lesions may be adenomatous or non-adenomatous with various histomorphologies. We aim to validate the newly proposed classification, to explore the neoplastic nature of the non-adenomatous lesions and to elucidate the molecular mechanisms underlying the different histomorphologies. METHODS: 44 background precursor lesions identified in 53 cases of surgically resected IBD-associated colorectal and ileal carcinomas were reviewed for the histomorphological features (classified into adenomatous, mucinous, sessile serrated adenoma (SSA)-like, traditional serrated adenoma-like, differentiated, eosinophilic and serrated not otherwise specified (NOS)) and analysed for a key panel of colonic cancer-related molecular markers. RESULTS: Approximately 60% of the lesions were adenomatous, of which some had mixed serrated, mucinous or eosinophilic changes. The remaining non-adenomatous lesions, including all other types except SSA-like type, mostly showed mixed features and focal adenomatous dysplasia. KRAS mutation and p53 mutant-type expression were found in about half cases across all types, while PIK3CA mutation only in some of adenomatous and eosinophilic lesions and MLH1/PMS2 loss in a subset of adenomatous, mucinous and eosinophilic but not in differentiated and serrated lesions. SAT-B2 or PTEN loss and IMP3 overexpression were seen in a small subset of lesions. No BRAF, NRAS or EGFR gene mutation was detected in any type. Certain molecular-morphological correlations were demonstrated; however, no single or combined molecular alteration(s) was specific to any particular morphological type. CONCLUSIONS: IBD-associated precancerous lesions are heterogeneous both histologically and molecularly. True colitis-associated adenomatous lesions are unlikely conventional adenomas. Non-adenomatous lesions without frank cytologic dysplasia should also be regarded as neoplastic.

A randomized trial of desflurane or sevoflurane on postoperative quality of recovery after knee arthroscopy.

BACKGROUND: Studies have described different recovery profiles of sevoflurane and desflurane typically early after surgery. METHODS: We conducted a randomized superiority trial to determine whether Overall Recovery 3 days after knee arthroscopy would be superior with desflurane. Adult participants undergoing knee arthroscopic surgery with general anesthesia were randomized to either desflurane or sevoflurane general anesthesia. Intraoperative and postoperative drugs and analgesics were administered at the discretion of the anesthesiologist. Postoperative quality of recovery was assessed using the "Postoperative Quality of Recovery Scale". The primary outcome was Overall Recovery 3 days after surgery and secondary outcomes were individual recovery domains at 15 minutes, 40 minutes, 1 day, 3 days, 1 month, and 3 months. Patients and researchers were blinded. RESULTS: 300 patients were randomized to sevoflurane or desflurane (age 51.7±14.1 vs. 47.3±13.5 years; duration of anesthesia 24.9±11.1 vs. 23.3±8.3 minutes). The proportion achieving baseline or better scores in all domains increased over the follow-up period in both groups but was not different at day 3 (sevoflurane 43% vs. desflurane 37%, p = 0.314). Similarly, rates of recovery increased over time in the five subdomains, with no differences between groups for physiological, p = 0.222; nociceptive, p = 0.391; emotive, p = 0.30; Activities-of-daily-living, p = 0.593; and cognitive recovery, p = 0.877. CONCLUSION: No significant difference in the quality of recovery scale could be shown using sevoflurane or desflurane general anesthesia after knee arthroscopy in adult participants.

21-Year Survival of Left Internal Mammary Artery-Radial Artery-Y Graft.

BACKGROUND: In 1999, Royse et al. reported on the left internal mammary artery, radial artery, Y-graft technique (LIMA-RA-Y), which achieves total arterial revascularization (TAR). However, the most common coronary reconstruction remains LIMA and supplementary saphenous vein grafts (LIMA + SVG). OBJECTIVES: The goal of this study was to conduct a survival comparison of LIMA-RA-Y versus the conventional LIMA + SVG. METHODS: Of the original 464 LIMA-RA-Y patients reported (1996 to 1998), 346 were from the Royal Melbourne Hospital. Survival at June 2017 was compared with a group of 534 patients from 1996 to 2003 from the same institution who received LIMA + SVG, or 5,800 patients who received TAR with different grafting configurations. Propensity score matching (PSM) was performed with 1:1 matching using 26 variables. Comparisons used Kaplan-Meier (KM) and Cox proportional hazards methods. LIMA-RA-Y was compared with LIMA + SVG in which all non-left anterior descending artery grafts were performed with either composite RA or aorta-coronary SVG with no use of right internal mammary artery. We also conducted a comparison of LIMA-RA-Y versus TAR. RESULTS: Baseline characteristics of the LIMA-RA-Y group (n = 346) compared with LIMA + SVG (n = 534) after PSM (n = 232 pairs) did not differ (3.3 ± 0.8 grafts per patient). Survival was worse for LIMA + SVG in the unmatched groups (KM, p < 0.001) and for PSM groups (KM, p = 0.043; Cox proportional hazards ratio: 1.3; 95% confidence interval: 1.0 to 1.6; p = 0.038). Survival did not differ between LIMA-RA-Y and other TAR (n = 5,800) patients before, or after, PSM (n = 332 pairs). CONCLUSIONS: Use of LIMA + SVG has worse survival than LIMA-RA-Y in achieving total arterial revascularization.

