Risk factors for failure of limb salvage among veterans with foot ulcers.

BACKGROUND: There is limited available information to guide early discussions involving limb salvage for patients with non-traumatic foot ulcers. We hypothesized patient, wound and treatment factors identifiable at initial operative treatment would be associated with failure of attempted limb salvage. METHODS: We retrospectively assessed United States military veterans treated operatively for non-traumatic foot ulcers at a Veteran's Administration (VA) hospital from 2008 to 2018. Cox proportional hazard analysis assessed for independent associations with eventual above ankle amputation. RESULTS: Limb salvage failed for 52 of 461 patients (11.0%). Univariable associations included initial wound area ≥1 cm (p < .001), immediate TMA (p < .001), diagnosis of PVD (p < .001) or diabetes (p = .005), nonpalpable pulse (p = .006), CKD (p = .023), creatine ≥ 1.5 (p = .004), and HgA1c ≥ 6.2 (p < .001). Independent associations consisted of initial wound area ≥1 cm (HR 6.0, 95% CI 1.4-25.1, p = .014), immediate TMA (HR 3.5, 95% CI 1.9-6.4, p < .001), and PVD (HR 3.5, 95% CI 1.6-7.5, p = .001). When <2 risk factors were present, 99.1% and 96.8% retained their hindfoot at 5 and 10 years, respectively. However, this decreased to 87.3% and 80.1% with two risk factors and fell to 63.3% and 43.3% with three risk factors. CONCLUSION: Failure of limb salvage was increasingly likely as the number of identified independent risk factors increased. These results may assist in prognostication and shared decision making between patients and providers. LEVEL OF EVIDENCE: Prognostic, Level III.

Generation and characterization of mice with mesenchyme-specific deletion of the entire ESET histone methyltransferase protein.

ESET protein (also known as SETDB1) catalyzes methylation of histone H3 at lysine 9 (H3-K9). In addition to the full-length transcript, mouse ESET gene also gives rise to alternative spicing variants encoding truncated proteins capable of retaining interaction with other epigenetic enzymes. To completely eliminate full-length ESET and its splicing variants, we have generated a conditional ESET allele with exon 4 flanked by two loxP sites for Cre-mediated DNA deletion and downstream frame-shift mutation of the entire coding region. Mating with Prx1-Cre mice and analysis of the resultant embryos revealed that mesenchyme-specific knockout of exon 4 completely eliminates full-length ESET and its truncated protein products, leading to profound defects in both the flat bones and long bones, ectopic hypertrophy of growth plate chondrocytes and downregulation of Indian hedgehog protein. In addition, exon 4 deletion results in reduced thickness of articular cartilage in E17.5 embryos, whereas deletion of exons 15-16 fails to do so. These findings offer us a useful tool to further study epigenetic regulation in a truly ESET-null background, and demonstrate that ESET plays a critical role in the control of chondrocyte hypertrophy and skeletal development.

ESET histone methyltransferase is essential to hypertrophic differentiation of growth plate chondrocytes and formation of epiphyseal plates.

The ESET (also called SETDB1) protein contains an N-terminal tudor domain that mediates protein-protein interactions and a C-terminal SET domain that catalyzes methylation of histone H3 at lysine 9. We report here that ESET protein is transiently upregulated in prehypertrophic chondrocytes in newborn mice. To investigate the in vivo effects of ESET on chondrocyte differentiation, we generated conditional knockout mice to specifically eliminate the catalytic SET domain of ESET protein only in mesenchymal cells. Such deletion of the ESET gene caused acceleration of chondrocyte hypertrophy in both embryos and young animals, depleting chondrocytes that are otherwise available to form epiphyseal plates for endochondral bone growth. ESET-deficient mice are thus characterized by defective long bone growth and trabecular bone formation. To understand the underlying mechanism for ESET regulation of chondrocytes, we carried out co-expression experiments and found that ESET associates with histone deacetylase 4 to bind and inhibit the activity of Runx2, a hypertrophy-promoting transcription factor. Repression of Runx2-mediated gene transactivation by ESET is dependent on its H3-K9 methyltransferase activity as well as its associated histone deacetylase activity. In addition, knockout of ESET is associated with repression of Indian hedgehog gene in pre- and early hypertrophic chondrocytes. Together, these results provide clear evidence that ESET controls hypertrophic differentiation of growth plate chondrocytes and endochondral ossification during embryogenesis and postnatal development.

Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes.

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Prolonged opioid use after knee arthroscopy in military veterans.

BACKGROUND: Chronic postoperative pain occurs with an appreciable incidence after elective surgery. Known risk factors include perioperative pain and posttraumatic stress disorder (PTSD). Military veterans are a population at particular risk for PTSD and hence may be at increased risk for chronic pain after surgery. Our goal was to identify risk factors for chronic postoperative pain in young veterans after minor elective surgery, including the contribution of PTSD. METHODS: We reviewed the medical and pharmacy records of veterans (18-50 years old), undergoing elective knee arthroscopy from 2007 to 2010 at the Veteran's Administration Puget Sound Health Care System. The data included demographics, ASA physical status class, comorbidities, anesthesia medications, and opioid prescriptions starting 3.5 months before surgery and ending 3.5 months after surgery. We documented the presence of PTSD based on either the patient's problem list or the clinical notes. We used prolonged postoperative opioid prescription longer than 3 months after surgery as a surrogate for chronic postoperative pain. RESULTS: We identified 145 patients who met inclusion criteria. The median age was 39 ± 8 years old. Eighty-seven percent of the patients were men. The prevalence of PTSD was 32% (95% confidence interval, 25%-41%). PTSD was associated with increased incidence of smoking (P = 0.009) and preoperative opioid use (P = 0.0006). Preoperative opioids were prescribed in 44% (63 of 145) of the patients: in 64% (30 of 47) of patients with PTSD, compared with 34% (33 of 98) in patients without PTSD (P = .0006). Chronic postoperative pain was identified in 30% (43 of 145) of patients. The strongest independent predictor of chronic postoperative pain was an opioid prescription before surgery (odds ratio = 65.3; 95% confidence interval, 014.5-293.0). In patients older than 27.5 years who did not receive opioids before surgery, PTSD may also have been a risk factor for chronic postoperative pain. CONCLUSIONS: This single-center retrospective study suggests that the most important predictor of chronic postoperative pain is preoperative opioid use. For patients not taking opioids preoperatively, PTSD may increase the risk of prolonged postoperative opioid prescriptions and chronic postoperative pain, potentially related to patient age.

Impact of Extended Prophylactic Antibiotics on Risk of Prosthetic Joint Infection in Primary Total Hip Arthroplasty: A Matched Cohort Analysis.

BACKGROUND: Extended oral prophylactic antibiotics have been increasingly used in arthroplasty with the goal of reducing the risk of prosthetic joint infection (PJI). While a reduction in the rate of PJI has been noted with extended oral antibiotic regimens in high-risk patients, no large database study has assessed infection risk after primary total hip arthroplasty among well-balanced cohorts receiving and not receiving postoperative extended oral antibiotics. METHODS: A retrospective cohort study was conducted using a national database, TriNetX, to identify patients who underwent primary total hip arthroplasty. This cohort was stratified by oral antibiotic prescription within one day of procedure. A one-to-one propensity score matching based on age, sex, class of obesity, and medical comorbidities was conducted. Outcomes explored in this study were 90-day risk of PJI, superficial skin infection, deep skin infection, and all-cause revision. RESULTS: 90-day postoperative infection complications of PJI were higher in the group receiving antibiotics (hazard ratio: 1.83, P -value = 0.012). Other complications such as superficial skin infection, deep skin infection, and all-cause revision showed no statistically significant differences. CONCLUSION: This database analysis of 5,476 patients demonstrated no decrease in complications of PJI, superficial or deep skin infection, or revision at 90 days. Future randomized controlled trials are needed to evaluate the efficacy of extended oral antibiotics. LEVEL OF EVIDENCE: III.

Using Wearable Technology to Measure the Association Between Neck Posture and Pain During Urologic Open and Robotic Surgery.

