Yeast ORC sumoylation status fine-tunes origin licensing.

Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability.

Dysphagia is associated with increased mortality risk after hip fracture surgery.

PURPOSE: This study aims to explore the prevalence of dysphagia, as well as mortality associated with dysphagia in the elderly population receiving surgical treatment for a hip fracture. METHODS: A retrospective cohort study was completed at an academic level 1 tertiary care center. Patients older than or equal to 65 admitted with a hip fracture diagnosis from January 2015 to December 2020 (n = 617) were included. The main outcome was the prevalence of dysphagia and association with mortality. Secondary analysis included timing of dysphagia and contributions to mortality. RESULTS: Fifty-six percent of patients had dysphagia, and the mortality rates were higher in patients with dysphagia (8.9%) versus those without dysphagia (2.6%), chi-square p = 0.001, and odds ratio 3.69 (CI 1.6-8.5). Mortality rates in patients with acute dysphagia were also higher (12.4%) than those with chronic dysphagia (5%) and chi-squared p = 0.02. Mortality rates in patients with a perioperative dysphagic event (13.9%) were higher than those with non-perioperative dysphagia (4%) and chi-squared p = 0.001. Mortality rates in patients who had acute perioperative dysphagia (21.2%) were higher than those with chronic dysphagia that presented perioperatively (6.8%) and chi-squared p = 0.006. CONCLUSIONS: This study demonstrates high rates of dysphagia in the elderly hip fracture population and a significant association between dysphagia and mortality. Timing and chronicity of dysphagia were relevant, as patients with acute perioperative dysphagia had the highest mortality rate. Unlike other identified risk factors, dysphagia may be at least partially modifiable. More research is needed to determine whether formal evaluation and treatment of dysphagia lowers mortality risk.

The Fkh1 Forkhead associated domain promotes ORC binding to a subset of DNA replication origins in budding yeast.

The pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognitioncomplex (ORC). The ORC-DNA complex directs the formation of origins, the specific chromosomal regions where DNA synthesis initiates. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (Forkhead homolog) protein and its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. Several of the FHA-dependent origins examined required a distinct Fkh1 binding site located 5' of and proximal to their ORC sites (5'-FKH-T site). Genetic and molecular experiments provided evidence that the Fkh1-FHA domain promoted origin activity directly through Fkh1 binding to this 5' FKH-T site. Nucleotide substitutions within two relevant origins that enhanced their ORC-DNA affinity bypassed the requirement for their 5' FKH-T sites and for the Fkh1-FHA domain. Significantly, assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism was relevant at ∼25% of yeast origins. Thus, the FHA domain of the conserved cell-cycle transcription factor Fkh1 enhanced origin selection in yeast at the level of ORC-origin binding.

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early- and late-replicating euchromatin.

A eukaryotic chromosome relies on the function of multiple spatially distributed DNA replication origins for its stable inheritance. The spatial location of an origin is determined by the chromosomal position of an MCM complex, the inactive form of the DNA replicative helicase that is assembled onto DNA in G1-phase (also known as origin licensing). While the biochemistry of origin licensing is understood, the mechanisms that promote an adequate spatial distribution of MCM complexes across chromosomes are not. We have elucidated a role for the Sir2 histone deacetylase in establishing the normal distribution of MCM complexes across Saccharomyces cerevisiae chromosomes. In the absence of Sir2, MCM complexes accumulated within both early-replicating euchromatin and telomeric heterochromatin, and replication activity within these regions was enhanced. Concomitantly, the duplication of several regions of late-replicating euchromatin were delayed. Thus, Sir2-mediated attenuation of origin licensing within both euchromatin and telomeric heterochromatin established the normal spatial distribution of origins across yeast chromosomes important for normal genome duplication.

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins.

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.

Myoepithelial carcinoma with RB1 mutation: remarkable chemosensitivity to carcinoma of unknown origin therapy.

BACKGROUND: Myoepithelial carcinoma of soft tissue is a rare, malignant neoplasm that is morphologically and immunophenotypically similar to its counterpart in salivary gland. It demonstrates myoepithelial differentiation, possessing both epithelial and myogenic characteristics. Thought to be chemotherapy insensitive, the optimal treatment regimen of this tumor has yet to be established and only a select few cases in the literature discuss treatment efficacy in detail. CASE PRESENTATION: Here we present a case of a young adult with metastatic myoepithelial carcinoma with an initial excellent response to systemic therapy utilizing carboplatin and paclitaxel with continued complete response after 3 years. The patient also underwent complete surgical excision and received adjuvant radiation to the primary site of disease. Exome sequencing revealed an inactivating mutation in RB1 which we believe to be the first such mutation to be reported in this cancer type. CONCLUSIONS: Given increasing evidence suggesting RB1 loss is associated with responsiveness to conventional chemotherapies, particularly platinum-based regimens, we hypothesize that this genetic feature predisposed chemosensitivity in our patient's tumor.

Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases.

In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here, we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC) and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call "3-box" due to its facilitating Cul3 binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3 and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.

A Link between ORC-origin binding mechanisms and origin activation time revealed in budding yeast.

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as 'chromatin-dependent' when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at 'chromatin-dependent' origins. Finally, the 'chromatin-dependent' class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time.

The budding yeast Fkh1 Forkhead associated (FHA) domain promoted a G1-chromatin state and the activity of chromosomal DNA replication origins.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.

High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements.

In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.

The Use of the Piriformis Fossa Radiographic Landmark to Predict "In-Out-In" Placement of the Posterosuperior Femoral Neck Screw.

OBJECTIVES: To investigate the correlation between a screw's radiographic relationship to the piriformis fossa with position on CT in the clinical setting. METHODS: Intraoperative fluoroscopic images of patients treated with cannulated screw fixation of a femoral neck fracture, who also had a postoperative CT scan, were retrospectively evaluated by 4 fellowship-trained orthopaedic trauma surgeons. The posterosuperior screw on the AP fluoroscopic view was determined to be above the piriformis fossa (APF) or below the piriformis fossa (BPF). Using CT scan to determine IOI placement, the ability to predict IOI position based on fluoroscopic imaging was evaluated by calculating accuracy, sensitivity, specificity, and interobserver reliability. RESULTS: 73 patients met inclusion criteria. The incidence of IOI screw placement was 59% on CT evaluation. The use of the PF landmark accurately predicted CT findings in 89% of patients. A screw placed APF was 90% sensitive and 88% specific in predicting cortical breach, with near-perfect interobserver agreement (κ = 0.81). CONCLUSION: The use of the PF radiographic landmark is highly sensitive and specific in predicting the placement of an IOI posterosuperior femoral neck screw. LEVEL OF EVIDENCE: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Osteoporosis management in the United States.

There are numerous organizational osteoporosis initiatives in the United States offering a variety of recommendations and guidelines. A common method of implementing these goals is centered around multidisciplinary provider teams with the broad task of diagnosis, treatment, and prevention of current and future osteoporosis related fractures. These teams have generally proved to be successful even though it remains debated, which specific provider specialty is ultimately responsible for osteoporosis care. The current United States healthcare infrastructure represents the significant obstacle in widespread adoption of successful treatment programs. The development of further quality standards and incorporation of fracture liaison services into reimbursement and funding models will allow for continued improvement in osteoporosis care.

Outcomes Following Total Hip Arthroplasty for Osteonecrosis of the Femoral Head in Patients on Hemodialysis.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a potentially debilitating condition, often requiring total hip arthroplasty (THA). Patients on hemodialysis (HD) are at increased risk for complications after THA for osteoarthritis, however there is limited information on outcomes of THA for ONFH in patients on HD. With increasing prevalence of chronic kidney disease (CKD) requiring HD, studies are needed to characterize the risk of complications in these patients. Therefore, the purpose of this study was to evaluate HD as a potential risk factor for complication after THA in patients with ONFH on HD. METHODS: Patients on HD with ONFH who underwent THA with at least 2 years of follow-up were identified using a combination of ICD-9 and CPT codes in a national insurance database. A 10:1 matched control cohort of patients with ONFH not on HD was created for comparison. A logistic regression analysis was used to evaluate rates of death, hospital readmission, emergency room (ER) visit, infection, revision, and dislocation between cohorts. Differences in hospital charges, reimbursement, and length of stay between the two groups were also assessed. RESULTS: One thousand one hundred thirty-seven patients on HD who underwent THA for ONFH were compared to a matched control cohort of 11,182 non-HD patients who underwent THA for ONFH. Patients on HD experienced higher rates of death (HD 4.1%, non-HD 0.9%; odds ratio [OR] 3.35, p < 0.01), hospital readmission (HD 16.1%, non-HD 5.9%; OR 2.69, p < 0.01) and ER visit (HD 10.4%, non-HD 7.4% OR 1.5, p < 0.01). Hemodialysis was not associated with higher risk of infection, revision, or dislocation, but was associated with significantly higher charges (p < 0.01), reimbursement (p < 0.01), and hospital length of stay (p < 0.01). CONCLUSIONS: While patients on HD do not have increased risk of implant-related complications, they are at increased risk of developing medical complications following THA for ONFH and subsequently may require more resources. Orthopedic surgeons and nephrologists should be cognizant of the increased risk in this population to provide appropriate preoperative counseling and enhanced perioperative medical management. LEVEL OF EVIDENCE: Therapeutic Level III.

