Empagliflozin inhibits increased Na influx in atrial cardiomyocytes of patients with HFpEF.

AIMS: Heart failure with preserved ejection fraction (HFpEF) causes substantial morbidity and mortality. Importantly, atrial remodeling and atrial fibrillation is frequently observed in HFpEF. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have recently been shown to improve clinical outcomes in HFpEF, and post-hoc analyses suggest atrial antiarrhythmic effects. We tested if isolated human atrial cardiomyocytes from patients with HFpEF exhibit an increased Na influx, which is known to cause atrial arrhythmias, and if that is responsive to treatment with the SGTL2i empagliflozin. METHODS AND RESULTS: Cardiomyocytes were isolated from atrial biopsies of 124 patients (82 with HFpEF) undergoing elective cardiac surgery. Na influx was measured with the Na-dye Asante Natrium Green-2 AM (ANG-2). Compared to patients without heart failure (NF), Na influx was doubled in HFpEF patients (NF vs HFpEF: 0.21±0.02 vs 0.38±0.04 mmol/L/min (N=7 vs 18); p=0.0078). Moreover, late INa (measured via whole-cell patch clamp) was significantly increased in HFpEF compared to NF. Western blot and HDAC4 pulldown assay indicated a significant increase in CaMKII expression, CaMKII autophosphorylation, CaMKII activity, and CaMKII-dependent NaV1.5 phosphorylation in HFpEF compared to NF, whereas NaV1.5 protein and mRNA abundance remained unchanged. Consistently, increased Na influx was significantly reduced by treatment with the CaMKII inhibitor autocamtide-2 related inhibitory peptide (AIP), late INa inhibitor tetrodotoxin (TTX) but also with NHE1 inhibitor cariporide. Importantly, empagliflozin abolished both increased Na influx and late INa in HFpEF. Multivariate linear regression analysis, adjusting for important clinical confounders, revealed HFpEF to be an independent predictor for changes in Na handling in atrial cardiomyocytes. CONCLUSION: We show for the first time increased Na influx in human atrial cardiomyocytes from HFpEF patients, partly due to increased late INa and enhanced NHE1-mediated Na influx. Empagliflozin inhibits Na influx and late INa, which could contribute to antiarrhythmic effects in patients with HFpEF.

Switching between foods is reliably associated with intake across eating events in children.

Emerging evidence suggests switching between foods during an eating event is positively associated with intake. However, it is unclear whether switching is a stable behavior that predicts consumption across multiple eating events. The current study explored whether switching is consistent within children and reliably associated with intake across varied eating events. We analyzed data from 88 (45 F), 7-8-year-old children without obesity participating in a 7-visit prospective cohort study (ClinicalTrials.gov NCT03341247). Amount consumed and energy intake were measured at 4 separate meals of foods that varied by portion sizes served. Meals included macaroni and cheese, chicken nuggets, broccoli, and grapes (all 0.7-2.5 kcal/g). Children's intake was also assessed during 2 eating in the absence of hunger (EAH) paradigms separated by ≥ 1 year. The EAH paradigm included 9 sweet and savory snack foods (all 1.9-5.7 kcal/g). All eating events were video-recorded and switching was assessed by counting the number of times a child shifted between different food items. Results demonstrated that switching was reliably associated with intake at both the meals and the EAH paradigms (ps < 0.01). Specifically, at meals each additional switch was associated with 11.7 ± 1.3 kcal (7.7 ± 0.8 g) more consumed, and during EAH each additional switch was associated with 8.1 ± 2.1 kcal (2.1 ± 0.5 g) more consumed. Switching behavior was also moderately consistent across meals (ICC = 0.70) and EAH paradigms (ICC = 0.50). However, switching at meals was not related to switching at EAH paradigms. This study demonstrates the consistency of switching behavior and its reliable association with intake across eating events, highlighting its potential to contribute to chronic overconsumption and childhood obesity.

Etiologies and predictors of mortality in an all-comer population of patients with non-ischemic heart failure.

