Urinary Tract Infection after Total Hip Arthroplasty: A Retrospective Cohort Study.

Perioperative urinary tract infections (UTI) are a relatively common occurrence after total hip arthroplasty (THA). The purpose of this study was to assess demographics, outcomes and trends in the development of UTI's after THA using the National Hospital Discharge Survey (NHDS). All patients undergoing THA were divided based on whether or not they developed a UTI, and data regarding demographics, outcome and complications were gathered and analyzed. No significant trend in rate of UTI after THA was found. Patients who developed a UTI were more likely to be female, have more comorbidities and receive a transfusion. They had a longer hospitalization, lower rate of discharge directly home and an increased rate of discharge to a rehabilitation facility. Orthopaedists should identify those patients at increased risk who according to this study appear to be older, female patients with multiple co-morbidities who received a blood transfusion. (Journal of Surgical Orthopaedic Advances 29(3):162-164, 2020).

Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing PNPLA3 levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in PNPLA3 which, when co-inherited, reduces PNPLA3 mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the PNPLA3 gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and PNPLA3. These pathways may be exploited by drug modalities to "tune down" the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.

In Hospital Outcomes of Total Hip Arthroplasty in the Medicaid Population.

Total hip arthroplasty (THA) is one of the most common orthopaedic procedures. This study's purpose was to evaluate national trends, patient demographics and hospital outcomes for Medicaid patients who underwent a primary THA. The National Hospital Discharge Survey (NHDS) database was queried for patients undergoing THA from 2001-2010. Patients were stratified into two groups based on insurance. We found from 2001-2005, Medicaid accounted for 2.38% of all THA performed, increasing insignificantly to 2.61% between 2006-2010. The Medicaid group was younger (50.3 vs. 65.6 years, p < 0.01). Length of stay was longer for the Medicaid group (4.6 vs. 4.0 days, p < 0.01). Medicaid patients were more likely to be discharged home (53.7% vs. 47.2%, p < 0.01) and less likely to be discharged to rehabilitation facilities (24.4% vs. 29.0%, p < 0.05). In conclusion, we discovered that the rate of Medicaid insurance in patients undergoing primary THA was stable through 2010, prior to the Affordable Care Act. We found Medicaid THA patients had longer length of stay, despite being a mean 15 years younger than the non-Medicaid cohort. Medicaid insurance status should be factored into risk adjustment models to avoid creating additional disincentive to treat the Medicaid population. (Journal of Surgical Orthopaedic Advances 28(4):281-284, 2019).

Aspirin Alone Is Not Enough to Prevent Deep Venous Thrombosis After Total Joint Arthroplasty.

Thromboembolic events after total joint arthroplasty are potentially devastating complications. This study evaluated the efficacy of 4 different anticoagulants in preventing deep venous thrombosis and pulmonary embolism after total joint arthroplasty. The demographics and anticoagulant use (warfarin, enoxaparin, and aspirin with and without outpatient mechanical pumps) for patients who underwent primary unilateral total joint arthroplasties performed by a single surgeon from January 2013 to October 2014 were retrospectively reviewed. All patients underwent lower extremity ultrasound at the 3-week postoperative visit. A total of 613 primary unilateral total joint arthroplasties met the study inclusion criteria. There were 288 primary total knee arthroplasties and 325 primary total hip arthroplasties. The patients were 62.2% female, having a mean age of 67.6±10.6 years and a mean body mass index of 30.2±5.9 kg/m2. There were 119 patients in group 1 (aspirin alone), 40 patients in group 2 (aspirin plus pumps), 246 patients in group 3 (warfarin), and 208 patients in group 4 (enoxaparin). The overall 3-week symptomatic and asymptomatic deep venous thrombosis and symptomatic pulmonary embolism rates in the entire cohort were 5.7% and 0.3%, respectively. The venous thromboembolism rate was significantly affected by the anticoagulant of choice (P<.01). Compared with aspirin alone, warfarin decreased the risk of venous thromboembolism (P<.01). Increasing age led to increased risk of venous thromboembolism (P=.05). This study indicated that aspirin chemoprophylaxis alone was not as efficacious as warfarin and enoxaparin in preventing asymptomatic and symptomatic venous thromboembolism found during routine postoperative surveillance with lower extremity ultrasound. Aspirin alone may be inadequate and should be augmented with an outpatient mechanical pump as part of multimodal prophylaxis. [Orthopedics. 2019; 42(1):48-55.].

