Systemic artery to pulmonary artery aneurysm malformations associated with variants at MCF2L.

Arteriovenous malformations (AVM) are characterized by abnormal vessels connecting arteries and veins resulting in a disruption of normal blood flow. Hereditary hemorrhagic telangiectasia (HHT) is the most common cause of pulmonary AVM characterized by a right to left shunt. Here we describe a distinct malformation where the flow of blood was from a systemic artery to the pulmonary artery (PA) resulting in a left to right shunt instead of the right to left shunt seen in individuals with HHT. This distinct malformation was identified in seven probands, one from a multiplex family containing 10 affected individuals from five generations. To identify the molecular basis of this distinct malformation, we performed exome sequencing (ES) on the seven probands and the affected paternal female cousin from the multiplex family. PhenoDB was used to prioritize candidate causative variants along with burden analysis. We describe the clinical and radiological details of the new systemic artery to PA malformation with or without pulmonary artery aneurysm (SA-PA(A)) and recommend distinct treatment techniques. Moreover, ES analysis revealed possible causative variants identified in three families with variants in a novel candidate disease gene, MCF2L. Further functional studies will be necessary to better understand the molecular mechanisms involved on SA-PA(A) malformation, however our findings suggest that MCF2L is a novel disease gene associated with SA-PA(A).

Implementation and adaptation of the Re-Engineered Discharge (RED) in five California hospitals: a qualitative research study.

BACKGROUND: Project Re-Engineered Discharge (RED) is an evidence-based strategy to reduce readmissions disseminated and adapted by various health systems across the country. To date, little is known about how adapting Project RED from its original protocol impacts RED implementation and/or sustainability. The goal of this study was to identify and characterize contextual factors influencing how five California hospitals adapted and implemented RED and the subsequent impact on RED program sustainability. METHODS: Participant observation and key informant and focus group interviews with 64 individuals at five California hospitals implementing RED in 2012 and 2013 were conducted. These involved hospital leadership, personnel responsible for Project RED implementation, hospital staff, and clinicians. Interview transcripts were coded and analyzed using a modified grounded theory approach and constant comparative analysis. RESULTS: Both internal and external contextual factors were identified that influenced hospitals' decisions on RED adaptation and implementation. These also impacted RED sustainability. External factors included: impending federal penalties for hospitals with high readmission rates targeting specific diagnoses, and access to external funding and technical support to help hospitals implement RED. Internal or organizational level contextual factors included: committed leadership prioritizing Project RED; RED adaptations; depth, accountability and influence of the implementation team; sustainability planning; and hospital culture. Only three of the five hospitals continued Project RED beyond the implementation period. CONCLUSIONS: The sustainability of RED in participating hospitals was only possible when hospitals approached RED implementation as a transformational process rather than a patient safety project, maintained a high level of fidelity to the RED protocol, and had leadership and an implementation team who embraced change and failure in the pursuit of better patient care and outcomes. Hospitals who were unsuccessful in implementing a sustainable RED process lacked all or most of these components in their approach.

Time-resolved contrast-enhanced MRA (TWIST) with gadofosveset trisodium in the classification of soft-tissue vascular anomalies in the head and neck in children following updated 2014 ISSVA classification: first report on systematic evaluation of MRI and TWIST in a cohort of 47 children.

AIM: To evaluate the relative accuracy of contrast-enhanced time-resolved angiography with interleaved stochastic trajectories versus conventional contrast-enhanced magnetic resonance imaging (MRI) following International Society for the Study of Vascular Anomalies updated 2014-based classification of soft-tissue vascular anomalies in the head and neck in children. MATERIALS AND METHODS: Time-resolved angiography with interleaved stochastic trajectories versus conventional contrast-enhanced MRI of children with diagnosis of soft-tissue vascular anomalies in the head and neck referred for MRI between 2008 and 2014 were retrospectively reviewed. Forty-seven children (0-18 years) were evaluated. Two paediatric neuroradiologists evaluated time-resolved MRA and conventional MRI in two different sessions (30 days apart). Blood-pool endovascular MRI contrast agent gadofosveset trisodium was used. RESULTS: The present cohort had the following diagnoses: infantile haemangioma (n=6), venous malformation (VM; n=23), lymphatic malformation (LM; n=16), arteriovenous malformation (AVM; n=2). Time-resolved MRA alone accurately classified 38/47 (81%) and conventional MRI 42/47 (89%), respectively. Although time-resolved MRA alone is slightly superior to conventional MRI alone for diagnosis of infantile haemangioma, conventional MRI is slightly better for diagnosis of venous and LMs. Neither time-resolved MRA nor conventional MRI was sufficient for accurate diagnosis of AVM in this cohort. Conventional MRI combined with time-resolved MRA accurately classified 44/47 cases (94%). CONCLUSION: Time-resolved MRA using gadofosveset trisodium can accurately classify soft-tissue vascular anomalies in the head and neck in children. The addition of time-resolved MRA to existing conventional MRI protocols provides haemodynamic information, assisting the diagnosis of vascular anomalies in the paediatric population at one-third of the dose of other MRI contrast agents.