BACKGROUND: Despite progress in diagnosis and therapy of heart failure (HF), etiology and risk stratification remain elusive in many patients. METHODS: The My Biopsy HF Study (German clinical trials register number: DRKS22178) is a retrospective monocentric study investigating an all-comer population of patients with unexplained HF based on a thorough workup including endomyocardial biopsy (EMB). RESULTS: 655 patients (70.9% men, median age 55 [45/66] years) with non-ischemic, non-valvular HF were included in the analyses. 489 patients were diagnosed with HF with reduced ejection fraction (HFrEF), 52 patients with HF with mildly reduced ejection fraction (HFmrEF) and 114 patients with HF with preserved ejection fraction (HFpEF). After a median follow-up of 4.6 (2.5/6.6) years, 94 deaths were enumerated (HFrEF: 68; HFmrEF: 8; HFpEF: 18), equating to mortality rates of 3.3% and 11.6% for patients with HFrEF, 7.7% and 15.4% for patients with HFmrEF and 5.3% and 11.4% for patients with HFpEF after 1 and 5 years, respectively. In EMB, we detected a variety of putative etiologies of HF, including incidental cardiac amyloidosis (CA, 5.8%). In multivariate logistic regression analysis adjusting for age, sex and comorbidities only CA, age and NYHA functional class III + IV remained independently associated with all-cause mortality (CA: HRperui 3.13, 95% CI 1.5-6.51; p = 0.002). CONCLUSIONS: In an all-comer population of patients presenting with HF of unknown etiology, incidental finding of CA stands out to be independently associated with all-cause mortality. Our findings suggest that prospective trials would be helpful to test the added value of a systematic and holistic work-up of HF of unknown etiology.

Children's inhibitory control abilities in the presence of rewards are related to weight status and eating in the absence of hunger.

The Reflective-Impulsive Dual Processes Model suggests that overeating occurs when the temptation to consume food overrides inhibitory control processes. However, how rewards interact with inhibitory control and their relation to children's weight status and food intake is not understood. Here, 7-to-11-year-old children (n = 66; 32 overweight/obese) completed two versions (baseline [i.e., non-reward incentivized/control] and reward incentivized [food, money, no reward]) of a Go/Nogo task. Intake of palatable foods in the absence of hunger (i.e., eating in the absence of hunger-EAH) was measured following a standardized meal. A drift diffusion model was used to characterize children's performance parameters on the Go/Nogo. On the baseline Go/Nogo, children with higher weight status responded more cautiously, but on reward trials for food/money children were more cautions and made more false alarms relative to the no reward condition. Energy intake during EAH positively correlated with FA errors for food and money vs. no reward, but sex moderated this effect such that FA positively associated with EAH in girls but not boys. Independent of sex, FA for money vs. no reward and food vs. money were both positively associated with energy consumed during EAH. These results suggest that the presence of food and money rewards impair inhibitory control processing, especially in children with higher weight status. Further, increased inhibitory control impairment in response to food rewards, specifically, may be a risk factor for disinhibited eating in girls. Though preliminary, results may be useful in the development of targeted treatments to help moderate excess consumption in children.

Increased C reactive protein, cardiac troponin I and GLS are associated with myocardial inflammation in patients with non-ischemic heart failure.

Inflammatory cardiomyopathy diagnosed by endomyocardial biopsy (EMB) is common in non-ischemic heart failure (HF) and might be associated with adverse outcome. We aimed to identify markers predicting myocardial inflammation in HF. We screened 517 patients with symptomatic non-ischemic HF who underwent EMB; 397 patients (median age 54 [IQR 43/64], 28.7% females) were included in this study. 230 patients were diagnosed with myocardial inflammation, defined as ≥ 7.0 CD3+ lymphocytes/mm2 and/or ≥ 35.0 Mac1 macrophages/mm2 and were compared to 167 inflammation negative patients. Patients with myocardial inflammation were more often smokers (52.4% vs. 39.8%, p = 0.013) and had higher C-reactive protein (CRP) levels (5.4 mg/dl vs. 3.7 mg/dl, p = 0.003). In logistic regression models CRP ≥ 8.15 mg/dl (OR 1.985 [95%CI 1.160-3.397]; p = 0.012) and Troponin I (TnI) ≥ 136.5 pg/ml (OR 3.011 [1.215-7.464]; p = 0.017) were independently associated with myocardial inflammation, whereas no association was found for elevated brain natriuretic peptide (OR 1.811 [0.873-3.757]; p = 0.111). In prognostic performance calculation the highest positive predictive value (90%) was detected for the combination of Global longitudinal strain (GLS) ≥ -13.95% and TnI ≥ 136.5 pg/ml (0.90 (0.74-0.96)). Elevated CRP, TnI and GLS in combination with TnI can be useful to detect myocardial inflammation. Smoking seems to predispose for myocardial inflammation.

Non-specific rheumatic manifestations in patients with Hashimoto's thyroiditis: a pilot cross-sectional study.

