Molecular profiling of activated neurons by phosphorylated ribosome capture.

The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, thereby enriching directly for the mRNAs expressed in discrete subpopulations of activated cells. We use this approach to identify neurons in the hypothalamus regulated by changes in salt balance or food availability. We show that galanin neurons are activated by fasting and that prodynorphin neurons restrain food intake during scheduled feeding. These studies identify elements of the neural circuit that controls food intake and illustrate how the activity-dependent capture of cell-type-specific transcripts can elucidate the functional organization of a complex tissue.

Dissecting Ubiquitylation and DNA Damage Response Pathways in the Yeast Saccharomyces cerevisiae Using a Proteome-Wide Approach.

In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various posttranslational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture, followed by an enrichment of ubiquitylated peptides and LC-MS/MS. In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.

ISW1a modulates cohesin distribution in centromeric and pericentromeric regions.

Cohesin is a highly conserved, multiprotein complex whose canonical function is to hold sister chromatids together to ensure accurate chromosome segregation. Cohesin association with chromatin relies on the Scc2-Scc4 cohesin loading complex that enables cohesin ring opening and topological entrapment of sister DNAs. To better understand how sister chromatid cohesion is regulated, we performed a proteomic screen in budding yeast that identified the Isw1 chromatin remodeler as a cohesin binding partner. In addition, we found that Isw1 also interacts with Scc2-Scc4. Lack of Isw1 protein, the Ioc3 subunit of ISW1a or Isw1 chromatin remodeling activity resulted in increased accumulation of cohesin at centromeres and pericentromeres, suggesting that ISW1a may promote efficient translocation of cohesin from the centromeric site of loading to neighboring regions. Consistent with the role of ISW1a in the chromatin organization of centromeric regions, Isw1 was found to be recruited to centromeres. In its absence we observed changes in the nucleosomal landscape at centromeres and pericentromeres. Finally, we discovered that upon loss of RSC functionality, ISW1a activity leads to reduced cohesin binding and cohesion defect. Taken together, our results support the notion of a key role of chromatin remodelers in the regulation of cohesin distribution on chromosomes.

Mechanisms of genotoxicity and proteotoxicity induced by the metalloids arsenic and antimony.

Arsenic and antimony are metalloids with profound effects on biological systems and human health. Both elements are toxic to cells and organisms, and exposure is associated with several pathological conditions including cancer and neurodegenerative disorders. At the same time, arsenic- and antimony-containing compounds are used in the treatment of multiple diseases. Although these metalloids can both cause and cure disease, their modes of molecular action are incompletely understood. The past decades have seen major advances in our understanding of arsenic and antimony toxicity, emphasizing genotoxicity and proteotoxicity as key contributors to pathogenesis. In this review, we highlight mechanisms by which arsenic and antimony cause toxicity, focusing on their genotoxic and proteotoxic effects. The mechanisms used by cells to maintain proteostasis during metalloid exposure are also described. Furthermore, we address how metalloid-induced proteotoxicity may promote neurodegenerative disease and how genotoxicity and proteotoxicity may be interrelated and together contribute to proteinopathies. A deeper understanding of cellular toxicity and response mechanisms and their links to pathogenesis may promote the development of strategies for both disease prevention and treatment.

Return to Sport Following Distal Triceps Repair.