S.E. Mitchell Vascular Anomalies Flow Chart (SEMVAFC): a visual pathway combining clinical and imaging findings for classification of soft-tissue vascular anomalies.

Classification of vascular anomalies (VAs) is challenging due to overlapping clinical symptoms, confusing terminology in the literature and unfamiliarity with this complex entity. It is important to recognize that VAs include two distinct entities, vascular tumours (VTs) and vascular malformations (VaMs). In this article, we describe SE Mitchell Vascular Anomalies Flow Chart (SEMVAFC), which arises from a multidisciplinary approach that incorporates clinical symptoms, physical examination and magnetic resonance imaging (MRI) findings to establish International Society for the Study of Vascular Anomalies (ISSVA)-based classification of the VAs. SEMVAFC provides a clear visual pathway for physicians to accurately diagnose Vas, which is important as treatment, management, and prognosis differ between VTs and VaMs.

Limits to sustained energy intake. XX. Body temperatures and physical activity of female mice during lactation.

Lactating animals consume greater amounts of food than non-reproductive animals, but energy intake appears to be limited in late lactation. The heat dissipation limit theory suggests that the food intake of lactating mice is limited by the capacity of the mother to dissipate heat. Lactating mice should therefore have high body temperatures (Tb), and changes in energy intake during lactation should be reflected by variation in Tb. To investigate these predictions, 26 mice (Mus musculus) were monitored daily throughout lactation for food intake, body mass, litter size and litter mass. After weaning, 21 days postpartum, maternal food intake and body mass were monitored for another 10 days. Maternal activity and Tb were recorded every minute for 23 h a day using implanted transmitters (vital view). Energy intake increased to a plateau in late lactation (days 13-17). Daily gain in pup mass declined during this same period, suggesting a limit on maternal energy intake. Litter size and litter mass were positively related to maternal energy intake and body mass. Activity levels were constantly low, and mice with the largest increase in energy intake at peak lactation had the lowest activity. Tb rose sharply after parturition and the circadian rhythm became compressed within a small range. Tb during the light period increased considerably (1.1 ° C higher than in baseline), and lactating mice faced chronic hyperthermia, despite their activity levels in lactation being approximately halved. Average Tb increased in relation to energy intake as lactation progressed, but there was no relationship between litter size or litter mass and the mean Tb at peak lactation. These data are consistent with the heat dissipation limit theory, which suggests performance in late lactation is constrained by the ability to dissipate body heat.

Making a long story short: noncoding RNAs and chromosome change.

As important as the events that influence selection for specific chromosome types in the derivation of novel karyotypes, are the events that initiate the changes in chromosome number and structure between species, and likewise polymorphisms, variants and disease states within species. Although once thought of as transcriptional 'noise', noncoding RNAs (ncRNAs) are now recognized as important mediators of epigenetic regulation and chromosome stability. Here we highlight recent work that illustrates the influence short and long ncRNAs have as participants in the function and stability of chromosome regions such as centromeres, telomeres, evolutionary breakpoints and fragile sites. We summarize recent evidence that ncRNAs can facilitate chromosome change and present mechanisms by which ncRNAs create DNA breaks. Finally, we present hypotheses on how they may create novel karyotypes and thus affect chromosome evolution.

Chronic diarrhea, ascites, and protein-losing enteropathy in an infant with hepatic venous outflow obstruction after liver transplantation.

An 18-month-old female status post-orthotopic liver transplant for biliary atresia presented nine months after transplant with severe diarrhea and intolerance of feeds. She was found to have a PLE as evidenced by a low serum albumin and a persistent elevation of fecal A1AT. Investigation eventually revealed that the cause of the PLE was a stricture at the anastomosis site between the hepatic vein and inferior cava, supported by resolution of the PLE after venoplasty of the stricture. The patient has subsequently required several repeat venoplasties for recurrence of her symptoms correlating with recurrence of the stricture. This is a very rare presentation of hepatic venous outflow obstruction. Moreover, normal duplex ultrasound imaging of liver vasculature and her unusual presentation led to a delay in her diagnosis highlighting the need for an increased index of suspicion.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

Calories or protein? The effect of dietary restriction on lifespan in rodents is explained by calories alone.