PURPOSE: Hashimoto's thyroiditis (HT) is often associated with rheumatic disorders (arthritis, etc.), but many HT patients report non-specific rheumatic signs and symptoms in the absence of clinically evident rheumatic diseases. Aim of this study was to evaluate the prevalence of non-specific rheumatic manifestations (RMs) in HT subjects without classified autoimmune comorbidities. METHODS: 500 HT patients (467 F, 33 M; median age 41 years, range 14-69) and 310 age- and sex-matched controls, consecutively referred to the Endocrine Unit of Messina University Hospital, were evaluated for non-specific RMs. None took L-thyroxine. EXCLUSION CRITERIA: autoimmune comorbidities, infectious, and/or inflammatory diseases, history of neoplasia, BMI > 30 kg/m2. RESULTS: In our HT cohort, 100 patients (20%) complained of one or more RMs, vs 21 controls (6.8%; P < 0.001). There were minimal differences between the manifestations recorded in the two groups, the most common being polyarthralgias and myalgias/fibromyalgia, but non-specific RMs occurred threefold more in HT patients. Comparing HT patients with RMs (96 F and 4 M) with those affected by HT alone, female sex was prevalent (F:M ratio 24:1 vs 5:1) with higher age at diagnosis (median 43 vs 37 years; P < 0.001). HT patients with RMs (62%) were mostly euthyroid (median TSH 2.0 µIU/L) and only 7% overtly hypothyroid, discouraging a possible causal relationship between thyroid dysfunction per se and RMs. CONCLUSIONS: A significant percentage of HT patients complains of non-specific rheumatic signs and symptoms, in the absence of other diagnosed systemic comorbidities and regardless of thyroid functional status, deserving careful evaluation and prolonged follow-up.

Ligand-dependent Hedgehog pathway activation in Rhabdomyosarcoma: the oncogenic role of the ligands.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. The Hedgehog (HH) pathway is known to develop an oncogenic role in RMS. However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. METHODS: The expression of HH ligands was studied by qPCR, western blot and immunohistochemistry. Functional and animal model studies were carried out with cells transduced with shRNAs against HH ligands or treated with HH-specific inhibitors (Vismodegib and MEDI-5304). Finally, the molecular characterisation of an off-target effect of Vismodegib was also made. RESULTS: The results showed a prominent expression of HH ligands supporting an autocrine ligand-dependent activation of the pathway. A comparison of pharmacologic Smoothened inhibition (Vismodegib) and HH ligand blocking (MEDI-5304) is also provided. Interestingly, a first description of pernicious off-target effect of Vismodegib is also reported. CONCLUSIONS: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols. Moreover, the description of a pernicious off-target effect of Vismodegib, via unfolded protein response activation, may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers.

Post-traumatic stress disorder dimensions and asthma morbidity in World Trade Center rescue and recovery workers.

OBJECTIVE: Using data from a cohort of World Trade Center (WTC) rescue and recovery workers with asthma, we assessed whether meeting criteria for post-traumatic stress disorder (PTSD), sub-threshold PTSD, and for specific PTSD symptom dimensions are associated with increased asthma morbidity. METHODS: Participants underwent a Structured Clinical Interview for Diagnostic and Statistical Manual to assess the presence of PTSD following DSM-IV criteria during in-person interviews between December 2013 and April 2015. We defined sub-threshold PTSD as meeting criteria for two of three symptom dimensions: re-experiencing, avoidance, or hyper-arousal. Asthma control, acute asthma-related healthcare utilization, and asthma-related quality of life data were collected using validated scales. Unadjusted and multiple regression analyses were performed to assess the relationship between sub-threshold PTSD and PTSD symptom domains with asthma morbidity measures. RESULTS: Of the 181 WTC workers with asthma recruited into the study, 28% had PTSD and 25% had sub-threshold PTSD. Patients with PTSD showed worse asthma control, higher rates of inpatient healthcare utilization, and poorer asthma quality of life than those with sub-threshold or no PTSD. After adjusting for potential confounders, among patients not meeting the criteria for full PTSD, those presenting symptoms of re-experiencing exhibited poorer quality of life (p = 0.003). Avoidance was associated with increased acute healthcare use (p = 0.05). Sub-threshold PTSD was not associated with asthma morbidity (p > 0.05 for all comparisons). CONCLUSIONS: There may be benefit in assessing asthma control in patients with sub-threshold PTSD symptoms as well as those with full PTSD to more effectively identify ongoing asthma symptoms and target management strategies.

