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Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 ( MECP2 ) on the X chromosome in young females. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target single-guide RNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation-edited MECP2 locus by dCpf1-CTCF (a catalytically dead Cpf1 fused with CCCTC-binding factor) with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of Methyl CpG-binding Protein 2 (MECP2) on the X chromosome in girls. Reactivation of the silent wild-typeMECP2allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of theMECP2promoter by dCas9-Tet1 with target sgRNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation editedMECP2locus by dCpf1-CTCF with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.One-Sentence SummaryReactivation ofMECP2from the inactive X chromosome by multiplex epigenome editing rescues Rett syndrome neurons in vitro.

Dysregulation of the transcription factor MYC is involved in many human cancers. The dimeric transcription factor complexes of MYC/MAX and MAX/MAX activate or inhibit, respectively, gene transcription upon binding to the same enhancer box DNA. Targeting these complexes in cancer is a long-standing challenge. Inspired by the inhibitory activity of the MAX/MAX dimer, we engineered covalently linked, synthetic homo- and heterodimeric protein complexes to attenuate oncogenic MYC-driven transcription. We prepared the covalent protein complexes (∼20 kDa, 167–231 residues) in a single shot via parallel automated flow synthesis in hours. The stabilized covalent dimers display DNA binding activity, are intrinsically cell-penetrant, and inhibit cancer cell proliferation in different cell lines. RNA sequencing and gene set enrichment analysis in A549 cancer cells confirmed that the synthetic dimers interfere with MYC-driven transcription. Our results demonstrate the potential of automated flow technology to rapidly deliver engineered synthetic protein complex mimetics that can serve as a starting point in developing inhibitors of MYC-driven cancer cell growth., Via parallel automated flow synthesis, we prepared covalent dimeric protein analogues of the transcription factor MYC. The dimers can enter cells and inhibit MYC-driven cancer cell proliferation.

The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. To test this idea, we focused our study on nuclear condensates reported in cell lines, demonstrated they occur in normal human and malignant breast cancer, and developed assays to test clinically active antineoplastic small molecule drugs relative to these condensates.To study the behavior of drugs within condensates, these were modeled in vitro with purified proteins and visualized by fluorescent confocal microscopy. We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. For each drug, the small molecule partitioned into the same condensate in vitro in which its established target resides in vivo. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration.Alkylating agents are a class of commonly used antineoplastic compounds, of which cisplatin is a prominent example. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Thus, we conclude that cisplatin preferentially modifies transcriptional condensate-associated DNA in cells, and that this causes selective condensate disruption. The mechanisms that produce drug resistance can provide clues to drug activity in the clinical setting. Investigating the behavior of tamoxifen within ER transcriptional condensates demonstrated that it disrupts these condensates in vitro and on oncogenes in cells; hormonal therapy resistant ESR1 mutations render these condensates resistant. MED1 overexpression, a poorly understood mechanism of tamoxifen resistance, increased the size of ER-MED1 condensates, thereby rending tamoxifen more dilute and ineffective when concentrated therein. This suggest that altering the size and nature of transcription condensates in breast cancer can mediate drug resistance in the clinical setting.Our results show that antineoplastic drugs partition selectively into condensates, that this can occur through physicochemical properties independent of their molecular targets, and that resistance to drugs may occur through condensate altering mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment. Citation Format: Isaac Klein, Ann Boija, Lena Afeyan, Susana Wilson Hawken, Mengyang Fan, Alessandra Dall'Agnese, Ozgur Oksuz, Jonathan Henninger, Krishna Shrinivas, Benjamin Sabari, Ido Sagi, Victoria Clark, Jesse Platt, Mrityunjoy Kar, Patrick McCall, Alicia Zamudio, John Mantiega, Eliot Coffey, Charles Li, Nancy Hannett, Yang Guo, Tim-Michael Decker, Tong Lee, Tinghu Zhang, Jing-Ke Weng, Dylan Taatjes, Arup Chakraborty, Phillip Sharp, Young Tae Chang, Anthony Hyman, Nathanael Gray, Richard Young. Partitioning of cancer therapeutics in nuclear condensates [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-10.

Methyl CpG binding protein 2 (MeCP2) is a key component of constitutive heterochromatin, which is crucial for chromosome maintenance and transcriptional silencing1–3. Mutations in the MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome3–5, which is associated with severe mental disability and autism-like symptoms that affect girls during early childhood. Although previously thought to be a dense and relatively static structure1,2, heterochromatin is now understood to exhibit properties consistent with a liquid-like condensate6,7. Here we show that MeCP2 is a dynamic component of heterochromatin condensates in cells, and is stimulated by DNA to form liquid-like condensates. MeCP2 contains several domains that contribute to the formation of condensates, and mutations in MECP2 that lead to Rett syndrome disrupt the ability of MeCP2 to form condensates. Condensates formed by MeCP2 selectively incorporate and concentrate heterochromatin cofactors rather than components of euchromatic transcriptionally active condensates. We propose that MeCP2 enhances the separation of heterochromatin and euchromatin through its condensate partitioning properties, and that disruption of condensates may be a common consequence of mutations in MeCP2 that cause Rett syndrome. The chromatin protein MeCP2 is a component of dynamic, liquid-like heterochromatin condensates, and the ability of MeCP2 to form condensates is disrupted by mutations in the MECP2 gene that occur in the neurodevelopmental disorder Rett syndrome.