The effect on survival from the use of a saphenous vein graft during coronary bypass surgery: a large cohort study.

OBJECTIVES: Saphenous vein graft (SVG) remains the predominant conduit used in coronary surgery. The internal mammary artery has higher later term patency and confers superior survival. Current debate focuses on the increased use of arterial conduits rather than eradication of venous conduits. METHODS: Patient data extracted from the Australian and New Zealand Society of Cardiothoracic Surgeons database from 2001-2013 were linked to the national death registry held by the Australian Institute of Health and Welfare for all-cause mortality with censor date 7 October 2014. The dataset was divided according to use of SVG rather than the arterial conduit. Analyses of SVG ≥ 1 or SVG = 1 were compared to SVG = 0. Additionally, groups of 3, 4 or 5 grafts were subjected to multiple analyses testing the mortality hazard with increasing use of SVG. Propensity score matched analyses were conducted using 24 variables. RESULTS: Of 51 113 primary coronary surgery patients, unmatched survival at up to 12.5 years was significantly lower for SVG ≥ 1, n = 33 359, mortality hazard ratio (HR) 1.24 [95% confidence interval (CI) 1.18-1.30], P < 0.001; and for SVG = 1, mortality HR 1.19 (95% CI 1.12-1.26), P < 0.001. Similar results were present for the propensity score matched groups; SVG ≥ 1, n = 14 355 pairs, HR 1.22 (95% CI 1.15-1.30), P < 0.001; and for SVG = 1, n = 12 316 pairs, HR 1.22 (95% CI 1.14-1.30), P < 0.001. All matched analyses within restricted graft groups had increasing HR with increased number of SVG used. CONCLUSIONS: Any use of SVGs is independently associated with reduced survival after coronary artery bypass surgery.

Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.

Visualization of a Mammalian Mitochondrion by Coherent X-ray Diffractive Imaging.

We report a three dimensional (3D) quantitative visualization of a mammalian mitochondrion by coherent x-ray diffractive imaging (CXDI) using synchrotron radiation. The internal structures of a mitochondrion from a mouse embryonic fibroblast cell line (NIH3T3) were visualized by tomographic imaging at approximately 60 nm resolution without the need for sectioning or staining. The overall structure consisted of a high electron density region, composed of the outer and inner membranes and the cristae cluster, which enclosed the lower density mitochondrial matrix. The average mass density of the mitochondrion was about 1.36 g/cm3. Sectioned images of the cristae reveal that they have neither a baffle nor septa shape but were instead irregular. In addition, a high resolution, about 14 nm, 2D projection image was captured of a similar mitochondrion with the aid of strongly scattering Au reference objects. Obtaining 3D images at this improved resolution will allow CXDI to be an effective and nondestructive method for investigating the innate structure of mitochondria and other important life supporting organelles.

Impact of Methylprednisolone on Postoperative Quality of Recovery and Delirium in the Steroids in Cardiac Surgery Trial: A Randomized, Double-blind, Placebo-controlled Substudy.