The effect of residency training on arthroscopic knot tying and knot stability: which knot is best tied by Orthopaedic surgery residents?

BACKGROUND: The aim of this study is to evaluate which of three arthroscopic knots are most reliably taught to and executed by residents at varying levels of training. METHODS: Three arthroscopic knots, the Samsung Medical Center (SMC), the Weston, and the surgeon's knot, were taught to 16 orthopaedic surgery residents. Each knot was tied in triplicate at two sessions 1 week apart. The knots were then biomechanically tested for strength. Corresponding knots tied by a sports medicine fellow served as the respective controls. RESULTS: Comparing all knots regardless of year of training, the SMC knot failed at significantly higher loads (237.2 ± 66.6 N) than the surgeon's knot (203.7 ± 45.3 N, p = 0.049) and the Weston knot (193.5 ± 56.1 N, p = 0.013). No significant differences in knot strength were found when comparing knots tied by residents at different levels of training and when comparing residents to the sports medicine fellow. There was no difference in conditioning elongation between surgeon's (p = 0.343), Weston (p = 0.486), or SMC knots (p = 0.200) tied by post-graduate year one and five residents. CONCLUSIONS: We report the first study that evaluates the loop strength of an arthroscopically tied knot performed by orthopaedic surgery residents in various levels of training. In our cohort, the SMC knot required a higher load to failure, when compared to the Surgeon's and Weston knot, after a simple arthroscopic knot tying curriculum. Based on these findings, he SMC knot should be considered as a part of future orthopaedic surgery resident arthroscopic training programs.

Percutaneous or Open Reduction of Closed Tibial Shaft Fractures During Intramedullary Nailing Does Not Increase Wound Complications, Infection or Nonunion Rates.

OBJECTIVE: To compare the incidence of complications (wound, infection, and nonunion) among those patients treated with closed, percutaneous, and open intramedullary nailing for closed tibial shaft fractures. DESIGN: Retrospective review. SETTING: Multiple trauma centers. PATIENTS: Skeletally mature patients with closed tibia fractures amenable to treatment with an intramedullary device. INTERVENTION: Intramedullary fixation with closed, percutaneous, or open reduction. MAIN OUTCOME MEASUREMENTS: Superficial wound complication, deep infection, nonunion. RESULTS: A total of 317 tibial shaft fractures in 315 patients were included in the study. Two-hundred fractures in 198 patients were treated with closed reduction, 61 fractures in 61 patients were treated with percutaneous reduction, and 56 fractures in 56 patients were treated with formal open reduction. The superficial wound complication rate was 1% (2/200) for the closed group, 1.6% (1/61) for the percutaneous group, and 3.6% (2/56) for the open group with no statistical difference between the groups (P = 0.179). The deep infection rate was 2% (4/200) for the closed group, 1.6% (1/61) for the percutaneous group, and 7.1% (4/56) for the open group with no significant difference between the groups (P = 0.133). Nonunion rate was 5.0% (10/200) for the closed group, 4.9% (3/61) for the percutaneous group, and 7.1% (4/56) for the open group, with no statistical difference between the groups (P = 0.492). CONCLUSIONS: This is the largest reported series of closed tibial shaft fractures nailed with percutaneous and open reduction. Percutaneous or open reduction did not result in increased wound complications, infection, or nonunion rates. Carefully performed percutaneous or open approaches can be safely used in obtaining reduction of difficult tibial shaft fractures treated with intramedullary devices. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements.

The protein kinase C orthologue PkcA plays a role in cell wall integrity and polarized growth in Aspergillus nidulans.

The calC2 mutation in Aspergillus nidulans causes hypersensitivity to Calcofluor White, along with other drug sensitivities that indicate a defect in cell wall integrity. We have cloned CalC by complementation, isolating the A. nidulans orthologue of protein kinase C (PkcA). The pkcA allele of the calC2 strain contains a mutation predicted to introduce a charged arginine residue in place of neutral glycine at a conserved site located immediately beside the C1B regulatory domain. Both PkcA and calC2 map to the same region of chromosome VIII. A PkcA::GFP chimera localizes to hyphal apices and growing septa, as well as to the conidiogenous apices of phialides, indicating a role for PkcA in polarized cell wall growth. These observations support the hypothesis that the role of PkcA in A. nidulans, is comparable to that played by Pkc1p in the Saccharomyces cerevisiae cell wall integrity pathway.