The incidence of posttransplant hepatic arterial stenosis (HAS) has been reported in 5 to 10% of orthotopic liver transplants and, left untreated, can lead to hepatic arterial thrombosis. Most vascular complications develop less than 3 months after initial transplant, with thrombosis representing over half of all complications. There has been a trend toward minimally invasive, endovascular techniques for treating HAS with angioplasty and stenting. In one review of endovascular therapies for HAS, primary technical success was achieved in 95% of the interventions. Complication rates following endovascular repair of HAS have been reported to be between 0 and 23% in the literature. The main risk factors for complications include tortuosity of the hepatic artery and history of a second liver transplant. Other associated risk factors include female gender, age greater than 60 years, prior history of transarterial chemoembolization, and multiple arterial graft anastomoses. The case presented here is representative of a complication of balloon rupture and fragmentation in a patient undergoing hepatic arterial stent placement post–liver transplant.

DICOM viewers must fulfill roles beyond primary diagnostic interpretation, including serving as presentation tools in teaching and multidisciplinary conferences, thereby enabling multiple individuals to review images collaboratively in real time. When in-person gathering is not possible, a variety of solutions have been deployed to maintain the ability for spatially separated users to view medical images simultaneously. These approaches differ in their backend architectures, utilization of application-specific optimizations, and ultimately in their end user satisfaction. In this work, we systematically compare the performance of conventional screensharing using a videoconferencing application with that of a custom, synchronized DICOM viewer linked using Web Real Time Communications (WebRTC) technology. We find superior performance for the WebRTC method with regard to image quality and latency across a range of simulated adverse network conditions, and we show how increasing the number of conference participants differentially affects the bandwidth requirements of the two viewing solutions. In addition, we compare these two approaches in a real-world teaching scenario and gather the feedback of trainee and faculty radiologists, who we found to favor the WebRTC method for its decreased latency, improved image quality, ease of setup, and overall experience. Ultimately, our results demonstrate the value of application-specific solutions for the remote synchronized viewing of medical imaging, which, given the recent increase in reliance on remote collaboration, may constitute a significant consideration for future enterprise viewer procurement decisions.

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors

Drug partitioning in nuclear condensates There is increasing interest in the function of phase-separated biomolecular condensates in cells because of their distinct properties and expanding roles in important biological processes. Klein et al. considered the fate of small-molecule therapeutics in the context of nuclear condensates (see the Perspective by Viny and Levine). They show that certain antineoplastic drugs have physicochemical properties that cause them to concentrate preferentially in condensates, both in vitro and in cancer cells. This property influences drug activity, and protein mutations that alter condensate formation can lead to drug resistance. Optimizing condensate partitioning may be valuable in developing improved therapeutics. Science , this issue p. 1386 ; see also p. 1314

Highlights • Video-conference platforms (VCPs) have rapidly become utilized in the current COVID-19 pandemic to promote social distancing. • Radiology trainee education, as regulated by the ACGME, necessitates active learning based on image review and VCPs can facilitate and support this. • Patient protected health information and HIPAA requirements must be adhered to even with VCPs and many platforms have the appropriate security measures to comply. • Tools within the VCP permit two-way interaction, screen control/sharing, and annotation features, which enable appropriate education even in a remote setting., Technologic advances have resulted in the expansion of web-based conferencing and education. While historically video-conferencing has been used for didactic educational sessions, we present its novel use in virtual radiology read-outs in the face of the COVID-19 pandemic. Knowledge of key aspects of set-up, implementation, and possible pitfalls of video-conferencing technology in the application of virtual read-outs can help to improve the educational experience of radiology trainees and promote potential future distance learning and collaboration.

Phase separation and gene control Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science , this issue p. eaar2555 , p. eaar3958 , p. 412 ; see also p. 329

The gene expression programs that define the identity of each cell are controlled by master transcription factors (TFs) that bind cell-type-specific enhancers, as well as signaling factors, which bring extracellular stimuli to these enhancers. Recent studies have revealed that master TFs form phase-separated condensates with the Mediator coactivator at super-enhancers. Here, we present evidence that signaling factors for the WNT, TGF-β, and JAK/STAT pathways use their intrinsically disordered regions (IDRs) to enter and concentrate in Mediator condensates at super-enhancers. We show that the WNT coactivator β-catenin interacts both with components of condensates and DNA-binding factors to selectively occupy super-enhancer-associated genes. We propose that the cell-type specificity of the response to signaling is mediated in part by the IDRs of the signaling factors, which cause these factors to partition into condensates established by the master TFs and Mediator at genes with prominent roles in cell identity., National Science Foundation (U.S.). (Grant PHY1743900), National Institutes of Health (U.S.) (Grants GM123511, GM117370 and T32CA009172), German Research Foundation Research Fellowship (DE 3069/1-1)

The coracoid process of the scapula is in close proximity to major neurovascular structures, including the brachial plexus and the axillary artery and vein. In addition, it serves as a major site of attachment for multiple tendons and ligaments about the shoulder. Isolated coracoid fractures are rare; however, they can be easily overlooked on routine shoulder radiographs. Importantly, when these fractures go undiagnosed, they are at high risk for nonunion. In this paper, we will review the relevant anatomy of the coracoid process, classification schemes for coracoid fractures, mechanisms of injury how these fractures typically present, multimodality imaging findings, and associated injuries. Finally, we will briefly discuss the clinical management of these fractures.