Purpose: Chronic neck pain is the most prevalent work-related musculoskeletal injury among surgeons. Urologists may be at higher risk of neck injury due to extended time spent operating in deep anatomical structures during open surgery. Our goal was to use wearable technology to quantify the relationship between neck posture and pain during open and robotic surgery. Materials and Methods: Urologic attendings and residents who spent at least 1 day per week performing surgery for >6 hours took part in this study. Neck posture was measured in real time during surgery using inertial measurement devices attached at the occipital protuberance and seventh cervical vertebrae. Self-reported neck pain scores were obtained throughout their workday. Results: Thirty participants and 202 hours of surgery were included in the study (21 attendings, 9 residents). There was a significant association between neck posture and pain (p = 0.04). Surgeons performing open procedures spent on average 147 minutes with their head in neck flexion postures of 30° or greater compared with 68 minutes for those performing robotic procedures (p = 0.007). Surgeons performing open procedures reported a mean change in neck pain of 2.0 on the numeric analogue scale, compared with 1.3 for those performing robotic procedures (p = 0.04). Conclusions: Real-time measurements of neck flexion during urologic surgery shows that greater duration and higher degree of neck flexion were associated with increased neck pain. Raising awareness about ergonomics in the operating room during residency will enable future generations of surgeons to make conscious decisions regarding their neck posture in surgery.

FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells.

Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

Proper expression of helix-loop-helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells.

The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes. Our results suggest that proper expression of Id2 is important to achieving a fine balance between growth and differentiation during chondrogenesis.

TLS-ERG leukemia fusion protein deregulates cyclin-dependent kinase 1 and blocks terminal differentiation of myeloid progenitor cells.

TLS-ERG fusion protein is derived from the t(16;21) translocation found in human myeloid leukemia. Here, we show that retroviral transduction of TLS-ERG confers a growth advantage to L-G myeloid progenitor cells and blocks terminal differentiation. We found that the level of cyclin-dependent kinase 1 (Cdk1) protein was significantly decreased in controls but unchanged in TLS-ERG-expressing cells after granulocyte colony-stimulating factor treatment or interleukin-3 withdrawal. Injection of TLS-ERG-expressing L-G cells induced rapid development of a leukemia-like disease in syngeneic mice. Through site-directed mutagenesis, we showed that transformation and deregulation of Cdk1 by TLS-ERG require an intact ets DNA-binding domain within the fusion protein. Interestingly, treatment of TLS-ERG-expressing L-G cells with 5-aza-2'-deoxycytidine (Decitabine) or trichostatin A resulted in down-regulation of Cdk1 and induction of terminal differentiation. To investigate whether Cdk1 deregulation is indeed responsible for transformation by TLS-ERG, we constructed lentiviral vectors for delivery of Cdk1 mutants and small interfering RNA (siRNA). Both dominant-negative inhibition and siRNA knockdown of Cdk1 were able to restore the ability of TLS-ERG-expressing L-G cells to undergo terminal differentiation. In addition, siRNA knockdown of Cdk1 in YNH-1 cells derived from a t(16;21) acute myelogenous leukemia patient also resulted in terminal differentiation. As restoration of terminal myeloid differentiation to TLS-ERG cells is dependent on cell cycle arrest, our findings suggest an important role for Cdk1 in cellular transformation and may be useful in the search for new treatments of TLS-ERG-associated myeloid leukemia.

The oncogenic TLS-ERG fusion protein exerts different effects in hematopoietic cells and fibroblasts.

The oncogenic TLS-ERG fusion protein is found in human myeloid leukemia and Ewing's sarcoma as a result of specific chromosomal translocation. To unveil the potential mechanism(s) underlying cellular transformation, we have investigated the effects of TLS-ERG on both gene transcription and RNA splicing. Here we show that the TLS protein forms complexes with RNA polymerase II (Pol II) and the serine-arginine family of splicing factors in vivo. Deletion analysis of TLS-ERG in both mouse L-G myeloid progenitor cells and NIH 3T3 fibroblasts revealed that the RNA Pol II-interacting domain of TLS-ERG resides within the first 173 amino acids. While TLS-ERG repressed expression of the luciferase reporter gene driven by glycoprotein IX promoter in L-G cells but not in NIH 3T3 cells, the fusion protein was able to affect splicing of the E1A reporter in NIH 3T3 cells but not in L-G cells. To identify potential target genes of TLS-ERG, the fusion protein and its mutants were stably expressed in both L-G and NIH 3T3 cells through retroviral transduction. Microarray analysis of RNA samples from these cells showed that TLS-ERG activates two different sets of genes sharing little similarity in the two cell lines. Taken together, these results suggest that the oncogenic TLS-ERG fusion protein transforms hematopoietic cells and fibroblasts via different pathways.