Almost exactly 100years ago Osborne and colleagues demonstrated that restricting the food intake of a small number of female rats extended their lifespan. In the 1930s experiments on the impact of diet on lifespan were extended by Slonaker, and subsequently McCay. Slonaker concluded that there was a strong impact of protein intake on lifespan, while McCay concluded that calories are the main factor causing differences in lifespan when animals are restricted (Calorie restriction or CR). Hence from the very beginning the question of whether food restriction acts on lifespan via reduced calorie intake or reduced protein intake was disputed. Subsequent work supported the idea that calories were the dominant factor. More recently, however, this role has again been questioned, particularly in studies of insects. Here we review the data regarding previous studies of protein and calorie restriction in rodents. We show that increasing CR (with simultaneous protein restriction: PR) increases lifespan, and that CR with no PR generates an identical effect. None of the residual variation in the impact of CR (with PR) on lifespan could be traced to variation in macronutrient content of the diet. Other studies show that low protein content in the diet does increase median lifespan, but the effect is smaller than the CR effect. We conclude that CR is a valid phenomenon in rodents that cannot be explained by changes in protein intake, but that there is a separate phenomenon linking protein intake to lifespan, which acts over a different range of protein intakes than is typical in CR studies. This suggests there may be a fundamental difference in the responses of insects and rodents to CR. This may be traced to differences in the physiology of these groups, or reflect a major methodological difference between 'restriction' studies performed on rodents and insects. We suggest that studies where the diet is supplied ad libitum, but diluted with inert components, should perhaps be called dietary or caloric dilution, rather than dietary or caloric restriction, to distinguish these potentially important methodological differences.

Oxidative costs of reproduction: Oxidative stress in mice fed standard and low antioxidant diets.

Lactation is one of the most energetically expensive behaviours, and trade-offs may exist between the energy devoted to it and somatic maintenance, including protection against oxidative damage. However, conflicting data exist for the effects of reproduction on oxidative stress. In the wild, a positive relationship is often observed, but in laboratory studies oxidative damage is often lower in lactating than in non-breeding animals. We hypothesised that this discrepancy may exist because during lactation food intake increases many-fold resulting in a large increase in the intake of dietary antioxidants which are typically high in laboratory rodent chow where they are added as a preservative. We supplied lactating and non-breeding control mice with either a standard or low antioxidant diet and studied how this affected the activity of endogenous antioxidants (catalase, superoxide dismutase; SOD, and glutathione peroxidise; GPx) and oxidative damage to proteins (protein carbonyls, PC) in liver and brain tissue. The low antioxidant diet did not significantly affect activities of antioxidant enzymes in brain or liver, and generally did not result in increased protein damage, except in livers of control mice on low antioxidant diet. Catalase activity, but not GPx or SOD, was decreased in both control and lactating mice on the low antioxidant diet. Lactating mice had significantly reduced oxidative damage to both liver and brain compared to control mice, independent of the diet they were given. In conclusion, antioxidant content of the diet did not affect oxidative stress in control or reproductive mice, and cannot explain the previously observed reduction in oxidative stress in lactating mammals studied in the laboratory. The reduced oxidative stress in the livers of lactating mice even under low antioxidant diet treatment was consistent with the 'shielding' hypothesis.

Limits to sustained energy intake XXIV: impact of suckling behaviour on the body temperatures of lactating female mice.

The objective of this study was to investigate the potential causes of high body temperature (Tb) during lactation in mice as a putative limit on energy intake. In particular we explored whether or not offspring contributed to heat retention in mothers while suckling. Tb and physical activity were monitored in 26 female MF1 mice using intraperitoneally implanted transmitters. In addition, maternal behaviour was scored each minute for 8 h d(-1) throughout lactation. Mothers that raised larger litters tended to have higher Tb while nursing inside nests (P < 0.05), suggesting that nursing offspring may have influenced heat retention. However, Tb during nursing was not higher than that recorded during other behaviours. In addition, the highest Tb during the observation period was not measured during nursing behaviour. Finally, there was no indication that mothers discontinued suckling because of a progressive rise in their Tb while suckling. Tb throughout lactation was correlated with daily increases in energy intake. Chronic hyperthermia during lactation was not caused by increased heat retention due to surrounding offspring. Other factors, like metabolic heat produced as a by-product of milk production or energy intake may be more important factors. Heat dissipation limits are probably not a phenomenon restricted to lactation.

Factors influencing individual variability in high fat diet-induced weight gain in out-bred MF1 mice.