[Historical Changes in Meniscus Surgery--The Changing Appreciation of Disease, Surgical Techniques and Complications]. / Geschichtlicher Wandel der Meniskuschirurgie - Veränderungen in Krankheitsverständnis, Operationstechniken und Komplikationen.

The menisci play an important role in normal function of the knee joint. Meniscal injuries are associated with pain, swelling, impairment in function and activity level as well as early arthrosis of the knee joint. Patients with meniscal tears have to undergo surgery frequently. The pathway from the beginning of meniscal surgery to the modern arthroscopic meniscal surgery was not straightforward. The clinical picture of meniscal injuries, which caused articular trouble, was controversial for a long time. Meniscal surgery developed from knee joint surgery of the loose articular bodies. The first meniscal tear was not described until 1731. More than 100 years later, in 1866 the first planned meniscal surgery was performed. The importance of surgical techniques of meniscal resection and meniscal repair was emphasised in 1885. The beginning of knee joint arthroscopy took place in the 2nd decade of the 20th century. However, arthroscopy did not start its triumphal phase not until the 1960s coursing from Japan over North America to Europe. Approximately 150 years had passed from the risky arthrotomy surgeries of the past to the modern arthroscopic meniscal surgeries with low complication rates. Today, arthroscopic knee surgeries and especially arthroscopic meniscal surgeries are one of the most frequently performed orthopaedic procedures.

Histone acetyltransferase inhibitors block neuroblastoma cell growth in vivo.

We have previously described novel histone acetyltransferase (HAT) inhibitors that block neuroblastoma cell growth in vitro. Here we show that two selected pyridoisothiazolone HAT inhibitors, PU139 and PU141, induce cellular histone hypoacetylation and inhibit growth of several neoplastic cell lines originating from different tissues. Broader in vitro selectivity profiling shows that PU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300, whereas PU141 is selective toward CBP and p300. The pan-inhibitor PU139 triggers caspase-independent cell death in cell culture. Both inhibitors block growth of SK-N-SH neuroblastoma xenografts in mice and the PU139 was shown to synergize with doxorubicin in vivo. The latter also reduces histone lysine acetylation in vivo at concentrations that block neoplastic xenograft growth. This is one of the very few reports on hypoacetylating agents with in vivo anticancer activity.

Fungi infection in honeybee hives in regions affected by Brazilian sac brood / Contaminação fúngica em colmeias de abelhas de regiões afetadas pela cria ensacada brasileira

The Brazilian Sac Brood is a disease that affects apiaries of Africanized bee hives in Brazil, thereby making them susceptible to high losses. This study investigated the pathogenicity of Africanized bee hives by the entomopathogenic fungi in a Brazilian Sac Brood endemic region. The degree of fungal contamination, presence of mycotoxins in beehive elements, and vulnerability of healthy beehives in environments subjected and not subjected to the disease were investigated. From the contaminating fungal load, species that are mycotoxin producers and pathogenic causing mortality in the bees have been isolated. The analysis of bee pollen and bee bread samples did not show the presence of the toxic pollen of Stryphnodendron (Fabaceae), which has been indicated as the causative agent of mortality in pre-pupal stage larvae. However, bee bread showed the highest correlation between substrate and fungal contamination...

A cria ensacada brasileira é uma doença que afeta apiários de colmeias de abelhas africanizadas no Brasil, tornando-os suscetíveis a perdas elevadas. Este estudo investigou a patogenicidade de fungos entomopatogênicos em colmeias de abelhas africanizadas de uma região endêmica de cria ensacada brasileira. O grau de contaminação fúngica, a presença de micotoxinas em elementos colmeia e a vulnerabilidade das colmeias saudáveis em ambientes sujeitos e não sujeitos à doença foram investigados. A partir da carga fúngica contaminante, espécies produtoras de micotoxinas e patogênicas, que provocam a mortalidade de abelhas, foram isoladas. A análise do pólen e do pão de abelha não demonstrou a presença do pólen tóxico de Stryphnodendron (Fabaceae), que tem sido apontado como agente causador da mortalidade de larvas em fase de pré-pupa. No entanto, o pão de abelha foi o substrato mais correlacionado com a contaminação fúngica...

Anterior spinal artery syndrome: reversible paraplegia after minimally invasive spine surgery.