Preoperative Opioid Use Is Associated with Early Revision After Total Knee Arthroplasty: A Study of Male Patients Treated in the Veterans Affairs System.

BACKGROUND: Opioid use is endemic in the U.S. and is associated with morbidity and mortality. The impact of long-term opioid use on joint-replacement outcomes remains unknown. We tested the hypothesis that use of opioids is associated with adverse outcomes after total knee arthroplasty (TKA). METHODS: We performed a retrospective analysis of patients who had had TKA within the U.S. Veterans Affairs (VA) system over a 6-year period and had been followed for 1 year postoperatively. The length of time for which an opioid had been prescribed and the morphine equivalent dose were calculated for each patient. Patients for whom opioids had been prescribed for >3 months in the year prior to the TKA were assigned to the long-term opioid group. A natural language processing-based machine-learning classifier was developed to classify revisions due to infectious and non-infectious causes on the basis of the postoperative note. Survival curves for the time to knee revision or manipulation were used to compare the long-term opioid group with the patients who did not take opioids long-term. Hazard and odds ratios for knee revision and manipulation were obtained as well. RESULTS: Of 32,636 patients (94.4% male; mean age [and standard deviation], 64.45 ± 9.41 years) who underwent TKA, 12,772 (39.1%) were in the long-term opioid group and 734 (2.2%) had a revision within a year after the TKA. Chronic kidney disease, diabetes, and long-term opioid use were associated with revision within 1 year-with odds ratios (95% confidence intervals [CIs]) of 1.76 (1.37 to 2.22), 1.11 (0.93 to 1.31), and 1.40 (1.19 to 1.64), respectively-and were also the leading factors associated with a revision at any time after the index TKA-with odds ratios (95% CIs) of 1.61 (1.34 to 1.92), 1.21 (1.08 to 1.36), and 1.28 (1.15 to 1.43), respectively. Long-term opioid use had a hazard ratio of 1.19 (95% CI = 1.10 to 0.24) in the analysis of its relationship with knee revision, but the hazard was not significant in the analysis of its association with knee manipulation. The accuracy of the text classifier was 0.94, with the area under the receiver operating characteristic curve being 0.99. There was no association between long-term use of opioids and the specific cause for knee revision. CONCLUSIONS: Long-term opioid use prior to TKA was associated with an increased risk of knee revision during the first year after TKA among predominantly male patients treated in the VA system. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Molecular cloning of ESET, a novel histone H3-specific methyltransferase that interacts with ERG transcription factor.

The ets-related gene erg encodes a transcription factor that is implicated in the control of cell growth and differentiation. To identify interacting partners of ERG, we screened a yeast two-hybrid cDNA library constructed from mouse hematopoietic cells using the N-terminal region of ERG as a bait. We isolated a 4.6 kb full-length mouse cDNA encoding a 1307-amino acid protein migrating as a 180 kD band, which was termed ESET (ERG-associated protein with SET domain). ESET is 92% identical to the human protein SETDB1 (SET domain, bifurcated 1). The interaction between ESET and ERG was supported by in vitro pull-down using glutathione S-transferase (GST) fusion protein, by transfection and co-immunoprecipitation experiments, and by association of endogenous SETDB1 with ERG. Since ESET possesses evolutionarily conserved SET, preSET, and postSET domains implicated in histone methylation, we tested the ability of ESET to methylate core histones. The results of these studies demonstrated that ESET is a histone H3-specific methyltransferase, and that mutations within ESET abolished its methyltransferase activity. Together, these findings raise the possibility that transcription factor ERG may participate in transcriptional regulation through ESET-mediated histone methylation.

Genomic structure and expression of the mouse ESET gene encoding an ERG-associated histone methyltransferase with a SET domain.