PURPOSE: The purpose of this investigation was to examine the timeline of return-to-sport following distal triceps repair; evaluate the degree of participation and function upon returning to sport; and identify risk factors for failure to return to sport. METHODS: Patients who underwent distal triceps repair with a minimum of 1 year of follow-up were retrospectively reviewed. Patients completed a subjective sports questionnaire and were scored on a visual analog scale for pain; the Mayo Elbow Performance Index; the Quick Disabilities of the Arm, Shoulder, and Hand; and the Single Assessment Numerical Evaluation. RESULTS: Out of 113 eligible patients who had a distal triceps repair, 81 patients (71.7%) were contacted. Sixty-eight patients (84.0%) who participated in sports prior to surgery were included at 6.0 ± 4.0 years after surgery, and the average age was 46.6 ± 11.5 years. Sixty-one patients (89.7%) resumed playing at least 1 sport by 5.9 ± 4.4 months following distal triceps repair. However, 18 patients (29.5%) returned to a lower level of activity intensity. The average postoperative Quick Disabilities of the Arm, Shoulder, and Hand; Mayo Elbow Performance; visual analog scale for pain; and Single Assessment Numerical Evaluation scores were 8.2 ± 14.0, 89.5 ± 13.4, 2.0 ± 1.7, and 82.2 ± 24.3, respectively. No patients underwent revision surgery at the time of final follow-up. CONCLUSIONS: Distal triceps repair enables 89.7% of patients to return to sport by 5.9 ± 4.4 months following surgery. However, 29.5% of patients were unable to return to their preinjury level of activity. It is imperative that patients are appropriately educated to manage postoperative expectations regarding sport participation following distal triceps repair. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Error-free DNA damage tolerance pathway is facilitated by the Irc5 translocase through cohesin.

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error-free recombination-like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single-stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4-dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error-free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5-mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.

Etp1 confers arsenite resistance by affecting ACR3 expression.

In a high-throughput yeast two-hybrid screen of predicted coiled-coil motif interactions in the Saccharomyces cerevisiae proteome, the protein Etp1 was found to interact with the yeast AP-1-like transcription factors Yap8, Yap1 and Yap6. Yap8 plays a crucial role during arsenic stress since it regulates expression of the resistance genes ACR2 and ACR3. The function of Etp1 is not well understood but the protein has been implicated in transcription and protein turnover during ethanol stress, and the etp1∆ mutant is sensitive to ethanol. In this current study, we investigated whether Etp1 is implicated in Yap8-dependent functions. We show that Etp1 is required for optimal growth in the presence of trivalent arsenite and for optimal expression of the arsenite export protein encoded by ACR3. Since Yap8 is the only known transcription factor that regulates ACR3 expression, we investigated whether Etp1 regulates Yap8. Yap8 ubiquitination, stability, nuclear localization and ACR3 promoter association were unaffected in etp1∆ cells, indicating that Etp1 affects ACR3 expression independently of Yap8. Thus, Etp1 impacts gene expression under arsenic and other stress conditions but the mechanistic details remain to be elucidated.

The ancillary N-terminal region of the yeast AP-1 transcription factor Yap8 contributes to its DNA binding specificity.

Activator protein 1 (AP-1) is one of the largest families of basic leucine zipper (bZIP) transcription factors in eukaryotic cells. How AP-1 proteins achieve target DNA binding specificity remains elusive. In Saccharomyces cerevisiae, the AP-1-like protein (Yap) family comprises eight members (Yap1 to Yap8) that display distinct genomic target sites despite high sequence homology of their DNA binding bZIP domains. In contrast to the other members of the Yap family, which preferentially bind to short (7-8 bp) DNA motifs, Yap8 binds to an unusually long DNA motif (13 bp). It has been unclear what determines this unique specificity of Yap8. In this work, we use molecular and biochemical analyses combined with computer-based structural design and molecular dynamics simulations of Yap8-DNA interactions to better understand the structural basis of DNA binding specificity determinants. We identify specific residues in the N-terminal tail preceding the basic region, which define stable association of Yap8 with its target promoter. We propose that the N-terminal tail directly interacts with DNA and stabilizes Yap8 binding to the 13 bp motif. Thus, beside the core basic region, the adjacent N-terminal region contributes to alternative DNA binding selectivity within the AP-1 family.

Traction Tenolysis for Flexor Tendon Adhesions: Outcomes in 97 Patients.