Percutaneous renal transplant biopsy (PRTB) is the gold standard for evaluating allograft rejection after renal transplant. Hemorrhage is the predominant complication. We describe the implementation of a standardized protocol for PRTB at a single institution, with the aim of reducing bleeding complications. Utilizing the plan-do-study-act model for quality improvement, we created and deployed a protocol centered on controlling patient's hypertension, platelet function, and anticoagulation status. The 4-year study encompassed a total of 880 PRTBs, before and after implementation of the protocol. Total complication rate, which was 5.8% in the 2 years leading up to implementation of the protocol, was reduced to 2.9% after the protocol was introduced ( P = .04). A standardized approach to PRTB can potentially lower complication rates; we present a framework for implementating a quality improvement protocol at other institutions.

The spread of bad neighborhoods Our genomes have complex three-dimensional (3D) arrangements that partition and regulate gene expression. Cancer cells frequently have their genomes grossly rearranged, disturbing this intricate 3D organization. Hnisz et al. show that the disruption of these 3D neighborhoods can bring oncogenes under the control of regulatory elements normally kept separate from them (see the Perspective by Wala and Beroukim). These novel juxtapositions can result in the inappropriate activation of oncogenes. Science , this issue p. 1454 ; see also p. 1398

Extracranial metastasis of glioblastoma multiforme (GBM) is rare, but has recently been reported with increasing frequency. GBM metastases typically present after a biopsy or resection of the primary tumor. An otherwise healthy 54year-old woman presented with recurring pleural effusions originally believed to be from a primary lung malignancy. The patient subsequently experienced a generalized tonic clonic seizure and a right temporal brain mass was discovered. The patient later developed weakness and radiculopathy, and an extramedullary extradural mass spreading from C7 to T6 was discovered. She underwent resection of both central nervous system lesions as well as a lung biopsy, and all pathologic specimens were consistent with GBM. The case presented is unique in that the patient's initial symptoms were related to her metastasis. Furthermore, a purely epidural spread of GBM that respects the leptomeninges and intramedullary parenchyma is highly unusual.

Background: Oral fludarabine and intravenous rituximab (FR) was the standard first-line therapy for CLL or small lymphocytic lymphoma (SLL) patients (pts) in BC from 2003-2015. Ibrutinib for relapsed/refractory (R/R) CLL was introduced and publicly funded in 2015. Our aim was to review long term outcomes of all CLL/SLL pts treated with FR in BC, including the impact of 2nd line therapy with ibrutinib versus chemoimmunotherapy and to report the risk of secondary malignancies in this population based cohort. Methods: The BC Provincial CLL Database was used to identify all CLL/SLL pts who received first-line FR from 2003-2017. The BC Cancer Registry was used to identify secondary malignancies occurring after FR. Primary outcomes were overall survival (OS) and treatment free survival (TFS), defined as start of FR to next-line therapy or death/last follow-up. Variables examined for impact on OS/TFS included age at FR, gender, primary diagnosis (CLL vs SLL), B symptoms, advanced stage (Rai stage 3-4 CLL, Ann Arbor 1-2 SLL), baseline hemoglobin, lymphocyte count, platelets, LDH and FISH abnormalities. All variables significant on univariate analyses (P Results: 673 pts were identified as receiving FR as first-line therapy for CLL (86%) or SLL (14%). Median time from CLL/SLL diagnosis to FR was 2.5 years (y) (range 0.1-27.3). Median age at FR was 67 y (range 26-91) with 73% ≥ 60 y and 39% ≥ 70 y. Most pts were male (66.1%), had early stage disease (84.2%) with no B symptoms (89.7%) and normal LDH (81.1%). Of 411 pts with pre-treatment FISH testing, prevalence of FISH abnormalities were: 48.5% del13q, 25.7% trisomy 12, 12.9% del11q, 8.0% del17p. Median number of FR cycles was 6 (range 1-10). Median follow-up of living pts from FR was 6.4 y (range 0.2-12.7). 2 y and 5 y OS were 89.4% (95% CI: 86.8-91.6) and 73% (95% CI: 69.0-76.6) respectively; median OS 11.6 y (95% CI: 4.6-13.7 y). 2 y and 5 y TFS were 72% (95% CI: 68-75%) and 37% (95% CI: 33 - 41) respectively, median TFS 3.8 y (95% CI: 1.78-7.09). Those with del17p had significantly worse OS and TFS compared to those without (median OS 5.7 vs 13.7 y, P During the follow up period, 351 pts (52%) went on to 2nd-line therapy: ibrutinib 87 (including 2 with BR and 1+R), cyclophosphamide-based (CVP/CHOP) +/- R 102, repeat FR 71, FCR 6, F alone 21, bendamustine +/-R 13, chlorambucil+/-R 38, steroids 3, R alone 3, alemtuzumab 2, other chemotherapy 3 and allotransplant 2. Median follow-up after 2nd-line therapy was 2.8 y (range 0.1-10.8). Median OS and TFS from 2nd-line treatment (TFS2) for ibrutinib (n=87) vs. for other treatments (n=264) was: OS not reached vs 5.3 y, P A total of 202 malignancies were recorded after initiation of FR in 166 pts (24.7%), Table 1. The median time from FR to 2nd malignancy was 2.3 y (range 0.1-13.5). Richter's transformation (RT) occurred in 36 pts (5.3%) at median 1.9 y (range 0.1-13.2) from FR. Most frequent 2nd malignancies were: non-melanoma skin cancer (11.7%), lung (2.5%), colon (2.1%), other heme (1.9%), and prostate (1.8%). There were 4 cases of acute myeloid leukemia (AML), 2 of which received alkylator therapy after FR prior to AML diagnosis. Conclusions: In this large, homogeneous cohort of CLL/SLL pts treated with first-line FR, including nearly 40% of pts ≥ age 70, we demonstrate a short median TFS of 3.8 y; however, a long OS of 11.6 y. Rates of 2nd malignancies are low after this non-alkylator based chemoimmunotherapy regimen. Ibrutinib for R/R CLL/SLL after FR resulted in significantly improved survival over alternate therapy, with excellent 2 yr OS 91% and TFS 78%. These data demonstrate the efficacy of FR and the benefit of ibrutinib over chemoimmunotherapy as second-line therapy for CLL/SLL in the real-world. Disclosures Bruyere: Jenssen: Other: Travel Grant; Celgene: Honoraria. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Scott:Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Savage:BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Connors:Takeda Pharmaceuticals: Honoraria; Seattle Genetics: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy. Sehn:TEVA Pharmaceuticals Industries: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; F. Hoffmann-La Roche/Genentech: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; F. Hoffmann-La Roche/Genentech: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; TEVA Pharmaceuticals Industries: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria. Gerrie:Lundbeck, Seattle Genetics: Consultancy, Honoraria.

Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well characterized, but little is known about the mechanisms by which ADs effect gene activation. Here, we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation., National Institutes of Health (U.S.) (Grant GM123511), National Institutes of Health (U.S.) (Grant GM117370), Swedish Research Council (Postdoctoral Fellowship VR 2017-00372), Damon Runyon Cancer Research Foundation ( Fellowship 2309-17)

Background Lumbar spinal stenosis (LSS) is the most common indication for spine surgery among the geriatric population. Although decompressive surgery is effective, older patients do not benefit as much as younger patients, and they are frequently excluded from studies assessing postoperative physiotherapy. We sought to evaluate the long-term outcomes after surgery when a novel postoperative physiotherapy regimen was included. Methods We performed a retrospective review of patients with LSS greater than 70 years old who underwent lumbar decompressive surgery by the senior author over the past five years. We evaluated patients who participated in a novel postoperative physiotherapy regimen involving four phases of rehabilitation aimed at progressively independent ambulation. The visual analog scale (VAS), lower extremity motor strength, and functional independence measure (FIM) were collected preoperatively and after physiotherapy to measure outcomes. Results Ten consecutive patients with an average age of 83 years (range: 71 - 96) met the inclusion criteria. Nine patients underwent minimally invasive laminotomies at L4-L5 and one underwent a laminotomy at L3-L4. The average follow-up time was 41.9 months. The preoperative mean VAS was 7.35, and at the end of the study, it was 1.7 (p = 0.005). Three of the four patients with preoperative motor deficits improved. The median transfer and locomotion subscores of the FIM were six preoperatively and increased to seven postoperatively. Neither of these improvements was significant. Conclusions Patients older than 70 years undergoing decompressive surgery and a novel postoperative physiotherapy regimen experienced significant reductions in pain. Independence also increased; however, this did not reach statistical significance.

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes., National Institutes of Health (U.S.) (Grant GM123511), National Institutes of Health (U.S.) (Grant P01-CA042063), National Science Foundation (U.S.) (Grant PHY-1743900), National Cancer Institute (U.S.) (Grant P30-CA14051)

Cancer arises from genetic alterations that produce dysregulated gene expression programs. Normal gene regulation occurs in the context of chromosome loop structures called insulated neighborhoods, and recent studies have shown that these structures are altered and can contribute to oncogene dysregulation in various cancer cells. We review the types of genetic and epigenetic alterations that influence neighborhood structures and contribute to gene dysregulation in cancer, present models for insulated neighborhoods associated with the most prominent human oncogenes, and discuss how such models may lead to further advances in cancer diagnosis and therapy. Keywords: chromosome structure; insulated neighborhood; oncogene regulation, National Institutes of Health (U.S.) (Grant HG002668)

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5′ UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS. Rescue of fragile X syndrome neurons by CRISPR-mediated DNA methylation editing of the FMR1 gene., National Institutes of Health (Grant HD045022), National Institutes of Health (Grant MH104610), National Institutes of Health (Grant R01NS088538), National Institutes of Health (Grant R01GM123511)

Summary Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events.We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers., United States. National Institutes of Health (HG002668), United States. National Institutes of Health (CA109901)

Groin pain is a common complaint in the primary care setting. The etiologies of groin pain are multifactorial and include neurological causes, fractures, malignancy, and sacroiliac joint (SI) dysfunction. Differentiating spinal and back pathologies from inguinal etiologies is challenging, but the characteristics, distribution, symptoms, signs, physical exam, and imaging help to appropriately guide the evaluation and subsequent therapy.