ESET (ERG-associated protein with a SET domain, also called SETDB1) is a novel histone methyltransferase that catalyzes methylation of histone H3-lysine 9 (H3-K9). Here we describe the genomic structure and expression of the mouse ESET gene that gives rise to ESET protein and its alternative splicing product. ESET is a 36-kb single copy gene and full-length ESET transcript consisting of 22 exons. The splicing variant retains only the first 12 exons and thus lacks sequences encoding the methyl CpG-binding domain and the catalytic SET domain. The U2 type conserved GT/AG consensus sequence is present at all of the splicing junctions within the ESET gene. The transcription initiation site of the ESET gene was determined by 5'-RACE experiment and by primer extension. The 5'-flanking sequence of the ESET gene does not contain the consensus TATA box. Instead, this ESET promoter region has features such as SP1-binding sites that are typical of housekeeping genes. The ESET promoter was functionally active when tested in transfection and luciferase assay. Full-length ESET transcript appears to be ubiquitously expressed. While the SET domain-deficient splicing variant is present in immortalized cell lines, it is undetectable by RT-PCR in the majority of normal mouse tissues.

Characterization and expression of the human gene encoding two translocation liposarcoma protein-associated serine-arginine (TASR) proteins.

Translocation liposarcoma protein (TLS)-associated serine-arginine (TASR)-1 and -2 are two newly identified serine-arginine splicing factors. Our recent studies suggest that disruption of TASR-mediated pre-mRNA splicing is involved in the pathogenesis of human leukemia and sarcomas. The mRNA transcripts for TASR-1 and -2 share an identical sequence at the 5' untranslated region (5' UTR) and in part of the coding region; however the other regions of the transcripts diverge from each other and it was not clear whether the differences resulted from alternative splicing or transcription from two distinct genes. Here we describe the assignment of both TASR cDNAs to the same 16 kb DNA segment located on chromosome 1. Despite the presence of at least three retroposed products of TASR-1 mRNA in the human genome, only the 16 kb structural TASR gene on chromosome 1 is actively transcribed. In addition, multiple polyadenylation sites and a rare U12-type intron were found within the TASR gene. Transcription initiation site of the TASR gene was determined by primer extension; analysis of the TASR promoter revealed that it lacks the TATA box but contains a GC-rich sequence. When cloned into a luciferase reporter and transfected into human cells, the TASR promoter construct generated luciferase activity that was at least 2000 fold greater than the promoterless plasmid. Northern blot analysis showed that at least five different TASR-1 and -2 transcripts are expressed in a broad range of human tissues.

Preoperative supraphysiological testosterone in older men undergoing knee replacement surgery.

OBJECTIVES: Older patients undergoing knee replacement surgery can recover more slowly than younger patients and require extended rehabilitation. Because administration of supraphysiological testosterone (T) dramatically increases strength, we hypothesized that preoperative T therapy would improve functional recovery and reduce hospital stay in older men undergoing knee replacement surgery. DESIGN: Double-blinded, placebo-controlled pilot trial. SETTING: A Veterans Affairs orthopedics clinic and inpatient postoperative unit. PARTICIPANTS: Twenty-five men, mean age 70, undergoing elective knee replacement. INTERVENTION: Preoperative, supraphysiological T administration (600 mg T enanthate intramuscularly weekly for 4 weeks) or sesame oil placebo. MEASUREMENTS: Length of hospital stay and functional ability by Functional Independence Measure (FIM) score. RESULTS: Mean length of hospital stay +/- standard deviation was nonsignificantly reduced in the T group (5.9 +/- 2.4 days vs 6.8 +/- 2.5 days; P =.15). At postoperative Day 3, there was a significant improvement in ability to stand (mean FIM score 5.2 +/- 1.0 vs 4.0 +/- 1.1; P =.04) and trends towards improvements in walking and stair climbing in the T group. There were no complications attributable to T therapy. CONCLUSIONS: In older men undergoing knee replacement surgery, preoperative supraphysiological T administration may confer some clinical benefit. Future studies using longer courses of preoperative T administration in larger numbers of older men undergoing knee replacement surgery are warranted.