Easy access to high-energy palatable foods has been suggested to have contributed to the world-wide obesity epidemic. However, within these 'obesogenic' environments many people manage to remain lean. Mice also show variability in their weight gain responses to high-fat diet (HFD) feeding and their weight loss responses to calorically restricted (CR) feeding. In this study we investigated which factors contribute to determining susceptibility to HFD-induced obesity in mice, and whether the responses in weight gain on HFD are correlated with the responses to CR. One-hundred twenty four mice were exposed to 30% CR for 28days followed by a 14day recovery period, and subsequent exposure to 60% HFD for 28days. Responses in various metabolic factors were measured before and after each exposure (body mass; BM, body composition, food intake; FI, resting metabolic rate; RMR, physical activity, body temperature and glucose tolerance; GT). Weight changes on HFD ranged from -1 to 26%, equivalent to -0.2g to 10.5g in absolute mass. Multiple regression models showed that fat free mass (FFM) of the mice before exposure to HFD predicted 12% of the variability in weight gain on HFD (p<0.001). Also, FI during the first week of HFD feeding predicted 20% of the variability in BM and fat mass (FM) gain 4weeks later. These data may point to a role for the reward system in driving individual differences in FI and weight gain. Weight gain on the HFD was significantly negatively correlated to weight loss on CR, indicating that animals that are poor at defending against weight gain on HFD, were also poor at defending against CR-induced weight loss. Changes in FM and FFM in response to HFD or CR were not correlated however.

Suppression of leptin during lactation: contribution of the suckling stimulus versus milk production.

Lactation in the rat is characterized by the suppression of pulsatile LH secretion, a large increase in food intake, and changes in energy balance due to the metabolic drain of milk production. The change in energy balance may be a major component in altering reproductive function. A number of factors may contribute to changing energy balance of a lactating animal; one is leptin, the product of adipose tissue, which is known to act partly as a satiety factor to decrease food intake. The aims of the present study were to determine whether there are changes in leptin levels during lactation, a state of high energy demand, and during periods of acute suckling in the presence or absence of changes in energy demand. Our goals were to determine whether lactation and the suckling stimulus influenced serum leptin levels and whether there was a potential role for leptin in the suppression of LH secretion during lactation. The first experiment was performed during diestrus of the estrous cycle, and chronic lactation, (day 9 post partum) in animals suckling 8 pups. The results showed that leptin levels were significantly decreased in both ovarian intact or ovariectomized lactators; this decrease parallels the suppression of pulsatile LH secretion. Serum insulin levels were not altered in the lactating animals. The second experiment was performed in ovariectomized lactators whose 8 pup litters were removed for 48 h, starting on day 9. On day 11, mothers received no pups or pups that were either nonfostered (resulting in no milk production) or fostered (resulting in milk production). The pups were allowed to suckle for 24 h. Following 24 h of acute suckling, serum leptin, and insulin levels correlated with the energy drain on the mother. The levels of leptin were normal and of insulin were elevated in mothers producing no milk. Conversely, leptin levels were suppressed and insulin levels normal in mothers producing milk. The third experiment used the same groups as described for the second experiment except that serial blood samples were collected for measurement of pulsatile LH secretion following 24 h of acute suckling. The results showed that regardless of whether leptin levels remained normal or were suppressed in response to acute suckling, pulsatile LH secretion was significantly inhibited compared with the nonsuckled control animals. In summary, these data suggest that the metabolic drain of milk production, and not the suckling stimulus itself, is the most likely factor responsible for the suppression of leptin secretion during lactation. Furthermore, although the decreased levels of leptin may be causally related to the inhibition of pulsatile LH secretion during chronic lactation, changes in leptin are not a prerequisite for the suppression of LH secretion in response to suckling.

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis.

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.

Squamous cell carcinoma of the anal canal.