Blood Transfusion After Primary Total Hip Arthroplasty: National Trends and Perioperative Outcomes.

The purpose of this study was to quantify the changes in transfusion rates, both allogeneic blood transfusion (ALBT) and autogenic blood transfusion (ATBT) on a national scale, and determine patient factors associated with transfusions. The National Hospital Discharge Survey was evaluated between 2001 and 2010 for primary total hip arthroplasty (THA) patients and categorized on the basis of transfusion necessity, type, and comorbidity burden. A logistic regression comparison of ALBT, ATBT, and nontransfused patients was performed with respect to patient demographics and in-hospital complications. The proportion of patients requiring any transfusion decreased from an average rate of 22.8% between 2001 and 2005 to 21.2% between 2006 and 2010 (p = .01). ATBT rates decreased (r = -.99) from 11.0% in 2001 to 2.8% in 2010. ALBT rates increased (r = .66) from 14% in 2001 to 16.6% in 2010. The number of patients requiring a blood transfusion after THA decreased in the United States with a trend shifting from ATBT to ALBT. (Journal of Surgical Orthopaedic Advances 26(4):216-222, 2017).

Total Hip Arthroplasty and Hemiarthroplasty: US National Trends in the Treatment of Femoral Neck Fractures.

There is controversy regarding whether total hip arthroplasty (THA) or hemiarthroplasty (HA) is the treatment preferred for displaced intracapsular femoral neck fractures (FNFs). Using the US National Hospital Discharge Survey, we found that, of 12,757 patients admitted for FNF between 2001 and 2010, 4.6% underwent THA and 52.5% underwent HA. More of both procedures were performed over time. Mean age was higher for HA patients. Hospitalization duration and blood transfusion rates were higher for THA. There were region-based differences in frequency of THA and significant hospital-size-based differences in frequency of HA, possibly because of differences in regional training and subspecialist availability. In addition, a larger proportion of THA patients was covered by private insurance.

Total Knee Arthroplasty in the Younger Patient.

The rate of total knee arthroplasty (TKA) utilization in younger patients (< 65 years old) is increasing. Little is known regarding demographics and in-hospital outcomes in this population. The National Hospital Discharge Survey (NHDS) database was searched using International Classification of Diseases, Ninth Revision (ICD-9) procedure codes for patients admitted to U.S. hospitals for unilateral primary TKA between 2001 and 2010. Patients were separated into young (< 65 years of age) and senior cohorts (≥ 65 years of age). ICD-9 diagnosis and procedure codes were used to identify demographics, hospital length of stay, in-hospital adverse events, mortality, and discharge disposition. Trends were evaluated by linear regression with Pearson correlation coefficient (r) and statistical comparisons were made using Student t-test and chi-square analysis. The young cohort accounted for 38.4% of TKAs performed from 2001 to 2005, increasing to 42.7% of TKAs from 2006 to 2010. They had a higher percentage of males (36.4 vs. 34.2%, p < 0.001). Rates of obesity (11.1 vs. 6.0%, p < 0.001) and morbid obesity (5.8 vs. 1.9%, p < 0.001) were significantly higher, yet they had less comorbidities (4.7 vs. 5.2, p < 0.001), and lower rates of transfusion (12.2 vs. 19.8%, p < 0.001), pulmonary embolism (PE) (0.31 vs. 0.49%, p < 0.020), and mortality (0.03 vs. 0.18%, p < 0.001). Patients < 65 years old undergoing TKA have almost double the rate of obesity of patients ≥ 65 years old. This could explain the higher rates of periprosthetic infection and aseptic mechanical failure seen in younger patients. However, the young cohort had a more favorable discharge disposition and lower mortality and risk of PE than elderly patients.