BACKGROUND: Inflammation after cardiopulmonary bypass may contribute to postoperative delirium and cognitive dysfunction. The authors evaluated the effect of high-dose methylprednisolone to suppress inflammation on the incidence of delirium and postoperative quality of recovery after cardiac surgery. METHODS: Five hundred fifty-five adults from three hospitals enrolled in the randomized, double-blind Steroids in Cardiac Surgery trial were randomly allocated to placebo or 250 mg methylprednisolone at induction and 250 mg methylprednisolone before cardiopulmonary bypass. Each completed the Postoperative Quality of Recovery Scale before surgery and on days 1, 2, and 3 and 1 and 6 months after surgery and the Confusion Assessment Method scale for delirium on days 1, 2, and 3. Recovery was defined as returning to preoperative values or improvement at each time point. RESULTS: Four hundred eighty-two participants for recovery and 498 participants for delirium were available for analysis. The quality of recovery improved over time but without differences between groups in the primary endpoint of overall recovery (odds ratio range over individual time points for methylprednisolone, 0.39 to 1.45; 95% CI, 0.08-2.04 to 0.40-5.27; P = 0.943) or individual recovery domains (all P > 0.05). The incidence of delirium was 10% (control) versus 8% (methylprednisolone; P = 0.357), with no differences in delirium subdomains (all P > 0.05). In participants with normal (51%) and low baseline cognition (49%), there were no significant differences favoring methylprednisolone in any domain (all P > 0.05). Recovery was worse in patients with postoperative delirium in the cognitive (P = 0.004) and physiologic (P < 0.001) domains. CONCLUSIONS: High-dose intraoperative methylprednisolone neither reduces delirium nor improves the quality of recovery in high-risk cardiac surgical patients.

Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration.

The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process.

Primary cilia in the developing pig testis.

In vertebrates, a variety of cell types generate a primary cilium. Cilia are implicated in determination and differentiation of a wide variety of organs and during embryonic development. However, there is little information on the presence or function of primary cilia in the mammalian testis. Therefore, the objective of this study was to characterize expression of primary cilia in the developing pig testis. Testicular tissue from pigs at 2-10 weeks of age was analyzed for primary cilia by immunocytochemistry. Expression of primary cilia was also analyzed in testicular tissue formed de novo from a single cell suspension ectopically grafted into a mouse host. Functionality of primary cilia was monitored based on cilia elongation after exposure to lithium. Analysis showed that the primary cilium is present in testis cords as well as in the interstitium of the developing pig testis. Germ cells did not express primary cilia. However, we identified Sertoli cells as one of the somatic cell types that produce a primary cilium within the developing testis. Primary cilium expression was reduced from the second to the third week of pig testis development in situ and during de novo morphogenesis of testis tissue from a single cell suspension after xenotransplantation. In vitro, primary cilia were elongated in response to lithium treatment. These results indicate that primary cilia on Sertoli cells may function during testicular development. De novo morphogenesis of testis tissue from single cell suspensions may provide an accessible platform to study and manipulate expression and function of primary cilia.

Targeting of CRMP-2 to the primary cilium is modulated by GSK-3ß.

CRMP-2 plays a pivotal role in promoting axon formation, neurite outgrowth and elongation in neuronal cells. CRMP-2's role in other cells is unknown. Our preliminary results showed CRMP-2 expression in cilia of fibroblasts. To localize CRMP-2, define its role and study the regulation of CRMP-2's expression in cilia we carried out the following experiments. We find that in fibroblasts CRMP-2 localizes to the centrosome and is associated with the basal body and -at a low level- is present in primary cilia. Phosphorylated pCRMP-2 can only be detected at the basal body. RNAi knockdown of CRMP-2 interfered with primary cilium assembly demonstrating a critical requirement for CRMP-2. Deletion analysis of CRMP-2 identified a 51 amino acid sequence in the C-terminus that is required for targeting to the basal body and primary cilium. This domain contains GSK-3ß phosphorylation sites as well as two repeats of the VxPx motif, previously identified as a cilium targeting signal in other primary cilium proteins. To our surprise, mutation of the CRMP-2 VxPx motifs did not eliminate primary cilium targeting. Instead, mutation of the GSK-3ß phosphorylation sites abolished CRMP-2 targeting to the primary cilium without affecting basal body localization. Treatment of cells with lithium, a potent GSK-3ß inhibitor, or with two specific GSK-3ß inhibitors (the L803-mts peptide inhibitor and CHIR99021) resulted in cilium elongation and decreased basal body levels of pCRMP-2 as well as increased levels of total CRMP-2 at the primary cilium. In summary, we identified CRMP-2 as a protein critically involved in primary cilia formation. To our knowledge this is the first demonstration of modulation of primary cilium targeting by GSK-3ß.

KLC3 is involved in sperm tail midpiece formation and sperm function.

Kinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear. Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ΔHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa.

