Feasibility and impact of a physical activity and lifestyle program for Aboriginal families with Machado-Joseph disease in the Top End of Australia.

INTRODUCTION: Physical activity and lifestyle programs are scarce for people with hereditary ataxias and neurodegenerative diseases. Aboriginal families in the Top End of Australia who have lived with Machado-Joseph disease (MJD) for generations co-designed a physical activity and lifestyle program called the Staying Strong Toolbox. The aim of the present study was to explore feasibility and impact of the program on walking and moving around. METHODS: A mixed-methods, multiple case study design was used to pilot the Staying Strong Toolbox. Eight individuals with MJD participated in the program for 4 weeks. Participants tailored their own program using the Toolbox workbook. Families, support workers and researchers facilitated each individual's program. Feasibility was determined through program participation, adherence, coinciding or serious adverse events, participant acceptability and cost. Impact was determined through measures of mobility, ataxia, steps, quality of life, wellbeing and goal attainment, assessed before and after the program. RESULTS: All participants completed the program, averaging five activity sessions per week, 66 minutes per session, of walking (63.5%), strengthening/balance-based activities (16%), cycling (11.4%) and activities of daily living, cultural and lifestyle activities (10.5%). Seven participants were assessed on all measures on three occasions (baseline, pre-program and post-program), while one participant could not complete post-program measures due to ceremonial responsibilities. All had significant improvements in mobility, steps taken and ataxia severity (p<0.05) after the program. Quality of life and wellbeing were maintained. CONCLUSION: The program helped participants remain 'strong on the inside and outside'. Participants recommended implementation in 4-week blocks and for the program to be shared internationally. The Staying Strong Toolbox program was feasible for families with MJD. The program had a positive impact on walking and moving around, with participants feeling stronger on the outside (physically) and inside (emotionally, spiritually, psychosocially). The program could be adapted for use by other families with MJD.

Correction: Common-variant associations with fragile X syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice.

BACKGROUND: Prolonged exposure to high oxygen concentrations in premature infants, although lifesaving, can induce lung oxidative stress and increase the risk of developing BPD, a form of chronic lung disease. The lung alveolar epithelium is damaged by sustained hyperoxia, causing oxidative stress and alveolar simplification; however, it is unclear what duration of exposure to hyperoxia negatively impacts cellular function. METHODS: Here we investigated the role of a very short exposure to hyperoxia (95% O2, 5% CO2) on mitochondrial function in cultured mouse lung epithelial cells and neonatal mice. RESULTS: In epithelial cells, 4 h of hyperoxia reduced oxidative phosphorylation, respiratory complex I and IV activity, utilization of mitochondrial metabolites, and caused mitochondria to form elongated tubular networks. Cells allowed to recover in air for 24 h exhibited a persistent global reduction in fuel utilization. In addition, neonatal mice exposed to hyperoxia for only 12 h demonstrated alveolar simplification at postnatal day 14. CONCLUSION: A short exposure to hyperoxia leads to changes in lung cell mitochondrial metabolism and dynamics and has a long-term impact on alveolarization. These findings may help inform our understanding and treatment of chronic lung disease. IMPACT: Many studies use long exposures (up to 14 days) to hyperoxia to mimic neonatal chronic lung disease. We show that even a very short exposure to hyperoxia leads to long-term cellular injury in type II-like epithelial cells. This study demonstrates that a short (4 h) period of hyperoxia has long-term residual effects on cellular metabolism. We show that neonatal mice exposed to hyperoxia for a short time (12 h) demonstrate later alveolar simplification. This work suggests that any exposure to clinical hyperoxia leads to persistent lung dysfunction.

Endothelial to mesenchymal transition during neonatal hyperoxia-induced pulmonary hypertension.

