Cell-free soluble expression of the membrane protein PsbS.

Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photochemical quenching for photoprotection of plants under high-light conditions. The activation mechanism of PsbS and its pH-induced conformational changes are currently unknown. For structural investigation of PsbS, effective synthesis of PsbS with selective isotope or electron-spin labels or non-natural amino acids incorporated would be a great asset. Here we present cell-free (CF) expression as a successful method for in vitro production of PsbS that would allow such incorporation. The addition of several detergents, liposomes and lipid nanodiscs was tested for achieving soluble CF expression of PsbS. We have optimized the CF method to yield soluble PsbS of ∼500 ng/µl using a continuous-exchange method at 30 °C, along with a successful purification and refolding of PsbS in n-Dodecyl ß-D-maltoside (ß-DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.

Red Blood Cell Distribution Width and the Platelet Count in Iron-deficient Children Aged 0.5-3 Years.

Early detection of iron deficiency (ID) and iron deficiency anemia (IDA) in young children is important to prevent impaired neurodevelopment. Unfortunately, many biomarkers of ID are influenced by infection, thus limiting their usefulness. The aim of this study was to investigate the value of red blood cell distribution width (RDW) and the platelet count for detecting ID(A) among otherwise healthy children. A multicenter prospective observational study was conducted in the Netherlands to investigate the prevalence of ID(A) in 400 healthy children aged 0.5-3 years. ID was defined as serum ferritin (SF) <12 µg/L in the absence of infection (C-reactive protein [CRP] <5 mg/L) and IDA as hemoglobin <110 g/L combined with ID. RDW (%) and the platelet count were determined in the complete blood cell count. RDW was inversely correlated with SF and not associated with CRP. Calculated cutoff values for RDW to detect ID and IDA gave a relatively low sensitivity (53.1% and 57.1%, respectively) and specificity (64.7% and 69.9%, respectively). Anemic children with a RDW >14.3% had a 2.7 higher odds (95% confidence interval [CI]: 1.2-6.3) to be iron deficient, compared with anemic children with a RDW <14.3%. The platelet count showed a large range in both ID and non-ID children. In conclusion, RDW can be helpful for identifying ID as the cause of anemia in 0.5- to 3-year-old children, but not as primary biomarker of ID(A). RDW values are not influenced by the presence of infection. There appears to be no role for the platelet count in diagnosing ID(A) in this group of children.

Anaesthesia and postoperative analgesia in surgical neonates with or without Down's syndrome: is it really different?

BACKGROUND: Reports conflict on optimal postoperative analgesic treatment in children with intellectual disability. We retrospectively compared postoperative analgesics consumption between neonates with and without Down's syndrome in relation to anaesthesia requirements and pain scores. METHODS: We analysed hypnotic and analgesic drug administration, pain scores [COMFORT-Behaviour (COMFORT-B) scale], and duration of mechanical ventilation during the first 48 h after surgical repair of congenital duodenal obstruction in neonates, between 1999 and 2011. Data of 15 children with Down's syndrome were compared with data of 30 children without Down's syndrome. RESULTS: General anaesthesia requirements did not differ. The median (inter-quartile range) maintenance dose of morphine during the first 24 h after operation was 9.5 (7.8-10.1) µg kg(-1) h(-1) in the Down's syndrome group vs 7.7 (5.0-10.0) µg kg(-1) h(-1) in the control group (P=0.46). Morphine doses at postoperative day 2 and COMFORT-B scores at day 1 did not significantly differ between the two groups. COMFORT-B scores at day two were lower in children with Down's syndrome (P=0.04). The duration of postoperative mechanical ventilation did not statistically differ between the two groups (P=0.89). CONCLUSIONS: In this study, neonates with and without Down's syndrome received adequate postoperative analgesia, as judged from comparable analgesic consumption and pain scores. We recommend prospective studies in children of different age groups with Down's syndrome and in other groups of intellectually disabled children to provide further investigation of the hypothesis that intellectual disability predisposes to different analgesic requirements.

Ultrasound as guidance for a combined bilateral supraclavicular and caudal block, in order to reduce the total anaesthetic dose in a two year old child after a pneumococcal sepsis.

We present a case of the combination of a bilateral supraclavicular block and a caudal block in a two year old boy who needed amputations of four extremities after a pneumococcal sepsis. With the use of ultrasound guidance, reduction of local anaesthetic dose could be obtained in order not to reach the toxic dose of the local anaesthetic. Amputations of four extremities is not common practice. A good postoperative pain management is more than a challenge.

The research gap in chronic paediatric pain: A systematic review of randomised controlled trials.

