Five top stories in anatomic pathology: stories from the faculty at UMass Medical School and UMass Memorial Medical Center, Worcester, Massachusetts
This month's issue of the Archives of Pathology & Laboratory Medicine introduces a new series of special sections featuring relevant and contemporary topics in Anatomic Pathology and Cytopathology, herein referred to as “Five Top Stories.”
CONTEXT: Several developments in genitourinary pathology are likely to change our understanding and management of some genitourinary cancers considerably.
OBJECTIVE: To review 5 stories in genitourinary pathology: (1) fusion in the ETS (E26) gene family in prostatic adenocarcinoma; (2) insulin-like growth factor II messenger RNA-binding protein 3 (IMP3), an important prognostic biomarker for kidney and bladder cancers; (3) translocation renal cell carcinoma; (4) UroVysion fluorescence in situ hybridization test in urine cytology for detection of bladder cancer; and (5) the use of triple immunostaining for diagnosis of prostate cancer.
DATA SOURCES: Literature review and authors' personal experiences.
CONCLUSIONS: Many scientific findings have contributed recently to the understanding of the natural pathogenesis and progression of genitourinary cancers. This translational research helps in diagnosing, predicting, and potentially, treating genitourinary cancers.
CONTEXT: Cytology relies heavily on morphology to make diagnoses, and morphologic criteria have not changed much in recent years. The field is being shaped predominantly by new techniques for imaging and for acquiring and processing samples, advances in molecular diagnosis and therapeutics, and regulatory issues.
OBJECTIVE: To review the importance of classical morphology in the future of cytopathology, to identify areas in which cytology is expanding or contracting in its scope, and to identify factors that are shaping the field.
DATA SOURCES: Literature review.
CONCLUSIONS: Five stories paint a picture in which classical cytomorphology will continue to have essential importance, both for diagnosis and for improving our understanding of cancer biology. New endoscopy and imaging techniques are replacing surgical biopsies with cytology samples. New molecularly targeted therapies offer a chance for cytology to play a major role, but they pose new challenges. New molecular tests have the potential to synergize with, but not replace, morphologic interpretation of thyroid fine-needle aspirations. Ultrasound-guided fine-needle aspiration performed by cytopathologists is opening a new field of "interventional cytopathology" with unique value. For the productive evolution of the field, it will be important for cytopathologists to play an active role in clinical trials that document the ability of cytology to achieve cost-effective health care outcomes.
Adaptive beta-cell proliferation increases early in high-fat feeding in mice, concurrent with metabolic changes, with induction of islet cyclin D2 expression
Type 2 diabetes (T2D) is caused by relative insulin deficiency, due in part to reduced beta-cell mass (11, 62). Therapies aimed at expanding beta-cell mass may be useful to treat T2D (14). Although feeding rodents a high-fat diet (HFD) for an extended period (3-6 mo) increases beta-cell mass by inducing beta-cell proliferation (16, 20, 53, 54), evidence suggests that adult human beta-cells may not meaningfully proliferate in response to obesity. The timing and identity of the earliest initiators of the rodent compensatory growth response, possible therapeutic targets to drive proliferation in refractory human beta-cells, are not known. To develop a model to identify early drivers of beta-cell proliferation, we studied mice during the first week of HFD exposure, determining the onset of proliferation in the context of diet-related physiological changes. Within the first week of HFD, mice consumed more kilocalories, gained weight and fat mass, and developed hyperglycemia, hyperinsulinemia, and glucose intolerance due to impaired insulin secretion. The beta-cell proliferative response also began within the first week of HFD feeding. Intriguingly, beta-cell proliferation increased before insulin resistance was detected. Cyclin D2 protein expression was increased in islets by day 7, suggesting it may be an early effector driving compensatory beta-cell proliferation in mice. This study defines the time frame and physiology to identify novel upstream regulatory signals driving mouse beta-cell mass expansion, in order to explore their efficacy, or reasons for inefficacy, in initiating human beta-cell proliferation.