PURPOSE: Traction tenolysis is an alternative, less invasive way of performing flexor tendon tenolysis by winding affected tendons around a surgical instrument. This study assessed outcomes and complications in a cohort of patients who underwent traction tenolysis to determine its effectiveness. METHODS: We retrospectively reviewed 97 patients who underwent traction tenolysis performed by 4 fellowship-trained hand surgeons from 2010 to 2019. We collected data on preoperative and postoperative ranges of motion, the number and type of prior ipsilateral hand surgeries, and the duration of therapy and follow-up. Cases of traditional open tenosynovectomy tenolysis were excluded. RESULTS: Approximately two-thirds of the patients achieved more than 75% of the normal total active motion, and 80% achieved at least 50% of the normal total active motion. The mean total active flexion increased significantly by 42° and passive flexion by 25°. The differences in active and passive flexion significantly decreased from 28° before the surgery to 9° after the surgery. The active and passive flexion of the distal interphalangeal and proximal interphalangeal joints improved similarly, at approximately 20° and 10°, respectively. The average duration of follow-up was 11 ± 8 weeks. The complication rate was 5%: 1 case of intraoperative flexor digitorum superficialis tendon rupture, 1 case of postoperative infection, and 3 reoperations because of failure to progress. CONCLUSIONS: Traction tenolysis is an alternative to traditional open tenolysis surgery in selected patients. LEVEL OF EVIDENCE: Therapeutic IV.

Relationship Between Preoperative and Postoperative Motion After Four-Corner Wrist Fusion for Osteoarthritis: Clustering and Regression Analyses.

PURPOSE: Four-corner fusion (4CF) is a surgical option for refractory scapholunate advanced collapse and scaphoid nonunion advanced collapse wrist arthritis. Preoperative range of motion (ROM) predicts outcomes in many orthopedic procedures. This study investigates ROM in a cohort of 4CF patients to examine the relationship between preoperative and postoperative motion and identifies different clinical patterns. METHODS: We performed a retrospective review of 4CF patients. Patients with a history of inflammatory arthritis and radiographic characteristics of inflammation were excluded. Demographics, prior wrist surgery history, and ROM data were collected at preoperative and postoperative intervals after cast removal at 8 weeks, 3 months, and 8 months. Regression analysis compared the motion before and after 4CF. Subsequent cluster analysis to reduce confounding compared postoperative motion differences in the top 20% to the bottom 20% of patients by preoperative motion. RESULTS: We included 148 patients; 27 had prior surgery on the ipsilateral wrist. Preoperative arc averaged 86° ± 28° (flexion 46° ± 17°, extension 40° ± 15°); 8-week arc 43° ± 19° (flexion 19° ± 12°, extension 24° ± 12°); 3-month arc 62° ± 17° (flexion 30° ± 12°, extension 32° ± 11°); and 8-month arc 74° ± 17° (flexion 36° ± 11°, extension 37° ± 12°). Preoperative and final arcs were (r = 0.39). Clustering by the preoperative arc, the top 20% (mean 124° ± 15°) achieved a mean final arc of 81° ± 16°, while the bottom 20% (mean 47° ± 16°) achieved a mean final arc of 65° ± 19°. Intercluster differences were statistically significant. The bottom 20% gained motion postoperatively. Most patients in the middle 60% did not differ significantly in postoperative motion. CONCLUSIONS: Although wrist motion following 4CF correlates positively with preoperative motion, most patients do not differ significantly in postoperative motion. Patients with substantial preoperative motion deficits gain motion after 4CF. This information is important when counseling patients, determining the timing of surgical intervention, and managing expectations related to motion outcomes. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic II.

Protein-fragment complementation assays for large-scale analysis of protein-protein interactions.

Protein-protein interactions (PPIs) orchestrate nearly all biological processes. They are also considered attractive drug targets for treating many human diseases, including cancers and neurodegenerative disorders. Protein-fragment complementation assays (PCAs) provide a direct and straightforward way to study PPIs in living cells or multicellular organisms. Importantly, PCAs can be used to detect the interaction of proteins expressed at endogenous levels in their native cellular environment. In this review, we present the principle of PCAs and discuss some of their advantages and limitations. We describe their application in large-scale experiments to investigate PPI networks and to screen or profile PPI targeting compounds.