Key oncogenes can be misregulated in tumor cells by the acquisition of tumor-specific enhancers that direct gene expression through physical contacts (1,2). Identifying the full complement of enhancers regulating each oncogene could identify additional vulnerabilities in tumor expression programs, but remains challenging, because a given enhancer can regulate multiple genes and each of its targets may be genomically distant. Here we use DNA interaction data to construct gene regulatory landscapes for all expressed genes in a tumor cell and demonstrate that sets of enhancers can be assigned to these oncogenes using direct looping and insulated neighborhood data. A subset of these landscapes contains an exceptional amount of transcriptional apparatus, reminiscent of SEs (3), so we term this subset 3D-SEs. Well-characterized oncogenes, including c-Myc, acquire 3D-SEs in tumor cells where a c-Myc¬-associated linear SE was not identified, suggesting SE acquisition by c-Myc and other oncogenes has been underestimated. Each gene's regulatory network can also surprisingly extend beyond the confines of insulated neighborhoods and incorporate additional distal enhancers. Visualizing instances of 3D-SEs with microscopy demonstrates that they are components of liquid-liquid phase-separated bodies in cells, suggesting gene regulatory landscapes underpin these transcriptional condensates. The interactions comprising gene regulatory networks and their target genes thus extend previous interpretations of the targets of enhancers, signaling pathways, and disease-associated enhancer variants. References: 1. Hnisz, Abraham, Lee, et al. and Young, Cell 2013. 2. Mansour, Abraham, et al. and Young and Look, Science 2014. 3. Whyte, Orlando, Hnisz, Abraham, et al. and Young, Cell 2013. Citation Format: Brian J. Abraham, Yang Eric Guo, Denes Hnisz, Charles H. Li, Abraham S. Weintraub, Daniel S. Day, Jurian Schuijers, Tong Ihn Lee, Richard A. Young. Three-dimensional gene regulatory landscapes in normal and cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 971.

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control. YY1 is a structural regulator of enhancer-promoter interactions and facilitates gene expression., National Institutes of Health (U.S.) (Grant HG002668/GM123511), National Institutes of Health (U.S.) (Grant R37HD045022/R01-NS088538/R01-MH104610), Virginia and Daniel K. Ludwig Graduate Fellowship, National Science Foundation (U.S.). Graduate Research Fellowship Program

BACKGROUND AND METHODS: Elevated serum ferritin is a common clinical finding. The etiology of hyperferritinemia in the Asia-Pacific population is less clear due to a low prevalence of knownHFEmutations such asC282YandH63D, as well as an increased prevalence of viral hepatitis and hereditary anemia. A retrospective case review of 80 patients of Asian ethnicity referred to three subspecialists in tertiary care teaching hospitals between January 1997 and March 2005 for assessment of hyperferritinemia was performed.RESULTS: Only four patients (5%) had iron overload on liver biopsy or quantitative phlebotomy. Forty-nine patients (61%) had secondary causes for their hyperferritinemia, of which 26 had liver disease; 16 of those patients also had viral hepatitis. Thirteen patients fulfilled criteria for the insulin resistance syndrome. Other causes included hematological disorders (n=10), malignancy (n=2) and inflammatory arthritis (n=2). Twenty-seven cases (34%) of unexplained hyperferritinemia were found. Of a total of 22 patients who underwent liver biopsy, significant iron deposition was found in one patient. Fifteen patients underwentC282YandH63Dgenotyping, with two cases ofH63Dheterozygosity. Fourteen patients had first-degree relatives with hyperferritinemia. Three families were identified with more than two members affected, which is suggestive of a possible hereditary hyperferritinemia syndrome.CONCLUSION: Secondary causes of elevated ferritin in the Asian population, particularly liver disease, are common, but primary iron overload syndromes appear to be rare. In a significant proportion of patients, the etiology remains unexplained. The genetic basis for hyperferritinemia in Asians is poorly defined and requires further study.

Recent trials suggest serious toxicity in HIV-associated non-Hodgkin's lymphoma (NHL) with rituximab (R) and chemotherapy (CT), offsetting the benefit of rituximab.We retrospectively reviewed experience with CHOP-R vs. CT in 40 patients with HIV-associated diffuse large B-cell lymphoma (DLBCL) diagnosed between December 1992 and February 2006, all of whom were treated with curative intent.In a univariate analysis, International Prognostic Index (IPI) score, prior AIDS, HAART, and rituximab were significant for overall survival (OS). In a multivariate analysis, IPI 0-1 (p.02), no prior AIDS (p.0002), and receiving CHOP-R (p.01) were significant for improved OS, and HAART use (p.09) retained a trend for improved OS. The hazard ratio (HR) for patients with high IPI receiving CHOP-R was 0.3 (95% CI 0.1-0.8). Patients without prior AIDS receiving CHOP-R had an HR of 0.5 (95% CI 0.1-1.7). The OS at 30 months in patients not receiving HAART was 0%. With HAART, OS was 33% for CT and 86% for CHOP-R; HR for CHOP-R was 0.4 (95% CI 0.1-1.2). Toxic deaths were 3 (33%) for CHOP-R and 6 (25%) for CT (p = ns); all toxic deaths with CHOP-R were in patients not receiving HAART. Rituximab-treated patients had a lower death rate from lymphoma (CHOP-R, 2 [16%] vs. CT, 15 [63%]; p.04), and overall mortality (CHOP-R, 5 [42%] vs. CT, 21 [88%]; p.01).These retrospective data suggest that fatal toxicity of rituximab in HIV-NHL is not increased provided HAART is used, that the addition of rituximab to CT improved outcome, and that further prospective trials investigating this issue are warranted.