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from mechanical ventilation and hyperoxia, amongst other factors. Although most BPD survivors can be weaned from supplemental oxygen, many show evidence of cardiovascular sequelae in adulthood, including pulmonary hypertension and pulmonary vascular remodeling. Endothelial-mesenchymal transition (EndoMT) plays an important role in mediating vascular remodeling in idiopathic pulmonary arterial hypertension. Whether hyperoxic exposure, a known mediator of BPD in rodent models, causes EndoMT resulting in vascular remodeling and pulmonary hypertension remains unclear. We hypothesized that neonatal hyperoxic exposure causes EndoMT, leading to the development of pulmonary hypertension in adulthood. To test this hypothesis, newborn mice were exposed to hyperoxia and then allowed to recover in room air until adulthood. Neonatal hyperoxic exposure gradually caused pulmonary vascular and right ventricle remodeling as well as pulmonary hypertension. Male mice were more susceptible to developing pulmonary hypertension compared to female mice, when exposed to hyperoxia as newborns. Hyperoxic exposure induced EndoMT in mouse lungs as well as in cultured lung microvascular endothelial cells (LMVECs) isolated from neonatal mice and human fetal donors. This was augmented in cultured LMVECs from male donors compared to those from female donors. Using primary mouse LMVECs, hyperoxic exposure increased phosphorylation of both Smad2 and Smad3, but reduced Smad7 protein levels. Treatment with a selective TGF-ß inhibitor SB431542 blocked hyperoxia-induced EndoMT in vitro. Altogether, we show that neonatal hyperoxic exposure caused vascular remodeling and pulmonary hypertension in adulthood. This was associated with increased EndoMT. These novel observations provide mechanisms underlying hyperoxia-induced vascular remodeling and potential approaches to prevent BPD-associated pulmonary hypertension by targeting EndoMT. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Evaluation of the Allied Health Rural Generalist Program 2017-2019.

OBJECTIVE: To evaluate the development and implementation of the Allied Health Rural Generalist Program, a two-level online post-graduate education program, which includes Level 1, an entry-level non-award pathway program, and Level 2, a Graduate Diploma in Rural Generalist Practice. DESIGN: A convergent mixed methodology evaluation in two overlapping stages: a process evaluation on quality and reach, together with a mixed method case study evaluation on benefits, of the program. SETTING: Rural and remote Australia across ten sites and seven allied health professions: dietetics; occupational therapy; pharmacy; physiotherapy; podiatry; radiography; speech pathology. PARTICIPANTS: Process evaluation included 91 participants enrolled in all or part of the Rural Generalist Program. Case study evaluation included 50 managers, supervisors and Rural Generalist Program participants from the ten study sites. INTERVENTIONS: The Allied Health Rural Generalist Program. MAIN OUTCOME MEASURES: Process evaluation data were derived from enrolment data and education evaluation online surveys. Case study data were gathered via online surveys and semi-structured interviews. Quantitative and qualitative data were collected concurrently, analysed separately and then integrated to identify consistency, expansion or discordance across the data. RESULTS: The Rural Generalist Program was viewed as an effective education program that provided benefits for Rural Generalist Program participants, employing organisations and consumers. Key improvements recommended included increasing profession-specific and context-specific content, ensuring Rural Generalist Program alignment with clinical and project requirements, strengthening support mechanisms within employing organisations and ensuring benefits can be sustained in the long term. CONCLUSION: The Rural Generalist Program offers a promising strategy for building a fit-for-purpose rural and remote allied health workforce.

Common-variant associations with fragile X syndrome.

Fragile X syndrome is rare but a prominent cause of intellectual disability. It is usually caused by a de novo mutation that occurs on multiple haplotypes and thus would not be expected to be detectible using genome-wide association (GWA). We conducted GWA in 89 male FXS cases and 266 male controls, and detected multiple genome-wide significant signals near FMR1 (odds ratio = 8.10, P = 2.5 × 10-10). These findings withstood robust attempts at falsification. Fine-mapping yielded a minimum P = 1.13 × 10-14, but did not narrow the interval. Comprehensive functional genomic integration did not provide a mechanistic hypothesis. Controls carrying a risk haplotype had significantly longer FMR1 CGG repeats than controls with the protective haplotype (P = 4.75 × 10-5), which may predispose toward increases in CGG number to the premutation range over many generations. This is a salutary reminder of the complexity of even "simple" monogenetic disorders.

Heme Oxygenase-1 Supports Mitochondrial Energy Production and Electron Transport Chain Activity in Cultured Lung Epithelial Cells.

