Comparison of gas-liquid chromatography and EMIT assay for serum valproic acid

We describe a simple direct extraction method for the gas-liquid chromatography determination of serum valproic acid. The working range for the assay is 2-180 mg/L and our within-run precision was 5.8 and 4.3% at the 40 and 90 mg/L concentrations respectively. Hemolyzed and lipemic sera as well as samples from patients with hyperbilirubinemia and from patients with decreased renal function were put through the assay and no interfering peaks were noted. Interference occurred when teflon-lined screw caps were used during the extraction step. The method was proven to be accurate by linear regression analysis of samples containing weighed-in amounts of valproic acid. The above assay was compared to an enzyme immunoassay technique (EMIT). The working range for the latter is 10-150 mg/L and the with-run precision was 10.8 and 5.9% and 90 mg/L concentration respectively. Samples were run by both the gas-liquid chromatograph and enzyme immunoassay methods and gave very similar results over the range 16-139 mg/L.     

Lack of directed Vα14-Jα281 rearrangements in NK1+ T cells

NK1.1⁺ T cells are an unusual subset of TCRαβ cells distinguished by their highly restricted Vβ repertoire and predominant usage of an invariant Vα14-Jα281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant α chain, we have analyzed Vα14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1⁺ T cells. As expected a high proportion (17/20) of the hybrids had rearranged Vα14 to Jα281. However, Vα14-Jα281 rearrangements always occurred on only one chromosome and were accompanied by other Vα-Ja rearrangements (not involving Vα14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of Vα14-Jα281 rearrangements in NK1.1⁺ T cells.     

Systolic arterial pressure determination by a new pulse monitor technique

The Doppler ultrasound (DUS) technique is a widely accepted non-invasive technique to estimate systolic blood pressure (SBP) accurately in paediatric patients. The DUS has a number of limitations. A new pulse monitor, Mr Pulse (MP), operating on the principle of a finger plethysmograph, was developed to offer an alternative technique to estimate SBP. From 104 paired SBP measurements taken in 16 paediatric patients undergoing general anaesthesia, SBP determined by the MP technique correlated closely with that by the standard DUS technique (r2 = 0.98). Analysis of degree of agreement performed indicated that there was good agreement between SBP obtained by the MP and the DUS techniques. The mean +/- standard deviation of differences in paired SBP values between the two measurement techniques was 0.55 +/- 3.59 mmHg. Mr Pulse is as accurate as the DUS technique in estimating SBP and has the advantage of less critical sensor positioning as it is not subject to electrical interference. It has no electrical hazard.     

Criterios específicos de respuesta tumoral

Recent advances in biology and genetics have accelerated our knowledge about the development, growth, and dissemination of cancer, generating great expectations that these new discoveries will be translated into effective treatments for patients. Imaging techniques play a central role in the care of oncologic patients, since they have become tools capable of evaluating important characteristics of tumors and the response of tumors to different treatments. The objective of this article is to evaluate the different imaging-based criteria for assessing tumor response, discussing their advantages and limitations and illustrating the possible contribution of new imaging techniques as biomarkers of tumor response.     

Comparative structural analysis of human Nav1.1 and Nav1.5 reveals mutational hotspots for sodium channelopathies

Among the nine subtypes of human voltage-gated sodium (Na_v) channels, the brain and cardiac isoforms, Na_v1.1 and Na_v1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure–function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Na_v1.1–β4 complex at 3.3 Å resolution here and the Na_v1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Na_v1.1 and Na_v1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Na_v1.1 and Na_v1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies.