Monoclonal B-cell lymphocytosis (MBL) is a clonal lymphoproliferation with the immunophenotype of chronic lymphocytic leukemia (CLL) but a B-lymphocyte count of less than 5 x 10(9)/l and no lymphadenopathy, organomegaly, cytopenias or symptoms. We performed a retrospective analysis of patients with MBL (n = 46), Rai stage 0 CLL (n = 112) and Rai stageor =1 CLL (n = 54). Median follow-up and range was 30 (0.1-120) months for MBL, 60 (0.1-309) months for stage 0 CLL and 54 (0.1-309) months for stageor =1 CLL. None of the MBL patients required treatment compared with 24 of 112 (21%) stage 0 CLL and 28 of 54 (52%) stageor =1 CLL patients (p0.0003). No MBL underwent aggressive transformation compared with 1 of 112 (0.8%) stage 0 CLL and 6 of 54 (11%) stageor =1 CLL patients (p0.0003). Progression-free survival (PFS) appeared improved in MBL compared to stage 0 CLL, although this did not reach statistical significant (p = 0.07) due to the relatively short follow-up in the MBL group; two year PFS was 97.2% for MBL, 93.1% for stage 0 CLL, and 68% for stageor =1 CLL patients (p0.0001 for stageor =1 CLL compared with MBL and stage 0 CLL). This is the first study of outcome in MBL which demonstrates that patients have an improved disease course compared to stage 0 CLL patients. Over a median 2.5 years of follow-up, no MBL patients required treatment or died of CLL-related causes.

This study introduces the use of multivariate linear regression (MLR) and support vector regression (SVR) models to predict postoperative outcomes in a cohort of patients who underwent surgery for cervical spondylotic myelopathy (CSM). Currently, predicting outcomes after surgery for CSM remains a challenge. We recruited patients who had a diagnosis of CSM and required decompressive surgery with or without fusion. Fine motor function was tested preoperatively and postoperatively with a handgrip-based tracking device that has been previously validated, yielding mean absolute accuracy (MAA) results for two tracking tasks (sinusoidal and step). All patients completed Oswestry disability index (ODI) and modified Japanese Orthopaedic Association questionnaires preoperatively and postoperatively. Preoperative data was utilized in MLR and SVR models to predict postoperative ODI. Predictions were compared to the actual ODI scores with the coefficient of determination ( R 2 ) and mean absolute difference (MAD). From this, 20 patients met the inclusion criteria and completed follow-up at least 3 months after surgery. With the MLR model, a combination of the preoperative ODI score, preoperative MAA (step function), and symptom duration yielded the best prediction of postoperative ODI ( R 2 = 0.452; MAD = 0.0887; p = 1.17 × 10 −3 ). With the SVR model, a combination of preoperative ODI score, preoperative MAA (sinusoidal function), and symptom duration yielded the best prediction of postoperative ODI ( R 2 = 0.932; MAD = 0.0283; p = 5.73 × 10 −12 ). The SVR model was more accurate than the MLR model. The SVR can be used preoperatively in risk/benefit analysis and the decision to operate.

Intravascular lymphoma (IVL) is a rare neoplastic disease characterized by the presence of large malignant lymphoid cells in small vessels. It is often diagnosed at autopsy. Clinical manifestations are typically neurologic and dermatologic. Karyotypic abnormalities have been described in a small number of cases and have revealed complex alterations in the majority of cases. We have identified three cases of IVL with varied clinicopathological findings. Karyotypic analysis was undertaken by standard G-banding and supplemented by multi-colored karyotyping (M-FISH) to decipher the chromosomal content of marker chromosomes and undefined additions. M-FISH clarified the chromosomal abnormalities in two cases and unveiled cryptic translocations der(10)t(10;22), der(17)t(17;22), and balanced t(11;14). Comparison with previously published karyotypes revealed prominent involvement of chromosomes 1, 3, 6, 11, 14, and 18, similar to the pattern of clonal evolution in other B-cell lymphomas. The most frequent alterations seen were -6 or 6q- and +18 or dup(18q), with a minimally deleted region located at 6q21-q23 and a commonly amplified region located at 18q13-q23, respectively. Few differences between the classical and Asian variant of this disease were apparent at the karyotypic level. Cytogenetic analysis of additional cases supplemented by multicolor karyotyping may help identify the full spectrum of genetic alterations associated with IVL and assist in the delineation of the critical mutations associated with initiation and progression of this disease.

Nature Communications 8: Article number:14385 (2017); Published: 9 February 2017; Updated: 1 June 2017 In the original version of Supplementary Data 1 associated with this Article, the list of predicted enhancer-associated insertions was inadvertently truncated. The HTML has now been updated to include the correct version of the Supplementary Data 1.