Heme oxygenase-1 is induced by many cellular stressors and catalyzes the breakdown of heme to generate carbon monoxide and bilirubin, which confer cytoprotection. The role of HO-1 likely extends beyond the simple production of antioxidants, for example HO-1 activity has also been implicated in metabolism, but this function remains unclear. Here we used an HO-1 knockout lung cell line to further define the contribution of HO-1 to cellular metabolism. We found that knockout cells exhibit reduced growth and mitochondrial respiration, measured by oxygen consumption rate. Specifically, we found that HO-1 contributed to electron transport chain activity and utilization of certain mitochondrial fuels. Loss of HO-1 had no effect on intracellular non-heme iron concentration or on proteins whose levels and activities depend on available iron. We show that HO-1 supports essential functions of mitochondria, which highlights the protective effects of HO-1 in diverse pathologies and tissue types. Our results suggest that regulation of heme may be an equally significant role of HO-1.

Comparative hindlimb myology of foot-propelled swimming birds.

Several groups of birds have convergently evolved the ability to swim using their feet despite facing trade-offs with walking. However, swimming relative to terrestrial performance varies across these groups. Highly specialized divers, such as loons and grebes, excel at swimming underwater but struggle to stand on land, whereas species that primarily swim on the water surface, such as Mallards, retain the ability to move terrestrially. The identification of skeletal features associated with a swimming style and conserved across independent groups suggests that the hindlimb of foot-propelled swimming birds has adapted to suit the physical challenges of producing propulsive forces underwater. But in addition to skeletal features, how do hindlimb muscles reflect swimming ability and mode? This paper presents the first comparative myology analysis associated with foot-based swimming. Our detailed dissections of 35 specimens representing eight species reveal trends in hindlimb muscle size and attachment location across four independent lineages of extant swimming birds. We expand upon our dissections by compiling data from historical texts and provide a key to any outdated muscle nomenclature used in these sources. Our results show that highly diving birds tuck the femur and proximal tibiotarsus next to the ribcage and under the skin covering the abdomen, streamlining the body. Several hindlimb muscles exhibit dramatic anatomical variation in diving birds, including the flexor cruris lateralis (FCL) and iliofibularis (IF), which reduce in size and shift distally along the tibiotarsus. The femorotibialis medius (FTM) extends along an expanded cnemial crest. The resulting increased moment arms of these muscles likely help stabilize the hip and knee while paddling. Additionally, distal ankle plantarflexors, including the gastrocnemius and digital flexors, are exceptionally large in diving birds in order to power foot propulsion. These patterns exist within distantly related lineages of diving birds and, to a lesser extent, in surface swimmers. Together, our findings verify conserved muscular adaptations to a foot-propelled swimming lifestyle. The association of muscle anatomy with skeletal features and biomechanical movement demands can inform functional interpretation of fossil birds and reveal selective pressures underlying avian diversification.

THE ROLE OF MITOCHONDRIAL FATTY ACID USE IN NEONATAL LUNG INJURY AND REPAIR.

In premature neonates, hyperoxic exposure contributes to lung injury characterized by simplified alveolarization and arrested vascularization. These are the hallmarks of bronchopulmonary dysplasia, a disease with long-term consequences on pulmonary and neurodevelopmental function. Lung vascular development and endothelial cell signals are synergistically important for normal alveolarization. It has been shown that metabolism of nutrients such as glucose, fatty acid, and glutamine is key in controlling proliferation, differentiation, apoptosis, autophagy, senescence, and inflammatory responses, which contribute to the pathogenesis of chronic lung diseases, including bronchopulmonary dysplasia. Recent studies show that metabolic reprogramming occurs in vitro in cells and in vivo in animal models and more importantly in patients with bronchopulmonary dysplasia, suggesting that metabolic dysregulation may participate in the pathogenesis and progression of these diseases. Although endothelial cells rely mainly on glycolysis for bioenergetics, they have the metabolic flexibility to maintain cell function under stress or nutrient deprivation. Others have shown that hyperoxia decreases glycolysis and oxidative phosphorylation in epithelial cells. Nevertheless, endothelial cells show enhanced mitochondrial fatty acid use after exposure to hyperoxia. This may serve to preserve endothelial cell proliferation and alveolarization, and thereby mitigate neonatal hyperoxic lung injury.

Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus.

UNLABELLED: The bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacterium Thermus thermophilus to examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals. IMPORTANCE: The thermophilic bacterium Thermus thermophilus has become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies of T. thermophilus ribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA.

Engineering the genome of Thermus thermophilus using a counterselectable marker.