Ablation of the X-linked retinitis pigmentosa 2 (Rp2) gene in mice results in opsin mislocalization and photoreceptor degeneration
PURPOSE: Mutations in the RP2 gene are associated with 10% to 15% of X-linked retinitis pigmentosa (XLRP), a debilitating disorder characterized by the degeneration of retinal rod and cone photoreceptors. The molecular mechanism of pathogenesis of photoreceptor degeneration in XLRP-RP2 has not been elucidated, and no treatment is currently available. This study was undertaken to investigate the pathogenesis of RP2-associated retinal degeneration.
METHODS: We introduced loxP sites that flank exon 2, a mutational hotspot in XLRP-RP2, in the mouse Rp2 gene. We then produced Rp2-null allele using transgenic mice that expressed Cre-recombinase under control of the ubiquitous CAG promoter. Electroretinography (ERG), histology, light microscopy, transmission electron microscopy, and immunofluorescence microscopy were performed to ascertain the effect of ablation of Rp2 on photoreceptor development, function, and protein trafficking.
RESULTS: Although no gross abnormalities were detected in the Rp2(null) mice, photopic (cone) and scotopic (rod) function as measured by ERG showed a gradual decline starting as early as 1 month of age. We also detected slow progressive degeneration of the photoreceptor membrane discs in the mutant retina. These defects were associated with mislocalization of cone opsins to the nuclear and synaptic layers and reduced rhodopsin content in the outer segment of mutant retina prior to the onset of photoreceptor degeneration.
CONCLUSIONS: Our studies suggest that RP2 contributes to the maintenance of photoreceptor function and that cone opsin mislocalization represents an early step in XLRP caused by RP2 mutations. The Rp2(null) mice should serve as a useful preclinical model for testing gene- and cell-based therapies.
Infections with DNA tumor viruses, including members of the polyomavirus family, often result in tumor formation in immune-deficient hosts. The complex control involved in antiviral and antitumor immune responses during these infections can be studied in murine polyomavirus (PyV)-infected mice as a model. We found that NK cells efficiently kill cells derived from PyV-induced salivary gland tumors in vitro in an NKG2D (effector cell)-RAE-1 (target cell)-dependent manner; but in T cell-deficient mice, NK cells only delay but do not prevent the development of PyV-induced tumors. In this article, we show that the PyV-induced tumors have infiltrating functional NK cells. The freshly removed tumors, however, lack surface RAE-1 expression, and the tumor tissues produce soluble factors that downregulate RAE-1. These factors include the proinflammatory cytokines IL-1alpha, IL-1beta, IL-33, and TNF. Each of these cytokines downregulates RAE-1 expression and susceptibility to NK cell-mediated cytotoxicity. CD11b(+)F4/80(+) macrophages infiltrating the PyV-induced tumors produce high amounts of IL-1beta and TNF. Thus, our data suggest a new mechanism whereby inflammatory cytokines generated in the tumor environment lead to evasion of NK cell-mediated control of virus-induced tumors.
Conserved chromosome 2q31 conformations are associated with transcriptional regulation of GAD1 GABA synthesis enzyme and altered in prefrontal cortex of subjects with schizophrenia
Little is known about chromosomal loopings involving proximal promoter and distal enhancer elements regulating GABAergic gene expression, including changes in schizophrenia and other psychiatric conditions linked to altered inhibition. Here, we map in human chromosome 2q31 the 3D configuration of 200 kb of linear sequence encompassing the GAD1 GABA synthesis enzyme gene locus, and we describe a loop formation involving the GAD1 transcription start site and intergenic noncoding DNA elements facilitating reporter gene expression. The GAD1-TSS(-50kbLoop) was enriched with nucleosomes epigenetically decorated with the transcriptional mark, histone H3 trimethylated at lysine 4, and was weak or absent in skin fibroblasts and pluripotent stem cells compared with neuronal cultures differentiated from them. In the prefrontal cortex of subjects with schizophrenia, GAD1-TSS(-50kbLoop) was decreased compared with controls, in conjunction with downregulated GAD1 expression. We generated transgenic mice expressing Gad2 promoter-driven green fluorescent protein-conjugated histone H2B and confirmed that Gad1-TSS(-55kbLoop), the murine homolog to GAD1-TSS(-50kbLoop), is a chromosomal conformation specific for GABAergic neurons. In primary neuronal culture, Gad1-TSS(-55kbLoop) and Gad1 expression became upregulated when neuronal activity was increased. We conclude that 3D genome architectures, including chromosomal loopings for promoter-enhancer interactions involved in the regulation of GABAergic gene expression, are conserved between the rodent and primate brain, and subject to developmental and activity-dependent regulation, and disordered in some cases with schizophrenia. More broadly, the findings presented here draw a connection between noncoding DNA, spatial genome architecture, and neuronal plasticity in development and disease.