Ornithine decarboxylase antizyme Oaz3 modulates protein phosphatase activity.

Ornithine decarboxylase antizyme 3 (Oaz3) is expressed in spermatids, makes up the antizyme family of Oaz genes with Oaz1 and Oaz2, and was proposed to encode a 22 kDa antizyme protein involved in polyamine regulation similar to the 22 kDa OAZ1 and OAZ2 proteins. Here we demonstrate however that the major product encoded by Oaz3 is a 12 kDa protein, p12, which lacks the antizyme domain that interacts with ornithine decarboxylase. We show that p12 does not affect ornithine decarboxylase levels, providing an explanation for the surprising observation made in Oaz3 knock-out male mice, which do not display altered testis polyamine metabolism. This suggested a novel activity for Oaz3 p12. Using immuno-electron microscopy we localized p12 to two structures in the mammalian sperm tail, viz. the outer dense fibers and fibrous sheath, as well as to the connecting piece linking head and tail. We identified myosin phosphatase targeting subunit 3 (MYPT3), a regulator of protein phosphatase PP1ß, as a major p12-interacting protein, and show that MYPT3 is present in sperm tails and that its ankyrin repeat binds p12. We show that MYPT3 can also bind protein phosphatase PP1γ2, the only protein phosphatase present in sperm tails, and that p12- MYPT3 interaction modulates the activity of both PP1ß and PP1γ2. This is, to our knowledge, the first demonstration of a novel activity for an Oaz-encoded protein.

Binding of nickel to testicular glutamate-ammonia ligase inhibits its enzymatic activity.

Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate-ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure.

Gene trap mutation of murine outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism.

BACKGROUND: Outer dense fiber protein 2, Odf2, is a major component of the outer dense fibers, ODF, in the flagellum of spermatozoa. ODF are associated with microtubule doublets that form the axoneme. We recently demonstrated that tyrosine phosphorylation of Odf2 is important for sperm motility. In the course of a study of Odf2 using Odf2 mouse knockout lines we observed that males of a high percentage chimaerism, made using XL169 embryonic stem cells, were infertile, whereas mice of low-medium percentage chimaerism were fertile. RESULTS: XL169 ES cells have a beta-geo gene trap cassette inserted in the Odf2 gene. To determine possible underlying mechanisms resulting in infertility we analyzed epididymal sperm and observed that >50% displayed bent tails. We next performed ultrastructural analyses on testis of high percentage XL169 chimaeric mice. This analysis showed that high percentage XL169 chimaeric mice produce elongating spermatids that miss one or more entire outer dense fibers in their midpiece and principal piece. In addition, we observed elongating spermatids that show thinning of outer dense fibers. No other obvious abnormalities or defects are present in elongating spermatids. Spermatozoa from the caput and cauda epididymis of XL169 mice of high percentage chimaerism show additional tail defects, including absence of one or more axonemal microtubule doublets and bent tails. Sperm with bent tails display abnormal motility. CONCLUSIONS: Our results document the possible impact of loss of one Odf2 allele on sperm tail structure and function, resulting in a novel sperm tail phenotype.

Primary cilia in fibroblast-like type B synoviocytes lie within a cilium pit: a site of endocytosis.

The synovium is a thin connective tissue that lines the joint space of free moving articulations. In this report, the expression, structure, and composition of non-motile (primary) cilia in fibroblast-like synoviocytes (FLS) that populate the synovium have been studied. Primary cilia are non-motile, microtubule-based organelles that have been found in a variety of vertebrate cell types. We document that primary cilia are expressed in normal human synovium FLS, cultured human FLS, and FLS cells present in human synovial fluid, and that the cellular region occupied by the primary cilium shows a similar and highly defined architecture within these FLS. This architecture includes the presence of a unique structure that surrounds the lower portion of the cilium shaft. This structure, given the term cilium-pit, includes a space, the pit reservoir. Actin filament bundles surround the cilium-pit, and when these bundles are removed experimentally the volume of the cilium-pit and its continuity with the extracellular environment changes. Finally, this study documents that the cilium-pit is a site of endocytosis and is also the site for the localization of receptors (TNF receptors TNFR1 and TNFR2) associated with synoviocyte function. Taken together, the results of the present study suggest that the FLS cilium-pit functions to regulate the exposure of the primary cilium, both spatially and temporally to extracellular molecules and to couple primary cilium based signaling pathways with those linked to endocytosis.