UNLABELLED: Thermus thermophilus is an extremely thermophilic bacterium that is widely used as a model thermophile, in large part due to its amenability to genetic manipulation. Here we describe a system for the introduction of genomic point mutations or deletions using a counterselectable marker consisting of a conditionally lethal mutant allele of pheS encoding the phenylalanyl-tRNA synthetase α-subunit. Mutant PheS with an A294G amino acid substitution renders cells sensitive to the phenylalanine analog p-chlorophenylalanine. Insertion of the mutant pheS allele via a linked kanamycin resistance gene into a chromosomal locus provides a gene replacement intermediate that can be removed by homologous recombination using p-chlorophenylalanine as a counterselective agent. This selection is suitable for the sequential introduction of multiple mutations to produce a final strain unmarked by an antibiotic resistance gene. We demonstrated the utility of this method by constructing strains bearing either a point mutation in or a precise deletion of the rrsB gene encoding 16S rRNA. We also used this selection to identify spontaneous, large-scale deletions in the pTT27 megaplasmid, apparently mediated by either of the T. thermophilus insertion elements ISTth7 and ISTth8. One such deletion removed 121 kb, including 118 genes, or over half of pTT27, including multiple sugar hydrolase genes, and facilitated the development of a plasmid-encoded reporter system based on ß-galactosidase. The ability to introduce mutations ranging from single base substitutions to large-scale deletions provides a potentially powerful tool for engineering the genome of T. thermophilus and possibly other thermophiles as well. IMPORTANCE: Thermus thermophilus is an extreme thermophile that has played an important part in the development of both biotechnology and basic biological research. Its suitability as a genetic model system is established by its natural competence for transformation, but the scarcity of genetic tools limits the kinds of manipulations that can currently be performed. We have developed a counterselectable marker that allows the introduction of unmarked deletions and point mutations into the T. thermophilus genome. We find that this marker can also be used to select large chromosomal deletions apparently resulting from aberrant transposition of endogenous insertion sequences. This system has the potential to advance the genetic manipulation of this important model organism.

The central role of protein S12 in organizing the structure of the decoding site of the ribosome.

The ribosome decodes mRNA by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.

Receptor Tyrosine Kinase Expression Predicts Response to Sunitinib in Breast Cancer.

BACKGROUND: Preliminary data indicate that tyrosine kinase inhibitors (TKIs) function through rearranged during transfection (RET) in breast cancer. However, TKIs are not specific and can block several receptor tyrosine kinases (RTKs). This study used cell lines and primary breast cancer specimens to determine factors associated with TKI response. METHODS: Proliferation was assessed after short interfering RNA knockdown with or without sunitinib in breast cancer cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Breast cancer tissue and matched normal breast was obtained from 30 women with invasive breast carcinoma. Gene expression was assessed by reverse transcriptase-polymerase chain reaction. Fresh tissue was treated in vitro with sunitinib or control media for 30 min, and response was assessed by phosphorylation-specific western blot. RESULTS: The RTKs including epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR1-3), platelet-derived growth factor receptor (PDGFRa/b), and Kit were overexpressed in triple-negative breast tumors relative to HER2- and estrogen receptor-alpha (ERα)-positive tumors and normal breast tissue. Knockdown of EGFR reduced in vitro proliferation in MCF-7 and MDA-MB-231 but not in SKBR-3 or ZR-75-1 breast cancer cells. With the exception of RET, response to sunitinib was independent of RTK expression in all four cell lines. Both ERα-positive and low-EGFR-expressing tumors had an increased in vitro sunitinib response, as determined by alteration of Erk activation. Expression of other RTKs and additional clinical factors were not associated with response. CONCLUSION: Triple-negative breast cancers overexpress RTKs but have decreased in vitro response to the TKI sunitinib. In addition to RET, TKIs that block EGFR may increase the therapeutic efficacy of TKIs in breast cancer.

Transposon mutagenesis of the extremely thermophilic bacterium Thermus thermophilus HB27.

Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however, the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms.

The "Octopus" Lymphaticovenular Anastomosis: Evolving Beyond the Standard Supermicrosurgical Technique.