Background Context. Percutaneous balloon kyphoplasty is an established minimally invasive technique to treat painful vertebral compression fractures, especially in the context of osteoporosis with a minor complication rate. Purpose. To describe the heparin anticoagulation treatment of paraplegia following balloon kyphoplasty. Study Design. We report the first case of an anterior spinal artery syndrome with a postoperative reversible paraplegia following a minimally invasive spine surgery (balloon kyphoplasty) without cement leakage. Methods. A 75-year-old female patient underwent balloon kyphoplasty for a fresh fracture of the first vertebra. Results. Postoperatively, the patient developed an acute anterior spinal artery syndrome with motor paraplegia of the lower extremities as well as loss of pain and temperature sensation with retained proprioception and vibratory sensation. Complete recovery occurred six hours after bolus therapy with 15.000 IU low-molecular heparin. Conclusion. Spine surgeons should consider vascular complications in patients with incomplete spinal cord syndromes after balloon kyphoplasty, not only after more invasive spine surgery. High-dose low-molecular heparin might help to reperfuse the Adamkiewicz artery.

[Muscle atrophy caused by limited mobilisation]. / Muskelatrophie durch Mobilisationseinschränkung.

BACKGROUND: Immobilisation and limited mobilisation are associated with negative adaptations of human body, like elevated risk for thrombosis, reduced fitness level and muscle atrophy. The objective of the presented study was to investigate the effect of limited, reduced mobility of a few days on the upper leg muscles. PATIENTS AND METHODS: 32 patients who underwent an elective arthroscopic surgery of the knee because of meniscus tear and/or cartilage damage were examined preoperatively and at days 4 and 7 after surgery. Examinations of the not injured and not treated leg focused on measurements of leg circumference and maximum strength of M. quadriceps femoris. RESULTS: The measurements of circumferences of the upper leg at 20 cm above the knee joint line decreased significantly between preoperative and both postoperative examinations. Also the changing of circumference of the upper leg at 10 cm above the joint line between preoperative and day 4 postoperative was significant. The circumference of lower leg and strength measurements showed no significant changing. CONCLUSIONS: Limited, reduced mobility lasting a few days is followed by reduction of muscle cross section of load bearing upper leg muscles.

Dental abnormalities in Schimke immuno-osseous dysplasia.

Described for the first time in 1971, Schimke immuno-osseous dysplasia (SIOD) is an autosomal-recessive multisystem disorder that is caused by bi-allelic mutations of SMARCAL1, which encodes a DNA annealing helicase. To define better the dental anomalies of SIOD, we reviewed the records from SIOD patients with identified bi-allelic SMARCAL1 mutations, and we found that 66.0% had microdontia, hypodontia, or malformed deciduous and permanent molars. Immunohistochemical analyses showed expression of SMARCAL1 in all developing teeth, raising the possibility that the malformations are cell-autonomous consequences of SMARCAL1 deficiency. We also found that stimulation of cultured skin fibroblasts from SIOD patients with the tooth morphogens WNT3A, BMP4, and TGFß1 identified altered transcriptional responses, raising the hypothesis that the dental malformations arise in part from altered responses to developmental morphogens. To the best of our knowledge, this is the first systematic study of the dental anomalies associated with SIOD.

[Severe airway distress following cervical spine operation: retrospective breakdown of the chain of errors]. / Dramatischer Atemwegsnotfall nach Operation an der Halswirbelsäule : Retrospektive Analyse der Fehlerkette.

A 71-year-old female patient received a prothesis due to a cervical disc prolapsed and bleeding into the collar soft tissues occurred postoperatively. Following a computed tomography examination severe peracute respiratory decompensation occurred while administering topical anesthesia to the pharynx in order to perform fiber optic intubation. Endotracheal intubation using conventional laryngoscopy was unsuccessful and the patient required immediate cricothyroidotomy. As an on-site cricothyrotomy set to establish a secure airway was not available the decision was taken to perform surgical cricothyroidotomy. As a conclusion to this life-threatening event in the case of symptoms, such as dyspnea, dysphonia and dysphagia after operations of the cervical spine the airway has to be secured early and according to the local algorithm.

[Adoptive T-cell therapy of rhabdomyosarcoma]. / Adoptive T-Zell-Therapie des Rhabdomyosarkoms.

AIMS: To improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen. METHODS: A "second generation" chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis. RESULTS: Co-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the "second generation" AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR. CONCLUSIONS: Data suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.

Stem cells in the trabecular meshwork: present and future promises.