Tumor and circulating biomarkers in patients with second-line hepatocellular carcinoma from the randomized phase II study with tivantinib.

ARQ 197-215 was a randomized placebo-controlled phase II study testing the MET inhibitor tivantinib in second-line hepatocellular carcinoma (HCC) patients. It identified tumor MET as a key biomarker in HCC.Aim of this research was to study the prognostic and predictive value of tumor (MET, the receptor tyrosine kinase encoded by the homonymous MNNG-HOS transforming gene) and circulating (MET, hepatocyte growth factor [HGF], alpha-fetoprotein [AFP], vascular endothelial growth factor [VEGF]) biomarkers in second-line HCC. Tumor MET-High status was centrally assessed by immunohistochemistry. Circulating biomarkers were centrally analyzed on serum samples collected at baseline and every 4-8 weeks, using medians as cut-off to determine High/Low status. Tumor MET, tested in 77 patients, was more frequently High after (82%) versus before (40%) sorafenib. A significant interaction (p = 0.04) between tivantinib and baseline tumor MET in terms of survival was observed. Baseline circulating MET and HGF (102 patients) High status correlated with shorter survival (HR 0.61, p = 0.03, and HR 0.60, p = 0.02, respectively), while the association between AFP (104 patients) or VEGF (103 patients) status and survival was non-significant. CONCLUSIONS: Tumor MET levels were higher in patients treated with sorafenib. Circulating biomarkers such as MET and HGF may be prognostic in second-line HCC. These results need to be confirmed in larger randomized clinical trials.

Transitioning to the Direct Anterior Approach in Total Hip Arthroplasty: Is It Safe in the Current Health Care Climate?

BACKGROUND: The direct anterior approach for total hip arthroplasty has recently gained interest for its quicker short-term recovery despite concerns about increased complications and operative time, especially during the steep learning curve period. The primary goal of this study was to determine if the transition from a posterior approach to a direct anterior approach for total hip arthroplasty can be achieved without harming patient safety. METHODS: This is a retrospective cohort single-surgeon study of consecutive primary total hip arthroplasties performed over a transition period from posterior to direct anterior (DA) approach. RESULTS: A total of 412 patients (211 anterior, 201 posterior) were included with no significant demographic differences between cohorts. There were no significant differences between the DA and posterior group in 30-day readmission rates (2.84% vs 2.49%, P = .823), 90-day readmission rates (4.27 vs 5.97, P = .432), complication rate (6.16% vs 3.48%, P = .206), or revision rate (1.90% vs 2.99%, P = .535). The DA group had a significantly lower percentage of cases outside the goal abduction angle range (35°-50°) than the posterior group (9.52%, n = 20 vs 19.6%, n = 39, P < .01). The number of cases outside the goal leg length discrepancy range (±10 mm) was not significantly different (P = .846) between the DA (12.9%) and posterior (13.6%) groups. CONCLUSION: This single-surgeon study demonstrates that the DA approach can be transitioned to safely with no significant increase in complications or readmissions over a high-volume experienced surgeon's first 200 cases. Furthermore, our results endorse the consistency of the DA approach during the learning curve period in regard to component positioning compared to posterior approach.

Total Knee Arthroplasty in Obesity: In-Hospital Outcomes and National Trends.

BACKGROUND: Both the prevalence of obesity and the utilization rate of total knee arthroplasty are increasing. The rate and proportion of total knee arthroplasty (TKA) performed in the setting of obesity/morbid obesity is increasing significantly over time. METHODS: Using International Classification of Diseases-Ninth Revision codes, we searched the National Hospitals Discharge Survey national database for patients admitted for primary TKA between 2001 and 2010. We then used International Classification of Diseases-Ninth Revision codes for obesity (body mass index = 30-40 kg/m2) and morbid obesity (body mass index, ≥ 40 kg/m2) to select the obese cohorts. RESULTS: We found 29,694 nonobese, 2645 obese, and 1150 morbidly obese patients. There was an increase in each group over time. The rate of obesity/morbid obesity was strongly correlated with time. Obese and morbidly obese patients were more likely to be younger, female, diabetic, and have Medicaid than nonobese patients. Obese and morbidly obese patients had shorter hospital stays and higher home discharge rates than nonobese patients. Obese and morbidly obese patients had lower transfusion rates, shorter hospital stays, and no increase in inpatient wound infection or venous thromboembolic complications than nonobese patients. The Midwest region saw a greater burden of obese TKA patients. CONCLUSION: With the right measures and precautions, satisfactory inhospital outcomes are possible in the obese patient after primary TKA. A limitation of this study is short inhospital stay of the index procedure as complications may present later after discharge.