BACKGROUND: Supermicrosurgical lymphaticovenular anastomosis (LVA) is a promising treatment modality for lymphedema. However, its practice is restricted by the surgeon/equipment-related factors, and its effectiveness limited by technical constraints. We conducted a pilot study to evaluate the feasibility of a modified "octopus" LVA technique in addressing the above problems. METHOD: Nine consecutive lymphedema patients underwent LVA procedure using the "octopus" technique. Six had the upper extremity disease; three had the lower extremity disease. Except for one patient having primary lower extremity lymphedema, all had secondary disease related to cancer treatment. Disease severity ranged from Campisi stage Ib to IV. Qualitative and quantitative assessments were performed preoperatively, at 1, 3, and 6 months. RESULTS: A total of 130 lymphaticovenular drainage pathways were created in 39 "octopus" LVAs. All patients experienced prompt relief of lymphedema symptoms during the 1st postoperative week and continued to improve during the study period. None had postoperative complications. All had disease regression as demonstrated by statistically significant decrease in limb measurements (p = 0.0003) and severity down-staging. The modified technique was found to be easier than the standard supermicrosurgical technique and could be performed using a standard surgical microscope. CONCLUSION: The "octopus" technique is a viable, effective technical alternative to the standard LVA technique. It may greatly simplify this technically challenging procedure.

Phenotypic interactions among mutations in a Thermus thermophilus 16S rRNA gene detected with genetic selections and experimental evolution.

During protein synthesis, the ribosome undergoes conformational transitions between functional states, requiring communication between distant structural elements of the ribosome. Despite advances in ribosome structural biology, identifying the protein and rRNA residues governing these transitions remains a significant challenge. Such residues can potentially be identified genetically, given the predicted deleterious effects of mutations stabilizing the ribosome in discrete conformations and the expected ameliorating effects of second-site compensatory mutations. In this study, we employed genetic selections and experimental evolution to identify interacting mutations in the ribosome of the thermophilic bacterium Thermus thermophilus. By direct genetic selections, we identified mutations in 16S rRNA conferring a streptomycin dependence phenotype and from these derived second-site suppressor mutations relieving dependence. Using experimental evolution of streptomycin-independent pseudorevertants, we identified additional compensating mutations. Similar mutations could be evolved from slow-growing streptomycin-resistant mutants. While some mutations arose close to the site of the original mutation in the three-dimensional structure of the 30S ribosomal subunit and probably act directly by compensating for local structural distortions, the locations of others are consistent with long-range communication between specific structural elements within the ribosome.

AT-101 downregulates BCL2 and MCL1 and potentiates the cytotoxic effects of lenalidomide and dexamethasone in preclinical models of multiple myeloma and Waldenström macroglobulinaemia.

Multiple myeloma, the second most common haematological malignancy in the U.S., is currently incurable. Disruption of the intrinsic apoptotic pathway by BCL2 and MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Thus, we investigated the anti-tumour effects of a pan-BCL2 inhibitor, AT-101, which has high binding specificity for BCL2 and MCL1 in preclinical models of plasma cell cancers (Multiple myeloma and Waldenström macroglobulinaemia). Gene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of BCL2 family members. AT-101 was able to downregulate BCL2 and MCL1 in all plasma cell cancer models and induced apoptotic cell death in a caspase-dependent manner by altering mitochondrial membrane permeability. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). NanoString nCounter mRNA quantification and Ingenuity Pathways Analysis revealed differential changes in the CCNA2, FRZB, FYN, IRF1, PTPN11 genes in LDA-treated cells. In summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy.

GIPR expression in gastric and duodenal neuroendocrine tumors.