Translation of the coding potential of a messenger RNA into a protein molecule is a fundamental process in all living cells and consumes a large fraction of metabolites and energy resources in growing cells. Moreover, translation has emerged as an important control point in the regulation of gene expression. At the level of gene regulation, translational control is utilized to support the specific life histories of plants, in particular their responses to the abiotic environment and to metabolites. This review summarizes the diversity of translational control mechanisms in the plant cytoplasm, focusing on specific cases where mechanisms of translational control have evolved to complement or eclipse other levels of gene regulation. We begin by introducing essential features of the translation apparatus. We summarize early evidence for translational control from the pre-Arabidopsis era. Next, we review evidence for translation control in response to stress, to metabolites, and in development. The following section emphasizes RNA sequence elements and biochemical processes that regulate translation. We close with a chapter on the role of signaling pathways that impinge on translation.
OBJECTIVES: Pancreaticoduodenal trauma (PDT) is associated with substantial mortality and morbidity. In this study, contemporary trends were analysed using national data.
METHODS: The Nationwide Inpatient Sample for 1998-2009 was queried for patients with PDT. Interventions including any operation (Any-Op) and pancreas-specific surgery (PSURG) were identified. Trends in treatment and outcomes were determined [complications, length of stay (LoS), mortality] for the Any-Op, PSURG and non-operative (Non-Op) groups. Analyses included chi-squared tests, Cochran-Armitage trend tests and logistic regression.
RESULTS: A total of 27 216 patients (nationally weighted) with PDT were identified. Over time, the frequency of PDT increased by 8.3%, whereas the proportion of patients submitted to PSURG declined (from 21.7% to 19.8%; P = 0.0004) and the percentage of patients submitted to non-operative management increased (from 56.7% to 59.1%; P = 0.01). In the Non-Op group, mortality decreased from 9.7% to 8.6% (P < 0.001); morbidity and LoS remained unchanged at approximately 40% and approximately 12 days, respectively. In the PSURG group, mortality remained stable at approximately 15%, complications increased from 50.2% to 71.8% (P < 0.0001) and LoS remained stable at approximately 21 days. For all PDT patients, significant independent predictors of mortality included: the presence of combined pancreatic and duodenal injuries; penetrating trauma, and age over 50 years. Having any operation (Any-Op) was associated with mortality, but PSURG was not a predictor of death.
CONCLUSIONS: The utilization of operations for PDT has declined without affecting mortality, but operative morbidity increased significantly over the 12 years to 2009. The development of an evidence-based approach to invasive manoeuvres and an early multidisciplinary approach involving pancreatic surgeons may improve outcomes in patients with these morbid injuries.
Survival after hospital discharge for ST-segment elevation and non-ST-segment elevation acute myocardial infarction: a population-based study
BACKGROUND: Limited recent data are available describing differences in long-term survival, and factors affecting prognosis, after ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), especially from the more generalizable perspective of a population-based investigation. The objectives of this study were to examine differences in post-discharge prognosis after hospitalization for STEMI and NSTEMI, with a particular focus on factors associated with reduced long-term survival.