Revision total hip arthroplasty in the United States: national trends and in-hospital outcomes.

PURPOSE: With the increasing number of primary total hip arthroplasties (THA) being performed, the frequency of revision surgery is also expected to increase. We analysed the immediate in-hospital complications and epidemiologic data of 3,469 revision and 18,186 primary THA cases. METHODS: The National Hospital Discharge Survey (NHDS) was evaluated between 2001 and 2010 for patients who underwent revision and primary THA. Patients were identified and included in our retrospective study based on ICD-9 procedure codes. RESULTS: The number of primary and revision THAs increased steadily from 2001 to 2010. The revision burden decreased for the same studied period (r = -0.92) to reach 13.9 % in 2010. The South region had higher revision burden of 17.4 % (p < 0.001). The primary THA group was more likely to be obese, morbidly obese, and have hypertension (p < 0.001). The revision THA group had an increased rate of blood transfusions (p < 0.001), deep venous thrombosis (p = 0.008), post-operative sepsis (p < 0.001), and wound complications (p < 0.001). The in-hospital mortality rate was also higher for the revision THA group (0.6 % versus 0.2 %, p < 0.001). CONCLUSIONS: The revision burden has undergone a steady decrease over the ten years studied and the reason for this is likely multifactorial. The South region had a significantly higher revision burden when compared to the rest of the United States. Larger hospitals tend to perform relatively more revisions. Revision THA patients are associated with longer hospital stay, higher complications rate, and higher in-hospital mortality rate.

Effect of lower extremity fasciotomy length on intracompartmental pressure in an animal model of compartment syndrome: the importance of achieving a minimum of 90% fascial release.

BACKGROUND: There has been an increase in minimally invasive surgery for chronic exertional compartment syndrome (CECS), despite the potential for incomplete compartment release and iatrogenic injuries. To our knowledge, no study has examined the effect of the length of fascial release on compartment pressures. PURPOSE/HYPOTHESIS: The purpose was to explain the high failure rate seen in fascial release for CECS by evaluating the effect of fasciotomy length on intracompartmental pressures. We hypothesized that complete fascial release would need to be performed to return pressures to baseline levels. STUDY DESIGN: Controlled laboratory study. METHODS: Five male swine (10 lower extremities) were anesthetized. A slit catheter, connected to a pressure monitor, was inserted into the anterior compartment and a solution containing 5% swine albumin was injected into the compartment until the compartment pressure was >25 mm Hg for 10 minutes. Pressures were measured at rest, after the injection, and after each 10% incremental fasciotomy release. RESULTS: The mean resting intracompartmental pressure was 3.2 mm Hg (range, 0-6 mm Hg), which increased after the injection to a mean of 37 mm Hg (range, 26-67 mm Hg). After complete fasciotomy, the mean pressure was 1.1 mm Hg (range, 0-4 mm Hg). There was a strong negative correlation (r=-0.693) between fasciotomy length and intracompartmental pressure. In 90% of the specimens, the pressures were <15 mm Hg after 80% fascial release, and after 90% release, all pressures were ≤8 mm Hg. CONCLUSION: This study demonstrates a strong correlation between fasciotomy length and a reduction in intracompartmental pressures in a swine model. Our study suggests that 90% fascial release may represent a possible watershed zone, returning the intracompartmental pressure to a value at or near baseline values. CLINICAL RELEVANCE: The results suggest that even in cases with near complete fascial release, intracompartmental pressures may decrease enough to provide symptomatic relief and avoid possible iatrogenic injuries associated with percutaneous release. It is unknown whether the swine model may adequately translate to the clinical setting; thus, recommendations should be taken with caution, and future studies should be performed to examine the correlation in a human model.

