Improved diagnostic effectiveness with a sequential diagnostic paradigm in idiopathic pediatric sensorineural hearing loss.

OBJECTIVES: To determine whether a stepwise diagnostic paradigm is more diagnostically efficient and cost-effective than a simultaneous testing approach in the evaluation of idiopathic pediatric sensorineural hearing loss (SNHL). DESIGN: Prospective prevalence study. SETTING: Tertiary referral children's hospital. PATIENTS: Consecutive children (n = 150) presenting with idiopathic SNHL in the last 2 years. INTERVENTIONS: All children were evaluated with full diagnostic evaluations including GJB2 screens, temporal bone computed tomography scans, and laboratory investigations. MAIN OUTCOME MEASURES: 1) Diagnostic yields of GJB2 screens, imaging, and laboratory results per SNHL category; 2) Cost analysis comparing a sequential versus a simultaneous testing approach. RESULTS: Overall, 12.0% of patients had biallelic mutations in the GJB2 gene, whereas 30% of patients had an abnormality on temporal bone scan. Laboratory testing did not reveal the SNHL etiology in any patient. While maintaining diagnostic accuracy, significant cost savings were inferred by using a sequential diagnostic algorithm. Our data show children with severe to profound SNHL should first be tested with a GJB2 screen, as opposed to those with milder SNHL, who should undergo imaging as the initial testing step. In patients with initially positive GJB2 or imaging screens, logistic regression analysis significantly predicted negative results on further testing. CONCLUSIONS: A stepwise diagnostic paradigm tailored to the level of the hearing loss in children with bilateral SNHL is more diagnostically efficient and cost effective than the more commonly used full, simultaneous testing approach. Laboratory investigation should not be routine but based on clinical history.     

Sequence and chromosomal localization of two PET genes required for cytochrome c oxidase assembly in Saccharomyces cerevisiae

Summary The nuclear genes PET117 and PET191 are required for the assembly of active cytochrome c oxidase in S. cerevisiae, yet their gene products are not subunits of the final assembled cytochrome c oxidase complex. Plasmids bearing PET117 or PET191 were isolated by their ability to complement the pet117-1 or pet191-1 mutations, respectively. By restriction mapping, subcloning, and deletion analysis of yeast DNA fragments that complement these mutations, the PET117 and PET191 genes were localized to smaller regions of DNA, which were then sequenced from both strands. The PET117 open reading frame is of 107 codons and the PET191 open reading frame is of 108 codons. Neither the PET191 nor PET117 DNA sequences have been reported previously, and the derived amino-acid sequences of the PET191 and PET117 open reading frames exhibit no significant primary amino-acid sequence similarity to other protein sequences available in the NBRF data base, or from translated Genbank sequences. By hybridization of PET117 or PET191 probes first to a chromosome blot and next to a library of physically mapped fragments of yeast genomic DNA, the map locations of the PET191 and PET117 genes were determined. PET117 is located on chromosome V near the HIS1 gene and PET191 is located on chromosome X near the CYC1 gene.     

Association between the TAP1 gene polymorphisms and recurrent respiratory papillomatosis in patients from Western Mexico: A pilot study

BackgroundRecurrent respiratory papillomatosis (RRP) is a respiratory tract disease that affects children and adults and is characterized by the recurrent proliferation of multiple papillomas. The etiologic agent is the human papillomavirus, mainly genotypes 6 and 11. Furthermore, polymorphisms in TAP1 appear to influence the selection of antigenic peptides and the transport process to the rough endoplasmic reticulum, for their subsequent presentation to T lymphocytes, an essential process against viral diseases and tumor processes. Previous studies have shown that individuals with those polymorphisms are susceptible to immune, infectious, and tumor‐related diseases. The present study aimed to determine the association between the TAP1 rs1057141 (c.1177A>G) and rs1135216 (c.2090A>G) single nucleotide polymorphisms (SNPs) and RRP.MethodsA case–control study was carried out on a group of 70 individuals (35 controls and 35 patients). RRP diagnosis, HPV genotyping, and viral load were determined through histology and PCR. SNPs rs1057141 and rs1135216 were identified through allelic discrimination, using real‐time PCR. The haplotypic analyses were performed using the Arlequin 3.5 program.ResultsHPV‐6 and HPV‐11 were the genotypes found in the samples. In the polymorphism analysis, rs1057141 showed no significant differences (p = 0.049, CI = 0.994–7.331). In contrast, a significant difference was found in rs1135216 (p = 0.039, OR = 2.4) in the allelic analysis, as well as in the dominant (p = 0.027, OR = 3.06), codominant (p = 0.033, OR = 3.06), and additive model (p = 0.043, OR = 2.505) in subjects with the G allele.ConclusionThe G allele in rs1135216 was associated with a genetic risk of susceptibility for RRP in a population in Western Mexico.     