BACKGROUND AND OBJECTIVE: Chronic pain is associated with significant functional and social impairment. The objective of this review was to assess the characteristics and quality of randomized controlled trials (RCTs) evaluating pain management interventions in children and adolescents with chronic pain. METHODS: We performed a systematic search of PubMed, Embase and the Cochrane Library up to July 2017. We included RCTs that involved children and adolescents (3 months-18 years) and evaluated the use of pharmacological or non-pharmacological intervention(s) in the context of pain persisting or re-occurring for more than 3 months. Methodological quality was evaluated using the Cochrane Risk of Bias (ROB) Tool. RESULTS: A total of 58 RCTs were identified and numbers steadily increased over time. The majority were conducted in single hospital institutions, with no information on study funding. Median sample size was 47.5 participants (Q1,Q3: 32, 70). Forty-five percent of RCTs included both adults and children and the median of the mean ages at inclusion was 12.9 years (Q1,Q3: 11, 15). Testing of non-pharmacological interventions was predominant and only 5 RCTs evaluated analgesics or co-analgesics. Abdominal pain, headache/migraine and musculoskeletal pain were the most common types of chronic pain among participants. Methodological quality was poor with 90% of RCTs presenting a high or unclear ROB. CONCLUSIONS: Evaluation of analgesics targeting chronic pain relief in children and adolescents through RCTs is marginal. Infants and children with long-lasting painful conditions are insufficiently represented in RCTs. We discuss possible research constraints and challenges as well as methodologies to circumvent them. SIGNIFICANCE: There is a substantial research gap regarding analgesic interventions for children and adolescents with chronic pain. Most clinical trials in the field focus on the evaluation of non-pharmacological interventions and are of low methodological quality. There is also a specific lack of trials involving infants and children and adolescents with long-lasting diseases.

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-cis retinol dehydrogenase.

PURPOSE: Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS: A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS: The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS: Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

Rhinomanometric detection rate of rhinoscopically-assessed septal deviations.

Normal values for the flow at a transnasal pressure of 150 Pa were established with active anterior rhinomanometry (with decongestion) in a group of 33 normal subjects. These values were used to detect abnormalities in a group of 193 patients whose septum anatomy had been evaluated with rhinoscopy. About 25% of the rhinoscopically normal patients were found to have significantly low ("abnormal") flow values on one side. The same was true for patients with a small septal deviation restricted to one anatomical area. An abnormal flow was measured in about 35% of the patients with a moderate (restricted) septal deviation. In the patients whose septal deviation was not restricted to one anatomical area, about 45% had an abnormal flow. The highest detection rate was about 80% in patients with major deviations in the region of the vestibule and the valve. Such deviations were found only in a minority of the patients with complaints of nasal obstruction, which limits the importance of rhinomanometric evaluation in clinical practice.

The value of Ret-Hb and sTfR in the diagnosis of iron depletion in healthy, young children.

OBJECTIVES: Reticulocyte hemoglobin (Ret-Hb) content and soluble transferrin receptor (sTfR) are described as promising biomarkers in the analysis of iron status. However, the value of Ret-Hb and sTfR in the early detection of iron depletion, as frequently observed in children in high-income countries, is unclear. We hypothesized that young children to iron depletion, using the WHO cutoff of ferritin <12 µg/l, would have lower Ret-Hb and higher sTfR concentrations compared to children with a ferritin ⩾level 12 µg/l. SUBJECTS/METHODS: In this cross-sectional study, we analyzed mean concentrations of Ret-Hb and sTfR in 351 healthy children aged 0.5-3 years in a high-income country. The Student's t-test was used to compare Ret-Hb and sTfR concentrations between groups. RESULTS: We showed that concentrations of Ret-Hb and sTfR are similar in children with and without iron depletion. A decrease in Ret-Hb concentration was present only when ferritin concentrations were <8 µg/l. sTfR concentrations were similar in children with ferritin concentrations <6 µg/l and ⩾12 µg/l. CONCLUSIONS: Our results showed that the discriminative value of Ret-Hb and sTfR for the detection of iron depletion is limited. Our findings suggest that ferritin is the most useful biomarker in the screening of iron depletion in healthy children in high-income countries. However, ideally, reference ranges of iron status biomarkers should be based on studies showing that children with concentrations outside reference ranges have poor neurodevelopmental outcomes.

A double or bilobar gallbladder as a cause of severe complications after (laparoscopic) cholecystectomy.

A double or bilobar gallbladder is a rare congenital anomaly. If not recognized during preoperative evaluation or operation, it can cause severe complications. We describe two cases in which a second operation had to be performed because of the presence of a second or bilobar gallbladder that was not recognized in the preoperative evaluation and during (laparoscopic) cholecystectomy. The types of anomalies, the concomitant pathology, and treatment are discussed.