Return to work following distal triceps repair.

PURPOSE: The purpose of this study was to evaluate the rate and duration of return to work in patients undergoing distal triceps repair (DTR). METHODS: Consecutive patients undergoing DTR from 2009 to 2017 at our institution were retrospectively reviewed at a minimum of 1 year postoperatively. Patients completed a standardized and validated work questionnaire; a visual analog scale for pain; the Mayo Elbow Performance Score; the short version of the Disabilities of the Arm, Shoulder and Hand questionnaire; and a satisfaction survey. RESULTS: Of 113 eligible patients who underwent DTR, 81 (71.7%) were contacted. Of these patients, 74 (91.4%) were employed within 3 years prior to surgery (mean age, 46.0 ± 10.7 years; mean follow-up, 5.9 ± 3.9 years). Sixty-nine patients (93.2%) returned to work by 2.2 ± 3.2 months postoperatively. Sixty-six patients (89.2%) were able to return to the same level of occupational intensity. Patients who held sedentary-, light-, medium-, and high-intensity occupations were able to return to work at a rate of 100.0%, 100.0%, 80.0%, and 76.9%, respectively, by 0.3 ± 0.5 months, 1.8 ± 1.5 months, 2.5 ± 3.6 months, and 4.8 ± 3.9 months, respectively, postoperatively. Of the workers' compensation patients, 15 (75%) returned to work by 6.5 ± 4.3 months postoperatively, whereas 100% of non-workers' compensation patients returned to work by 1.1 ± 1.6 months (P < .001). Seventy-one patients (95.9%) were at least somewhat satisfied, with 50 patients (67.6%) reporting excellent satisfaction. Seventy-two patients (97.3%) would undergo the operation again if presented the opportunity. A single patient (1.4%) required revision DTR. CONCLUSIONS: Approximately 93% of patients who underwent DTR returned to work by 2.2 ± 3.2 months postoperatively. Patients with higher-intensity occupations had an equivalent rate of return to work but took longer to return to their preoperative level of occupational intensity. Information regarding return to work is imperative in preoperative patient consultation to manage expectations.

Complex Mechanisms of Antimony Genotoxicity in Budding Yeast Involves Replication and Topoisomerase I-Associated DNA Lesions, Telomere Dysfunction and Inhibition of DNA Repair.

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.

Management of Periprosthetic Joint Infection in Total Elbow Arthroplasty.

Periprosthetic joint infection (PJI) is a potentially devastating complication after total elbow arthroplasty (TEA) that can lead to significant morbidity for the patient as well as increased health care-related costs. Despite the potential morbidity associated with TEA PJI, evidence is limited regarding an optimal treatment algorithm. Initial management typically consists of either irrigation and debridement or 2-stage revision. A stable implant, a functioning triceps, and an intact soft tissue envelope are necessary to perform irrigation and debridement. Irrigation and debridement is associated with a relatively high risk of infection recurrence especially in chronic infections. Two-stage revision offers a lower recurrence risk, although there is a 25% chance of not completing the second stage. Resection arthroplasty, arthrodesis, and amputation are salvage options, whereas medical treatment, in the form of antibiotics alone, is reserved for poor surgical candidates. Further multicenter prospective study and retrospective review of registry data focusing on different treatment algorithms, prevention strategies, and functional outcomes would be helpful to elucidate the ideal management of elbow PJI.

Restoration of peak strength and endurance following distal biceps reconstruction with allograft for chronic ruptures.