Comprehensive Evolutionary Analysis of Complete Epstein–Barr Virus Genomes from Argentina and Other Geographies

The sequence variability of the Epstein–Barr virus has been extensively studied throughout previous years in isolates from various geographic regions and consequent variations at both genetic and genomic levels have been described. However, isolates from South America were underrepresented in these studies. Here, we sequenced 15 complete EBV genomes that we analyzed together with publicly available raw NGS data for 199 EBV isolates from other parts of the globe by means of a custom-built bioinformatic pipeline. The phylogenetic relations of the genomes, the geographic structure and variability of the data set, and the evolution rates for the whole genome and each gene were assessed. The present work contributes to overcoming the scarcity of complete EBV genomes from South America and is the most comprehensive geography-related variability study, which involved determining the actual contribution of each EBV gene to the geographic segregation of the entire genome. Moreover, to the best of our knowledge, we established for the first time the evolution rate for the entire EBV genome based on a host–virus codivergence-independent assumption and assessed their evolution rates on a gene-by-gene basis, which were related to the encoded protein function. Considering the evolution of dsDNA viruses with a codivergence-independent approach may lay the basis for future research on EBV evolution. The exhaustive bioinformatic analysis performed on this new dataset allowed us to draw a novel set of conclusions regarding the genome evolution of EBV.     

Developing an interactive and illustrative laboratory management curriculum for cytopathology trainees

Laboratory management is a critically important but often overlooked portion of cytopathology training. Indeed, recent surveys of new‐in‐practice pathologists have consistently shown this to be an area of deficiency. Fortunately, there are a multitude of resources available to fill this need.^1‐6 These resources, such as government websites and publications by professional organizations, are often freely accessible to everyone; however, the information they provide is scattered across the vast expanse of cyberspace. We offer this educational module to bridge those gaps and string together a web of resources. The curriculum content includes a pretest to assess the trainee's knowledge and a tree of clickable subject headings covering basic laboratory management topics that a graduating cytopathology fellow should be familiar with. Each subject heading is linked to a summary of the subject using tables and visual diagrams with hyperlinks to specific online resources and additional detailed information. A posttest is included to provide instant feedback. Although by no means comprehensive, we hope this will provide a stepping stone for our readers to build a sound laboratory management foundation that may guide their practice.     

Dietary and nutraceutical approach to type 2 diabetes

Nutritional medical treatment is the first step to achieve adequate glycemic control and prevent diabetic complications. Lifestyle changes include moderate weight loss (7%) and regular physical activity (150 min/week). The appropriate diet composition is < 30% total fat, < 10% saturated fats, > 15 g/1000 kcal fiber, half soluble, 45–60% of carbohydrates with amoderate intake of sugar (50 g/day) and protein intake of 15–20% of the total calories a day. Patients need to limit the intake of saturated fats to < 7% of the daily calorie intake. Monounsaturated fatty acids such as olive oil and other vegetable oils are recommended. L-carnitine, α-lipoic acid, berberine and ω-3 fatty acids can be useful supplements.     