Retrospective case series.Large national inpatient databases estimate that approximately 200,000 lumbar fusions are performed annually in the United States alone. It is common for surgeons to routinely order postoperative hematologic studies to rule out postoperative anemia despite a paucity of data to support routine laboratory utilization.To describe quantitative criteria to guide postoperative utilization of hematologic laboratory assessments.A retrospective analysis of 490 consecutive lumbar fusion procedures performed at a single institution by 3 spine surgeons was performed. Inclusion criteria included instrumented and noninstrumented lumbar fusions performed for any etiology. Data were acquired on preoperative and postoperative hematocrit, platelets, and international normalized ratio as well as age, sex, number of levels undergoing operation, indication for surgery, and intraoperative blood loss. Multivariate logistic regression was performed to determine correlation to postoperative transfusion requirement.A total of 490 patients undergoing lumbar fusion were identified. Twenty-five patients (5.1%) required postoperative transfusion. No patients required readmission for anemia or transfusion. Multivariate logistic regression analysis demonstrated that reduced preoperative hematocrit and increased intraoperative blood loss were independent predictors of postoperative transfusion requirement. Intraoperative blood loss1000 mL had an odds ratio of 8.9 (P=0.013), and preoperative hematocrit35 had an odds ratio of 4.37 (P=0.008) of requiring a postoperative transfusion.Routine postoperative hematologic studies are not necessary in many patients. High intraoperative blood loss and low preoperative hematocrit were independent predictors of postoperative blood transfusion. Our results describe quantitative preoperative and intraoperative criteria to guide data-driven utilization of postoperative hematologic studies following lumbar fusion.

Background: There have been rare reports of intracranial subdural hematoma (SDH) that migrated into the spine. All previous cases have been surgically managed and in this case report, we describe the first case of conservatively managed spinal hematoma secondary to migratory intracranial SDH. Case Description: A 26-year-old male presented with a left tentorial SDH after blunt trauma. He was conservatively managed and discharged home. He presented 8 days later with worsening lower back pain that was found to be secondary to a spinal SDH. Conclusion: Spinal hematomas can be a serious sequelae of migrated intracranial hematomas. Tentorial and other caudally located intracranial hematomas may be more prone to this phenomenon.

Transcriptional super-enhancers drive expression of oncogenes in many cancers and are being targeted with novel transcriptional and epigenetic therapeutics[1,2,3,4]. Super-enhancers are acquired by cancers through multiple mechanisms, including DNA translocation of an extant super-enhancer and focal amplification. We recently discovered a novel mechanism by which super-enhancers are nucleated in T cell acute lymphoblastic leukemias (T-ALLs)[5]. In this case, a small, monoallelic insertion creates a DNA binding site for a master transcription factor protein, which binds and recruits additional factors to nucleate the super-enhancer, which in turn drives high levels of the TAL1 oncogene. We describe here a method for unbiased identification of similar genomic insertions that nucleate potentially oncogenic regulatory elements in cancers. This approach uses data from genome-wide ChIP-Seq studies that map locations of enhancer-binding proteins to identify short DNA sequences missing from reference genomes. We have found and catalogued many additional genomic insertions in 80 additional cancers. An additional insertion has been functionally characterized and determined to be a bona fide enhancer-nucleating insertion that exists in patient genomes. I will describe new insights into the regulation of cancer that occur due to nucleation of novel regulatory elements. [1] Hnisz, Abraham, Lee, et al., Cell 2013 [2] Loven, Hoke, Lin, et al., Cell 2013 [3] Kwiatkowski, et al., Nature, 2014 [4] Wang, et al., Cell, 2015 [5] Mansour et al., Science 2014 Citation Format: Brian J. Abraham, Denes Hnisz, Abraham S. Weintraub, Nicholas Kwiatkowski, Charles H. Li, Sunniyat Rahman, Zhaodong Li, Tong Ihn Lee, A Thomas Look, Marc Mansour, Richard A. Young. Nucleation of transcriptional super-enhancers at tumor oncogenes by small insertions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2004.

Upregulation of tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of several inflammatory conditions, including rheumatoid arthritis. Therapeutic agents such as antibodies or soluble TNF-alpha receptor analogs, which block TNF-alpha activity are a recent addition to the therapeutic armamentarium for the conditions. We describe a patient who developed aplastic anemia complicated by sepsis after receiving etanercept, a TNF-alpha receptor analog, for the treatment of rheumatoid arthritis. Pancytopenia resolved within 3 wk of discontinuing etanercept. To our knowledge, this is the first report of aplastic anemia associated with TNF-alpha blockade.

Clinical trials report that fludarabine and rituximab (FR) as initial therapy for chronic lymphocytic leukemia (CLL) improves progression-free and overall survival (OS) when compared historically to fludarabine alone. To determine whether similar results are achievable with oral FR in a community-based setting, we conducted a population-based analysis of patients treated for CLL or small lymphocytic lymphoma (SLL) in British Columbia, where FR is standard initial therapy. Ninety-eight patients received FR for CLL/SLL from 2004 to 2009. Two- and 4-year OS was 90% and 73%, respectively (median not reached); 2- and 4-year treatment-free survival (TFS) was 69% and 54% (median 4.0 years). Age ≥ 60 years or ≥ 70 years had no effect on OS or TFS. Toxicity led to treatment discontinuation in 13%. FR with oral fludarabine was safely, conveniently and successfully given to community-based patients, irrespective of age, for first-line therapy for CLL/SLL, achieving OS and TFS similar to those in clinical trials.

Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (−) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (−) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex., Author Summary Mucormycosis is recognized as an emerging infectious disease. Compared to other fungal infections, mucormycosis results in high mortality: ∼50% of overall infections and >90% in disseminated infections. There is therefore an ongoing need to study these fungal pathogens. However, surprisingly little is known about the pathogenesis of mucormycosis. Our findings reveal a correlation between sporangiospore size and virulence: larger sporangiospores are more virulent than small spores. Larger spores start invasive hyphal growth immediately upon phagocytosis by host immune cells, whereas smaller spores have a long period of isotropic growth. Differential host immune response explains their difference in virulence, whereby larger spores escape host immune cells by germinating inside of macrophages. Our findings revealed an example of adaptation through fungal cell gigantism, which enable pathogenic fungi to survive within and establish infection in the host. Knowledge of the mechanisms of pathogenicity and the molecular basis of the sexual cycle in M. circinelloides will contribute to advance our understanding of pathogenic zygomycetes.

OBJECTIVES Acute leukemia, particularly acute myeloid leukemia, occurs more frequently in the elderly, a growing segment of the North American population. To evaluate our progress in the diagnosis, treatment and outcome of this condition, we reviewed our experience of all patients > or =60 years of age diagnosed with acute leukemia over a 20-year period at Saint Paul's Hospital, a university-based hospital in Vancouver, Canada. METHODS A retrospective chart review was performed of 103 patients > or =60 years of age diagnosed with acute leukemia (acute myeloid leukemia-81; acute lymphoid leukemia-15; acute leukemia not otherwise specified-7). RESULTS Median age was 72 (range 60-88) years. Bone marrow aspirate yielded cytogenetic information on 57 patients and 18 (31.6%) had an unfavourable karyotype. Fifty-three (51%) patients received induction chemotherapy (treated) and 50 (49%) were palliated (untreated). Treated patients were younger [median 67 years (range 60-79)] than untreated patients [76 years (61-88)], (P < 0.0001). Of the treated patients, 33 (62%) achieved a complete remission. The median overall survival for the group was 104 (1-2689) days, and for treated versus untreated patients-219 (1-2689) and 39 (2-1229) days, respectively (P = 0.0021). Univariate variables predictive of prolonged survival included induction chemotherapy (P = 0.0027), de novo leukemia (P = 0.0420), and younger age, with a relative increase in death in older subgroups (60-69, 70-79, 80+), (P = 0.0311). Induction chemotherapy was the only predictor of prolonged survival in multivariate analysis (P = 0.0027). CONCLUSIONS The prognosis of acute leukemia in older patients remains poor, and even though induction chemotherapy seem to prolong survival in patients able to receive treatment, most ultimately die of leukemia.

To address concerns about regional physician shortages within British Columbia, the University of British Columbia began distributing its undergraduate medical curriculum across multiple campuses (ie, urban Vancouver, small urban Victoria, and rural Prince George) in 2005. The distribution of the pathology curriculum required meeting 3 specific challenges: (i) implementation of high-quality technologies to facilitate distribution; (ii) recruitment of pathologists to teach; and (iii) creation of an electronic pathology learning center. Technological needs were met by a state-of-the-art audiovisual system allowing simultaneous interactive didactic sessions across all 3 sites, and by the use of a digital "virtual slide" system. Recruitment of pathologist educators proved challenging owing to comparatively limited staffing levels at the rural site. A physical and virtual pathology learning center was developed to assist students in self-directed study. Student performance on pathology examinations has proven to be essentially identical pre- and post-distribution, and is equivalent across all 3 sites. Quantitative and qualitative student survey data show that distributed pathology instruction is overwhelmingly well received by medical students at all sites, that pathologists' expertise is very important to students, and that pathology is one of the most popular components of the distributed curriculum. Pathology education continues to be a vital part of a distributed undergraduate medical program, and student grades and feedback demonstrate the value of the teaching and the technologies we have used. To be implemented successfully, the distribution of pathology education requires considerable financial and infrastructure investment, and ongoing commitment from pathologists and university administrators.

Plasmablastic lymphoma (PBL) is a recently described type of non-Hodgkin's lymphoma (NHL) that occurs in up to 3% of patients with HIV infection. Although the clinical-pathological features of several patients with HIV-associated plasmablastic lymphoma are documented, detailed description of clinical outcome is limited to isolated case reports. Generally, the response to lymphoma therapy is poor and survival is short. Response to highly active anti-retroviral therapy (HAART), however, has also been described. In this report, we describe the clinical course of two patients diagnosed with HIV-associated PBL in the era of HAART. One patient had a complete response to HAART, with a response-duration of 14 months, followed by relapse in the gastrointestinal tract several months after an anti-retroviral holiday. He is currently in complete remission (CR) eight months from diagnosis of relapse after receiving a full course of combination chemotherapy with modified CHOP, and 25 months from initial diagnosis. A second patient responded to brief chemotherapy in conjunction with HAART and is in clinical CR ten months from diagnosis. These cases illustrate that immunologic and virologic control with HAART may be beneficial for treating PBL and may possibly maintain continued CR. We advocate a high index of suspicion for primary PBL or its recurrence in patients with HIV infection, a history of low CD4 counts or high viral load, and oral or gastrointestinal symptoms.

Upregulation of tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of several inflammatory conditions, including rheumatoid arthritis. Therapeutic agents such as antibodies or soluble TNF-alpha receptor analogs, which block TNF-alpha activity are a recent addition to the therapeutic armamentarium for the conditions. We describe a patient who developed aplastic anemia complicated by sepsis after receiving etanercept, a TNF-alpha receptor analog, for the treatment of rheumatoid arthritis. Pancytopenia resolved within 3 wk of discontinuing etanercept. To our knowledge, this is the first report of aplastic anemia associated with TNF-alpha blockade.