BACKGROUND: Compounds targeting somatostatin-receptor-type-2 (SSTR2) are useful for small bowel neuroendocrine tumor (SBNET) and pancreatic neuroendocrine tumor (PNET) imaging and treatment. We recently characterized expression of 13 cell surface receptor genes in SBNETs and PNETs, identifying three drug targets (GIPR, OXTR, and OPRK1). This study set out to characterize expression of this gene panel in the less common neuroendocrine tumors of the stomach and duodenum (gastric and duodenal neuroendocrine tumors [GDNETs]). METHODS: Primary tumors and adjacent normal tissue were collected at surgery, RNA was extracted, and expression of 13 target genes was determined by quantitative polymerase chain reaction. Expression was normalized to GAPDH and POLR2A internal control genes. Expression relative to normal tissue (ddCT) and absolute expression (dCT) were calculated. Wilcoxon tests compared median expression with false discovery rate correction for multiple comparisons. RESULTS: Gene expression was similar in two gastric and seven duodenal tumors, and these were analyzed together. Like SBNETs (n = 63) and PNETs (n = 51), GDNETs showed significant overexpression compared with normal tissue of BRS3, GIPR, GRM1, GPR113, OPRK1, and SSTR2 (P < 0.05 for all). Of these, SSTR2 had the highest absolute expression in GDNETs (median dCT 4.0). Absolute expression of BRS3, GRM1, GPR113, and OPRK1 was significantly lower than SSTR2 in GDNETs (P < 0.05 for all), whereas expression of GIPR was similar to SSTR2 (median 4.3, P = 0.4). CONCLUSIONS: As in SBNETs and PNETs, GIPR shows absolute expression close to SSTR2 but has greater overexpression relative to normal tissue (21.1 versus 3.5-fold overexpression). We conclude that GIPR could provide an improved signal-to-noise ratio for imaging versus SSTR2 and represents a promising novel therapeutic target in GDNETs.

Urethral prolapse in dogs: a retrospective study.

OBJECTIVE: To evaluate the signalment, clinical signs, treatment, and outcome of dogs with urethral prolapse and identify risk factors associated with prolapse or treatment. STUDY DESIGN: Retrospective case series. ANIMALS: Dogs (n = 48) with urethral prolapse. METHODS: Medical records (May 1995-June 2010) from 2 referral centers were reviewed. Retrieved data included signalment, clinical signs, laboratory findings, treatment, complications, results of long-term follow-up. Records from Veterinary Medical Data Base (VMDB) were evaluated to determine odds ratios. RESULTS: Odds ratio for urethral prolapse in English bulldogs compared to all breeds was 366.99 (95% CI: 265.83, 506.65). Of 48 affected dogs, 46 had either resection and anastomosis (43 dogs) or urethropexy (3 dogs). The most common early postoperative complication was hemorrhage (39%); postoperative hemorrhage was less common when a simple continuous pattern was used for resection and anastomosis. Prolapse recurred in 57% of dogs available for long-term follow-up; recurrence was less common in dogs that were administered postoperative butorphanol or acepromazine. Gender was not associated with urethral prolapse or postoperative complications. CONCLUSIONS: Urethral prolapse occurs most commonly in English bulldogs. Postoperative hemorrhage and prolapse recurrence may be reduced with use of a simple continuous pattern for urethral anastomosis and by administration of postoperative sedation, respectively. Castration status did not appear to affect prolapse development or outcome.

In vivo T cell costimulation blockade with abatacept for acute graft-versus-host disease prevention: a first-in-disease trial.

We performed a first-in-disease trial of in vivo CD28:CD80/86 costimulation blockade with abatacept for acute graft-versus-host disease (aGVHD) prevention during unrelated-donor hematopoietic cell transplantation (HCT). All patients received cyclosporine/methotrexate plus 4 doses of abatacept (10 mg/kg/dose) on days -1, +5, +14, +28 post-HCT. The feasibility of adding abatacept, its pharmacokinetics, pharmacodynamics, and its impact on aGVHD, infection, relapse, and transplantation-related mortality (TRM) were assessed. All patients received the planned abatacept doses, and no infusion reactions were noted. Compared with a cohort of patients not receiving abatacept (the StdRx cohort), patients enrolled in the study (the ABA cohort) demonstrated significant inhibition of early CD4(+) T cell proliferation and activation, affecting predominantly the effector memory (Tem) subpopulation, with 7- and 10-fold fewer proliferating and activated CD4(+) Tem cells, respectively, at day+28 in the ABA cohort compared with the StdRx cohort (P < .01). The ABA patients demonstrated a low rate of aGVHD, despite robust immune reconstitution, with 2 of 10 patients diagnosed with grade II-IV aGVHD before day +100, no deaths from infection, no day +100 TRM, and with 7 of 10 evaluable patients surviving (median follow-up, 16 months). These results suggest that costimulation blockade with abatacept can significantly affect CD4(+) T cell proliferation and activation post-transplantation, and may be an important adjunct to standard immunoprophylaxis for aGVHD in patients undergoing unrelated-donor HCT.