METHODS: We reviewed the medical records of residents of the Worcester, MA, USA metropolitan area hospitalized at eleven central Massachusetts medical centers for acute myocardial infarction (AMI) during 2001, 2003, 2005, and 2007.
RESULTS: A total of 3762 persons were hospitalized with confirmed AMI; of these, 2539 patients (67.5%) were diagnosed with NSTEMI. The average age of study patients was 70.3 years and 42.9% were women. Patients with NSTEMI experienced higher post-discharge death rates with 3-month, 1-year, and 2-year death rates of 12.6%, 23.5%, and 33.2%, respectively, compared to 6.1%, 11.5%, and 16.4% for patients with STEMI. After multivariable adjustment, patients with NSTEMI were significantly more likely to have died after hospital discharge (adjusted hazards ratio 1.28; 95% confidence interval 1.14-1.44). Several demographic (eg, older age) and clinical (eg, history of stroke) factors were associated with reduced long-term survival in patients with NSTEMI and STEMI.
CONCLUSIONS: The results of this study in residents of central Massachusetts suggest that patients with NSTEMI are at higher risk for dying after hospital discharge, and several subgroups are at particularly increased risk.
The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-A crystal structure of BlaS bound to a 70StRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3' terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation.
Successful treatment of mobile right atrial thrombus and acute pulmonary embolism with intravenous tissue plasminogen activator
An 89-year-old woman came with symptoms of progressively worsening dyspnoea at rest over the preceding week. She was normotensive, had elevated jugular venous pressure and clear lungs. ECG revealed atrial fibrillation with the rapid ventricular rate. Labs were significant for markedly elevated pro-brain natriuretic peptide of 43,000 pg/mL and troponin-T of 1 ng/mL. An urgent 2D echocardiogram was obtained, which revealed the severely dilated right atrium and a large linear mobile mass in the right atrium consistent with a thrombus. An emergent CT scan revealed multiple bilateral pulmonary emboli. She received intravenous tissue plasminogen activator. Repeat echocardiogram performed 6 h later showed no evidence of the right atrial thrombus. She was subsequently maintained on intravenous heparin and transitioned to Coumadin. Early recognition of this rare but potentially fatal complication is important as prompt treatment measures can help in preventing life-threatening complications of the right atrial thrombus.
Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA-binding protein IMP1 facilitates its trafficking into motor neuron axons
Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the beta-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the beta-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1.
Children with head injuries frequently present to emergency departments. Even though most of these children have minor injuries, head injury is the most common cause of traumatic deaths in pediatric patients. The pediatric GCS and decision rules for obtaining head CT imaging help the provider evaluate head-injured infants and children. The provider must be vigilant to diagnose those who have life-threatening intracranial injuries or are victims of abusive head trauma. The goal of the emergency physician is to diagnose and treat the consequences of the primary injury and to limit or prevent secondary injury.
The Legionella pneumophila GTPase activating protein LepB accelerates Rab1 deactivation by a non-canonical hydrolytic mechanism
GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.
Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation
The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
Self Advocacy is the ability to speak up for yourself and for the things that are important to you. This tip sheet offers advice and gives examples of how to self advocate at home, at school, at work and in the community.
Initiating Data Management Instruction to Graduate Students at the University of Houston Using the New England Collaborative Data Management Curriculum
The need for graduate instruction on data management best practices across disciplines is a theme that has emerged from two campus-wide data management needs assessments that have been conducted at the UH Libraries since 2010. Graduate students are assigned numerous data management responsibilities over the course of their academic careers, but rarely receive formal training in this area. To address this need, the UH Libraries offered a workshop entitled Research Data Management 101 in April, 2014, and all graduate and professional students on campus were invited to attend. The New England Collaborative Data Management Curriculum (NECDMC) served as the basis for the workshop, and two general sessions were planned. A research group in the College of Natural Sciences & Mathematics requested a special session after advertisements for the workshop were distributed. One hundred and five individuals registered for the event, sixty-five signed into the workshop, and sixty-three completed the end-of-workshop assessment. The results from this assessment, general lessons learned, and plans for future sessions will be discussed.