BACKGROUND: Distal biceps reconstruction for chronic rupture often requires a graft to recover length and allow for distal tendon reattachment to bone. Our purpose was to assess peak strength and endurance recovery following biceps reconstruction with tendon grafts. HYPOTHESIS: We hypothesized that allograft reconstruction would result in decreased flexion and supination peak strength and endurance. METHODS: Consecutive distal biceps reconstructions with allograft, performed for chronic ruptures between January 2008 and March 2018 at a single institution, were reviewed. Isokinetic dynamometry for peak strength and endurance testing was performed on the operative and contralateral arms in flexion and supination. Functional outcomes and overall satisfaction with the operation were determined. RESULTS: Eleven patients were available for a complete evaluation, including dynamometry, at a mean of 46 months postoperatively. Reconstructions demonstrated a nonsignificant trend toward decreased peak flexion strength (P = .06), and significantly decreased peak supination strength (P = .01) compared with the unaffected arm. There were no differences in flexion and supination endurance between the affected and unaffected arms. Using standardized outcome scales, patients reported excellent function. CONCLUSION: Chronic biceps ruptures undergoing reconstruction are highly functional and patients are satisfied. Somewhat surprisingly, supination and flexion endurance were equal to the contralateral, uninvolved arm. However, this procedure does not restore peak supination strength.

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast.

Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin.

Infections of the Upper Extremity: New Developments and Challenges.

Hand infections are common in all patient populations. However, because of variability in presentation and severity, they can be challenging to correctly diagnose and complex to manage. It is important to be aware of special populations such as children, individuals who are immunocompromised, those with diabetes, and intravenous drug users who may have uncommon pathogens or unusual types of infection. Atypical or rare bacterial and fungal infections, even in an immunocompetent host, can be equally challenging to manage. In each of these scenarios, it is critical to be familiar with associated conditions to avoid mismanagement and initiate an appropriate team-based approach for care involving surgery and consultation with an infectious disease specialist.

New insights into cohesin loading.

Cohesin is a conserved, ring-shaped protein complex that encircles sister chromatids and ensures correct chromosome segregation during mitosis and meiosis. It also plays a crucial role in the regulation of gene expression, DNA condensation, and DNA repair through both non-homologous end joining and homologous recombination. Cohesins are spatiotemporally regulated by the Scc2-Scc4 complex which facilitates cohesin loading onto chromatin at specific chromosomal sites. Over the last few years, much attention has been paid to cohesin and cohesin loader as it became clear that even minor disruptions of these complexes may lead to developmental disorders and cancers. Here we summarize recent developments in the structure of Scc2-Scc4 complex, cohesin loading process, and mediators that determine the Scc2-Scc4 binding patterns to chromatin.

Radial head replacement with a bipolar system: an average 10-year follow-up.

BACKGROUND: We report the long-term results of a cohort of patients after radial head replacement with a bipolar design and a smooth cementless stem at a mean follow-up of 10.4 years. METHODS: Of 17 possible patients from a previous minimum 2-year follow-up study, 16 were available for review. Patients were assessed using clinical and radiographic examination and with standardized outcome measures. Range of motion, stability, and radiographic evaluation of implant loosening and joint degeneration were assessed. Comparisons were performed using the Wilcoxon signed rank test for unequal groups. RESULTS: The average follow-up was 10.5 years (range, 8.5-12 years). The median visual analog scale was 1 (range, 0-5), Minnesota Elbow Performance Index was 93 (range, 70-100), and the Disabilities of the Arm, Shoulder and Hand was 7.5 (range, 0-53). Range of motion was decreased on the operative side compared with the nonoperative side for flexion/extension (P = .005) and pronation/supination (P = .015). Grip strength was decreased on the affected side (P = .045). No patients had elbow instability. Significant arthritic changes developed in 2 patients at the ulnohumeral joint. The median cantilever quotient was 0.4 (range, 0.30-0.50). Osteolysis in zones 1 to 7 was found in all but 2 patients. The median stem radiolucency was 0.5 mm (range, 0.2-0.9 mm). No reoperations occurred since our previous report. Implant survival in this cohort was 97%. CONCLUSION: Bipolar radial head prosthesis with a smooth cementless stem effectively restores elbow stability and function after comminuted radial head fractures with or without concomitant elbow instability. Our study demonstrates excellent long-term implant survival.

Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae.

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+ and Sb(III)/H+ antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.