Primary open-angle glaucoma is recognized as a disease of aging, and studies show a relationship between aging and trabecular meshwork (TM) cell density. Human TM cell division occurs primarily in the anterior, non-filtering region. A commonly used glaucoma treatment, laser trabeculoplasty (LTP), triggers and increases cell division, as well as cell migration of these anterior TM cells. These freshly-divided migrating cells repopulate the burned laser sites, suggesting that they are stem cells. Several studies concerning this putative TM stem cell will be discussed.

The impact of head position on the cuff and tube tip position of preformed oral tracheal tubes in young children.

Head and neck movements affect both the length of the trachea and the position of tracheal tubes. This is of relevance when using cuffed tubes because changes in the position of the tube tip may not be equal to changes in the position of the cuff. The aim of the study was to assess the impact of head and neck movement on the position of the tube tip and the cuff of newly designed, oral preformed tracheal tubes in children. The tracheas of 128 children aged 1-8 years were intubated with preformed oral tubes. The distances 'carina-to-tracheal tube tip' and 'vocal cords-to-tube tip' were measured endoscopically. These measurements were performed with the head and neck in a functional neutral position (110 degrees ), during neck flexion (80 degrees ) and neck extension (130 degrees ). Tracheal length was dependent on head and neck position: neck extension elongated the trachea (p < 0.0001), and neck flexion shortened the trachea (p < 0.0001). Neck flexion moved the tube inward and resulted in endobronchial displacement in two patients. Neck extension moved the tube outwards. While no cuff was positioned between the vocal cords, cuff movement to the cricoid area occurred frequently. Complex interactions during head and neck movement along with the fixed insertion depth of preformed tubes often cause inadvertent malpositioning of the tube tip and cuff. Further changes to tube and cuff lengths might improve the safety of oral preformed tubes in children.

High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome.

BACKGROUND: In patients with juvenile polyposis syndrome (JPS) the frequency of large genomic deletions in the SMAD4 and BMPR1A genes was unknown. METHODS: Mutation and phenotype analysis was used in 80 unrelated patients of whom 65 met the clinical criteria for JPS (typical JPS) and 15 were suspected to have JPS. RESULTS: By direct sequencing of the two genes, point mutations were identified in 30 patients (46% of typical JPS). Using MLPA, large genomic deletions were found in 14% of all patients with typical JPS (six deletions in SMAD4 and three deletions in BMPR1A). Mutation analysis of the PTEN gene in the remaining 41 mutation negative cases uncovered a point mutation in two patients (5%). SMAD4 mutation carriers had a significantly higher frequency of gastric polyposis (73%) than did patients with BMPR1A mutations (8%) (p<0.001); all seven cases of gastric cancer occurred in families with SMAD4 mutations. SMAD4 mutation carriers with gastric polyps were significantly older at gastroscopy than those without (p<0.001). In 22% of the 23 unrelated SMAD4 mutation carriers, hereditary hemorrhagic telangiectasia (HHT) was also diagnosed clinically. The documented histologic findings encompassed a wide distribution of different polyp types, comparable with that described in hereditary mixed polyposis syndromes (HMPS). CONCLUSIONS: Screening for large deletions raised the mutation detection rate to 60% in the 65 patients with typical JPS. A strong genotype-phenotype correlation for gastric polyposis, gastric cancer, and HHT was identified, which should have implications for counselling and surveillance. Histopathological results in hamartomatous polyposis syndromes must be critically interpreted.

The use of reverse transcriptase for efficient first- and second-strand cDNA synthesis from single- and double-stranded RNA templates.

Molecular characterization of eight distinct, difficult-to-clone RNA plant viruses was accomplished after the development of a reverse transcriptase-based first- and second-strand cDNA synthesis method. Double-stranded (ds) RNA templates isolated from strawberry and blackberry and several herbaceous hosts (mint, pea and tobacco) were cloned using this method. Templates, combined with random primers, were denatured with methyl mercuric hydroxide. Reverse transcriptase was added followed by the addition of RNase H. The resulting dsDNA was then digested with restriction endonucleases to produce shorter fragments that could be cloned efficiently into a T-tailed vector after adding an A-overhang using Taq polymerase. This procedure resulted in a high number of cloned fragments and allowed insert sizes up to three kilobase-pairs. Unlike traditional cDNA construction methods, there is no need for additional enzymes/steps for second-strand synthesis, PCR amplification or prior sequence information. Synthesis and cloning of cDNA derived from dsRNA templates is much more efficient than with previously described methods. This procedure also worked well for cloning gel-purified dsRNA and with single-stranded RNA templates.