The nine subtypes of human voltage-gated sodium (Na_v) channels are responsible for the initiation and transmission of electrical impulses in different tissues: Na_v1.1 to Na_v1.3 and Na_v1.6 mainly function in the central nervous system, Na_v1.7 to Na_v1.9 are mostly distributed in the peripheral nervous system, Na_v1.4 is specialized in skeletal muscle, and Na_v1.5 is the primary cardiac isoform (1–4). Abnormalities of these channels, hinging on their tissue specificity, are associated with a broad spectrum of channelopathies. To date, more than 1,000 disease mutations have been identified in the primary sequence of Na_v channels, among which Na_v1.1 and Na_v1.5 each host more than 400 missense mutations (5–8).Na_v1.1 is encoded by SCN1A, which may have the largest number of epilepsy-related mutations. Up to 900 SCN1A mutations, more than half of which result in truncations (9), have been identified in epilepsy syndromes with different severities. Nonsense and hundreds of missense mutations of SCN1A are found in 70 to 80% of patients with Dravet syndrome, which is also known as the severe myoclonic epilepsy of infancy (10–13) (SI Appendix, Table S1). Several dozen missense mutations are associated with generalized epilepsy with febrile seizures plus and intractable childhood epilepsy with generalized tonic-clonic seizures (10) (SI Appendix, Table S2). Although most of the Na_v1.1 disease mutations lead to loss of function to different degrees, some represent gain of function. In most cases, the pathogenic mechanism remains elusive.A brief summary of Na_v1.5 pathophysiology is presented in the companion paper (14). Mechanistic understanding of the sodium channelopathies entails high-resolution structures of human Na_v channels. In the past 4 y, we have reported the cryoelectron microscopy (cryo-EM) structures, at resolutions ranging between 2.6 and 4.0 Å, of Na_v channels from insect (Na_vPaS), electric eel (EeNa_v1.4), and finally human, including Na_v1.2, Na_v1.4, Na_v1.5, and Na_v1.7, in the presence of multiple modulators, such as β1 and β2 subunits, peptide toxins, and small-molecule toxins tetrodotoxin and saxitoxin (15–21). Structures of a truncated rat Na_v1.5 were recently reported (22). All structurally resolved eukaryotic Na_v channels except for Na_vPaS exhibit similar conformations of potentially inactivated state.Notwithstanding these advances, high-resolution structures of human Na_v1.1 and Na_v1.5 wild-type and representative disease variants are necessary to provide accurate templates to directly map the disease mutations and to facilitate drug discovery. Furthermore, as these two channels harbor 80% of all identified mutations related to sodium channelopathies, a comparative analysis of their structures may reveal potential mutational hotspots, offering invaluable insight into the function and disease mechanism of Na_v channels.Here we present the cryo-EM structure of human Na_v1.1 associated with a modulating auxiliary subunit β4. In the accompanying paper, we report the structure of human Na_v1.5 that carries a common disease variant E1784K. Comparative structural analyses have revealed several clusters of disease mutations that are common to both Na_v1.1 and Na_v1.5.     

Obesity and lipoprotein cholesterol in the Framingham offspring study

This study examines the relationship between obesity and low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and very-low-density lipoprotein (VLDL) cholesterol in 4260 young adult men and women. The strongest association between obesity and LDL cholesterol was found in 20–29 yr-old males, the weakest in 40–49-yr-old males. Conversely, in women the relationship between LDL cholesterol and obesity was modest except in the oldes (40–49 yr) age group. An inverse relationship between obesity and HDL cholesterol was of similar shape and strength in all sex and age-specific groups. When the ratio of total cholesterol (TCHOL) to HDL cholesterol was compared in lean and grossly obese 20–29-yr-old males, substantial differences were found. Since other data show this index of the lipoprotein profile to be the single best indicator of CHD risk, it would appear that the atherogenic potential of obesity is greater than would be suggested by the relatively weak association between obesity and TCHOL or any single lipoprotein cholesterol. These data also suggest that the impact of obesity as a risk factor for CHD may have been underestimated. The paucity of lean males 40–49-yr-old prevents firm conclusions about the CHD risk in such a group. Indirect evidence indicates that lean 40–49-yr-old men would have a markedly more favorable lipoprotein profile and consequently a much lower risk of CHD.     

PER extended-spectrum β-lactamases originate from Pararheinheimera spp

To investigate the origin of PER extended-spectrum β-lactamases, publicly available sequence databases were searched for bla_PER-like genes. Three genomes from Pararheinheimera, a genus associated with water and soil environments, were found to carry bla_PER-like genes but lacked the ISCR1/ISPa12/ISPa13 insertion sequences commonly associated with bla_PER in clinical isolates. Sequence analysis revealed 78–96% nucleotide identity and conserved synteny between the clinical mobile genetic elements (MGEs) encoding bla_PER-1 and the bla_PER locus in the Pararheinheimera genomes. Notably, bla_PER genes were only identified in 3 of 21 Pararheinheimera and Rheinheimera genomes, whereas the genetic environment of bla_PER genes as found in clinical MGEs was conserved in all Pararheinheimera and Rheinheimera genomes. These findings indicate that bla_PER genes were likely acquired by a branch of the Pararheinheimera genus long before the antibiotic era. Later, bla_PER genes were mobilised, likely through the involvement of insertion sequences, from one or several Pararheinheimera species, allowing their dissemination into human pathogens.