National trends and perioperative outcomes in primary and revision total shoulder arthroplasty: Trends in total shoulder arthroplasty.

INTRODUCTION: Total shoulder arthroplasty (TSA) is a highly successful procedure for management of glenohumeral arthritis, fractures and rotator cuff tears. The purpose of this study was to evaluate patient demographics, perioperative outcomes and assess recent national trends in both primary and revision TSA. METHODS: The National Hospital Discharge Survey database was searched for patients admitted to US hospitals for primary and revision TSA from 2001 to 2010. RESULTS: A total of 1,297 patients who underwent primary TSA and 184 patients who underwent revision TSA were identified. The rates of primary TSA (r = 0.88) and revision TSA (r = 0.85) both demonstrated a strong positive correlation with time. The mean patient age of the primary group was significantly higher than the revision group. Gender was not significantly different between the groups. There was no significant difference in the racial make-up between the revision and primary groups. African Americans accounted for 3.3 % of primaries versus 4.3 % of revisions (p = 0.615). Revision TSA patients had a significantly longer average LOS (3.06 days vs 2.46 days, p < 0.01), more medical comorbidities (6.0 vs 5.1 comorbidities, p < 0.01) and a higher rate of developing a myocardial infarction (2.2 % versus 0 %, p < 0.01) than the primary TSA group. CONCLUSIONS: This study demonstrates that the rate of TSA is rapidly increasing in the US, with over a four-fold increase in revisions and five-fold increase in primaries over the ten years studied.

CD133+ melanoma subpopulations contribute to perivascular niche morphogenesis and tumorigenicity through vasculogenic mimicry.

Tumor cell subpopulations that express cancer stem cell markers such as CD133 (prominin1) or ABCB5 are thought to be crucial for tumor initiation and heterogeneity, but their biological significance in melanoma has been controversial. Here, we report that CD133(+) and ABCB5(+) subpopulations are colocalized in melanomas in perivascular niches that contain CD144 (VE-cadherin)(+) melanoma cells forming vessel-like channels, a phenomenon termed vasculogenic mimicry (VM). RNAi-mediated attenuation of CD133 established its critical function in morphogenesis of these perivascular niches as well as in melanoma tumorigenicity. Niche-associated genes CD144 and ABCB5 were downregulated in tumors derived from CD133 knockdown (KD) melanoma cells compared with controls. CD133KD cells also lacked the ability to form CD144(+) VM-like channels in a manner that was associated with a depletion of the ABCB5(+) cell subpopulation. Finally, CD133 KD cells exhibited poorer tumor growth in vivo. Taken together, our findings corroborate models in which CD133(+)/ABCB5(+) melanoma cells reside in a complex anastomosing microvascular niche that encompasses CD144(+) VM channels as well as authentic endothelial cell-lined blood vessels. Further, they indicate that CD133(+) cells act as stem-like cells, which drive tumor growth by promoting VM and the morphogenesis of a specialized perivascular niche in melanoma.

Differentiation of NUT midline carcinoma by epigenomic reprogramming.

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC.

Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains.

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in-frame fusion of the double bromodomain protein, BRD4, with a testis-specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4-NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient-derived cell line, we show that p300 sequestration into the BRD4-NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4-NUT released p300 and restored p53-dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off-context activity of a testis-specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation.

Transcriptional and developmental functions of the H3.3 histone variant in Drosophila.

