Linear and nonlinear optical properties of transfer ribonucleic acid (tRNA) thin solid films

We successfully obtained transfer ribonucleic acid (tRNA) thin solid films (TSFs) using an aqueous solution precursor in an optimized deposition process. By varying the concentration of RNA and deposition process parameters, uniform solid layers of solid RNA with a thickness of 30 to 46 nm were fabricated consistently. Linear absorptions of RNA TSFs on quartz substrates were experimentally investigated in a wide spectral range covering UV–VIS–NIR to find high transparency for λ > 350 nm. We analyzed the linear refractive indices, n(λ) of tRNA TSFs on silicon substrates by using an ellipsometer in the 400 to 900 nm spectral range to find a linear correlation with the tRNA concentration in the aqueous solution. The thermo-optic coefficient (dn/dT) of the films was also measured to be in a range −4.21 × 10⁻⁴ to −5.81 × 10⁻⁴ °C⁻¹ at 40 to 90 °C. We furthermore characterized nonlinear refractive index and nonlinear absorption of tRNA TSFs on quartz using a Z-scan method with a femtosecond laser at λ = 795 nm, which showed high potential as an efficient nonlinear optical material in the IR spectral range.

Optical measurements of one of the vital biological molecules (RNA) in the human body.     

Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.     

The thickness of facial alveolar bone overlying healthy maxillary anterior teeth

Background: A facial bone (<2 mm) overlying maxillary anterior teeth may be prone to resorptive processes after extraction and immediate implant placement. A thin bone contributes to risk of bone fenestration, dehiscence, and soft‐tissue recession. This study measures the distance between the cemento‐enamel junction (CEJ) and alveolar bone crest and the thickness of facial alveolar bone at points 1 to 5 mm from the bone crest for the six maxillary anterior teeth. Methods : Sixty‐six tomographic scans (31 males and 35 females; aged 17 to 69 years; mean age: 39.9 years) of intact anterior maxilla were randomly selected and evaluated by two calibrated and independent examiners (MG and TP). Results: A high variation of CEJ–bone crest (0.8 to 7.2 mm) was detected. A significantly larger CEJ–bone crest was measured in smokers (P <0.05) and patients who were ≥50 years old (P <0.05). The average bone thickness at 3 mm from the CEJ for the maxillary right central incisor was 1.41 mm and for the maxillary left central incisor was 1.45 mm. For the maxillary right and left lateral incisors, the crestal bone thickness averaged 1.73 and 1.59 mm, respectively. For the maxillary right and left canines, the crestal bone thickness averaged 1.47 and 1.60 mm, respectively. Conclusions : The present study supports the finding of a predominantly thin facial bone overlying the six maxillary anterior teeth. Therefore, it is essential to make informed treatment decisions based on thorough site evaluation before immediate implant placement.     

Impairment of movement initiation and execution but not preparation in idiopathic dystonia

Imaging studies have reported impaired activation of the striatum and their frontal projection sites in dsytonia, areas which are considered to play a role in motor preparation, movement initiation and execution. The aim of this study was to investigate the processes of motor preparation, response initiation and execution in patients with idiopathic torsion dystonia (ITD). We assessed 12 patients with ITD and 12 age-matched controls on a number of reaction time (RT) tasks that differed in degree of motor preparation possible. Subjects performed a visual simple RT (SRT) task, an uncued four-choice reaction time (CRT) task and a fully precued four-choice RT task. A stimulus 1-stimulus 2 (S1-S2) paradigm was used. The warning signal/precue (S1) preceded the imperative stimulus (S2) by either 0 ms (no warning signal or precue) 200 ms, 800 ms, 1,600 ms or 3,200 ms. The patients with ITD had significantly slower RTs and movement times than normals across all RT tasks. The unwarned SRT trials were significantly faster than the uncued CRT trials for both groups. For both groups, precued CRTs were significantly faster than the uncued CRTs. The results show that while response initiation and execution are significantly slower in patients with ITD than normals, movement preparation is not quantitatively or qualitatively different. The results are discussed in relation to previous imaging, behavioural and electrophysiological studies and models of fronto-striatal dysfunction in ITD.     

Functional analysis of novel KCNQ2 mutations found in patients with Benign Familial Neonatal Convulsions

Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant α-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.     

Silencing the major apple allergen Mal d 1 by using the RNA interference approach

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.     

Identification and susceptibility testing of Enterobacteriaceae and Pseudomonas aeruginosa by direct inoculation from positive BACTEC blood culture bottles into Vitek 2

Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the "gold standard." We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture.     

Hermansky-Pudlak syndrome type 4 in a patient from Sri Lanka with pulmonary fibrosis

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism and a platelet storage pool deficiency. Some patients also develop fatal pulmonary fibrosis and some have granulomatous colitis. Six human genes HPS1, ADB3A, HPS3, HPS4, HPS5, and HPS6 have been identified as cause of the six known subtypes of HPS. While there exist nearly 500 Puerto Rican and non-Puerto Rican HPS-1 patients, very few HPS-4 patients have been reported, and most of these have not been described in detail. We now delineate the clinical characteristics of an HPS-4 patient homozygous for a novel HPS-4 mutation, P685delC. The patient, the first individual with HPS reported from Sri Lanka, had severe pulmonary fibrosis, typical of HPS-1 disease, without granulomatous colitis. We conclude that pulmonary fibrosis occurs as part of HPS-4 and that HPS should be considered in all ethnic groups.