PURPOSE: To report the results of primary radiotherapy for treatment of anal canal carcinoma from the University of Florida series and review issues related to treatment of this disease. METHODS AND MATERIALS: Forty-nine patients were treated with primary radiation therapy (RT) for cure. Patients had a minimum 2-year follow-up (median, 9.8 years). After 1990, patients with lesions of at least 3 cm also received chemotherapy with fluorouracil (1000 mg/m(2)) plus cisplatin (100 mg/m(2)) or mitomycin (10-15 mg/m(2)) if medically fit (n = 26). RT was delivered with a 4-field box technique to deliver 45 Gy in 25 fractions. The inguinal nodes were treated daily using electrons to supplement the dose in that region to a total dose of 45 Gy if clinically negative or about 60 Gy if involved. There were no planned breaks. A 10- to 15-Gy boost was delivered using interstitial iridium 192 implant (n = 32), en face (60)Co field (n = 5), or external-beam photon fields (n = 11). RESULTS: Local control rates at 5 years were 100% for T1N0, 92% for T2N0 or N1, 75% for T3N0, 67% for T4N0, 88% for T4N(pos) or T(any)N2-3, and 85% overall. With surgical salvage, ultimate local control rates were 100%, 100%, 81%, 100%, and 88%, respectively, with 92% overall. Cause-specific survival rates at 5 years were 100% for Stage I, 88% for Stage II, 100% for Stage IIIA, and 70% for Stage IIIB. Absolute survival rates at 5 years were 62%, 68%, 100%, and 70%. Sphincter preservation rates were 83%, 79%, 75%, and 100% by stage and 81% overall. There was an improvement in local control with the addition of chemotherapy in more advanced disease, but it was not significant. There was an increase in acute toxicity with the addition of chemotherapy (12% > or = Grade 4) but not long-term toxicity. Late toxicity requiring colostomy occurred in 6% of patients and consisted of soft tissue necrosis. CONCLUSIONS: The majority of patients with anal canal carcinoma can be treated with curative intent using a sphincter-sparing approach of radiation with or without chemotherapy even with advanced disease. With the addition of chemotherapy to radiation, there is an increased risk of acute toxicity and about 1-2% incidence of toxic death. Smaller tumors (T1 and early T2) probably do not require the addition of chemotherapy.

Phase I study of twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas.

PURPOSE: To determine the maximum tolerated dose and dose-limiting toxicity associated with twice-weekly gemcitabine and concomitant external-beam radiotherapy in patients with adenocarcinoma of the pancreas. METHODS AND MATERIALS: Twenty-one patients with biopsy-proven adenocarcinoma of the pancreas were treated with external-beam radiotherapy to a dose of 50.4 Gy in 28 fractions, concurrent with gemcitabine, infused over 30 min before irradiation on a Monday and Thursday schedule. The dose of gemcitabine was escalated in 5 cohorts of 3--6 patients each. Initial gemcitabine dose was 10 mg/m(2), with dose escalation until dose-limiting toxicity was observed. RESULTS: The maximum tolerated dose of gemcitabine was 50 mg/m(2), when given in a twice-weekly schedule with radiation. Dose-limiting toxicity was seen in 2 patients at 60 mg/m(2), and consisted of severe upper gastrointestinal bleeding approximately 1 month after completion of treatment. Six patients had radiographic evidence of response to treatment, and 5 of these underwent complete surgical resection. Three patients who underwent complete resection had been deemed to have unresectable tumors before enrollment on trial. Four patients are alive, including 2 without evidence of disease more than 1 year after resection. CONCLUSION: The combination of external-beam radiation and twice-weekly gemcitabine at a dose of 50 mg/m(2) is well tolerated and shows promising activity for the treatment of pancreatic cancer. Our data suggest a higher maximum tolerated dose and different dose-limiting toxicity than previously reported. Further investigation of this regimen is warranted.

Solid-phase PCR in microwells: effects of linker length and composition on tethering, hybridization, and extension.

During the solid-phase PCR (SP-PCR), DNA oligonucleotides complementary to a soluble template and immobilized on a surface are extended in situ. Although primarily used for pathogen detection, SP-PCR has the potential for much broader application, including disease diagnostics, genotyping, and expression studies. Current protocols for SP-PCR in microwells are suitable for enzymatic detection of immobilized products, but yields are generally insufficient for direct detection of products using conventional fluorescent probes. Here, we quantitatively measure the outcome of tethering, hybridization, and solid-phase extension, and examine the effect of composition and length of the spacer at the 5' end of tethered oligonucleotides. Our results indicate that steric hindrance primarily affects polymerase activity rather than the efficiency of hybridization between the template and the tethered oligonucleotide. SP-PCR yields are significantly higher for a five-unit hexaethyleneglycol (HEG) spacer than for the more commonly used 10-residue deoxythymidine spacer. The optimal 5' HEG spacer resulted in a 60-fold increase in extension efficiency relative to a previously reported value for SP-PCR on a glass surface. Thus, optimized spacers should allow direct quantification of SP-PCR products, providing a simple, quantitative, and cost effective means of sample analysis for a variety of applications.

Porcine model for the development of interventional techniques in the gallbladder.

A technique that permits repeated access to the porcine gallbladder through a surgically placed 18 French latex catheter is described and experience with this system in 22 swine is discussed. This porcine gallbladder model produces a reliable and reproducible means for studying clinically applicable interventional procedures.