Changes in chromatin composition accompany cellular differentiation in eukaryotes. Although bulk chromatin is duplicated during DNA replication, replication-independent (RI) nucleosome replacement occurs in transcriptionally active chromatin and during specific developmental transitions where the genome is repackaged. In most animals, replacement uses the conserved H3.3 histone variant, but the functions of this variant have not been defined. Using mutations for the two H3.3 genes in Drosophila, we identify widespread transcriptional defects in H3.3-deficient animals. We show that mutant animals compensate for the lack of H3.3 in two ways: they upregulate the expression of the major histone H3 genes, and they maintain chromatin structure by using H3 protein for RI nucleosome replacement at active genes. Rescue experiments show that increased expression of H3 is sufficient to relieve transcriptional defects. In contrast, H3.3 is essential for male fertility, and germline cells specifically require the histone variant. Defects without H3.3 first occur around meiosis, resulting in a failure to condense, segregate, and reorganize chromatin. Rescue experiments with mutated transgenes demonstrate that H3.3-specific residues involved in RI nucleosome assembly-but not major histone modification sites-are required for male fertility. Our results imply that the H3.3 variant plays an essential role in chromatin transitions in the male germline.

Diagnosis of NUT midline carcinoma using a NUT-specific monoclonal antibody.

NUT midline carcinoma (NMC) is a uniformly lethal malignancy that is defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14. NMCs are morphologically indistinguishable from other poorly differentiated carcinomas, and the diagnosis is usually made currently by fluorescence in situ hybridization (FISH). As normal NUT expression is confined to testis and ovary, we reasoned that an immunohistochemical (IHC) stain for NUT would be useful in diagnosing NMC. To this end, we raised a highly specific rabbit monoclonal antibody, C52, against a recombinant NUT polypeptide, and developed an IHC staining protocol. The sensitivity and specificity of C52 staining was evaluated in a panel of 1068 tissues, predominantly diverse types of carcinomas (n=906), including 30 NMCs. Split-apart FISH for NUT rearrangement was used as a "gold standard" diagnostic test for NMC. C52 immunoreactivity among carcinomas was confined to NMCs. IHC staining had a sensitivity of 87%, a specificity of 100%, a negative predictive value of 99%, and a positive predictive value of 100%. Two new cases of NMC containing BRD4-NUT fusions were detected by C52 IHC, but missed by conventional FISH. In both instances, these tumors contained cryptic BRD4-NUT rearrangements, as confirmed by FISH using a refined set of probes. Some germ cell tumors, including 64% of dysgerminomas, showed weak NUT immunoreactivity, consistent with the expression of NUT in normal germ cells. We conclude that IHC staining with the C52 monoclonal antibody is a highly sensitive and specific test that reliably distinguishes NMC from other forms of carcinoma. The NUT antibody is being prepared for commercial release and will be available in the near future.

2. Chromatin assembly with H3 histones: full throttle down multiple pathways.

The typical eukaryotic genome packages roughly 6 feet of DNA into a nucleus about 5 mum in diameter, yet this compaction blocks access to the DNA. At the first level of compaction, DNA is wrapped around octamers of core histone proteins to form arrays of nucleosomes. Nucleosomes are sufficient to block access to DNA, and cells must therefore manipulate nucleosomes in the course of activating the genome. Dramatic progress has been made in understanding the mechanisms by which nucleosomes are manipulated. In addition to the major core histones, most eukaryotic genomes also encode additional variant histones, which have some structural similarity. These are targeted to specific loci by coupling specialized nucleosome assembly pathways to DNA replication, transcription, or to developmental processes. We review evidence that nucleosome assembly pathways are interlinked with histone-modification systems, and may thereby perpetuate epigenetic chromatin states.

Transcriptional activation triggers deposition and removal of the histone variant H3.3.

DNA in eukaryotic cells is packaged into nucleosomes, the structural unit of chromatin. Both DNA and bulk histones are extremely long-lived, because old DNA strands and histones are retained when chromatin duplicates. In contrast, we find that the Drosophila HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones as they are induced. Histone replacement does not occur at artificial HSP70 promoter arrays, demonstrating that transcription is required for H3.3 deposition. The H3.3 histone is enriched in all active chromatin and throughout large transcription units, implying that deposition occurs during transcription elongation. Strikingly, we observed that the stability of chromatin-bound H3.3 differs between loci: H3.3 turns over at continually active rDNA genes, but becomes stable at induced HSP70 genes that have shut down. We conclude that H3.3 deposition is coupled to transcription, and continues while a gene is active. Repeated histone replacement suggests a mechanism to both maintain the structure of chromatin and access to DNA at active genes.