Cross-linkers at growing microtubule ends generate forces that drive actin transport

The actin and microtubule cytoskeletons form active networks in the cell that can contract and remodel, resulting in vital cellular processes such as cell division and motility. Motor proteins play an important role in generating the forces required for these processes, but more recently the concept of passive cross-linkers being able to generate forces has emerged. So far, these passive cross-linkers have been studied in the context of separate actin and microtubule systems. Here, we show that cross-linkers also allow actin and microtubules to exert forces on each other. More specifically, we study single actin filaments that are cross-linked to growing microtubule ends, using in vitro reconstitution, computer simulations, and a minimal theoretical model. We show that microtubules can transport actin filaments over large (micrometer-range) distances and find that this transport results from two antagonistic forces arising from the binding of cross-linkers to the overlap between the actin and microtubule filaments. The cross-linkers attempt to maximize the overlap between the actin and the tip of the growing microtubules, creating an affinity-driven forward condensation force, and simultaneously create a competing friction force along the microtubule lattice. We predict and verify experimentally how the average transport time depends on the actin filament length and the microtubule growth velocity, confirming the competition between a forward condensation force and a backward friction force. In addition, we theoretically predict and experimentally verify that the condensation force is of the order of 0.1 pN. Thus, our results reveal an active mechanism for local actin remodeling by growing microtubules that relies on passive cross-linkers.

Vital cellular processes such as cell division and motility are driven by contraction and remodeling of active networks within the cell: the actin and microtubule cytoskeletons. These contraction and remodeling processes require the generation of forces and relative movement of filaments. It is well known that motor proteins can cross-link filaments and organize the network by driving the relative movements of the filaments (1). Besides the well-appreciated role of motor proteins, also polymerization dynamics have been shown to generate forces required for self-organization and remodeling (2–4). In addition, the importance of passive (nonmotor) cross-linkers has recently been increasingly recognized. It is now clear that passive cross-linkers can generate driving forces too, as has been shown for anillin in the contractile ring (5) and Ase1 in the spindle (6). These forces are generated via the entropy associated with the cross-linker’s diffusion within the overlap region (6) or via the condensation (or preferential binding) of cross-linkers to the overlap between filaments (5, 6), therefore named “condensation force.” In addition, passive cross-linkers create frictional forces (6, 7), which not only affect the speed at which cytoskeletal structures are remodeled, but can even be essential for their stability by opposing the motor proteins (8, 9).Prior studies on force generation by passive cross-linkers have focused on individual cytoskeletal systems. However, passive cross-linkers could also be a way for two systems to exert forces on each other. In particular, there is a growing body of work showing the importance of microtubule/actin crosstalk in vital cellular functions (10). Besides crosstalk via signaling pathways, it has been shown that crosstalk via motor proteins such as myosin-X (Myo10) is among the drivers of remodeling the microtubule spindle network during cell division (11, 12). In addition, various passive cross-linking proteins have been identified to enable crosstalk, such as the family of spectraplakins, which are proteins that can bind both actin and microtubules. The most prominent and best-studied member is ACF7 (MACF1) and its Drosophila homolog Short stop (Shot), which contain both microtubule lattice- and end-binding activities. These ubiquitous and conserved cross-linkers (traced back to lower metazoans; ref. 13) play a crucial role in a number of cellular processes, such as cell migration, cell–cell connections, vesicular transport, cell polarity, and cell division (10, 14). For example in the context of cell migration, ACF7 is shown to anchor microtubules at the cell’s leading edge (15) and to guide microtubules along actin bundles toward focal adhesions, where ACF7 depletion resulted in a phenotype with lost coalignment and failure of microtubules to target focal adhesions (16). In addition to ACF7, also other passive cross-linking proteins such as tau and Gas2Like1 have been shown to result in coalignment and stabilization of the actin and microtubule networks (16–19). Another example of a microtubule end-binding cross-linker that links actin to microtubule ends via EB3-mediated interactions is drebrin, which is required for sprouting neurites from a neuronal cell (20).Recent experiments suggest that the driving condensation forces and friction forces associated with passive cross-linkers can also couple to the growth dynamics of filaments and thereby allow passive cross-linkers to play a central role not only in contraction but also in transport. It was shown that an engineered passive cross-linker called TipAct can mediate transport of actin filaments by binding to the tips of growing microtubules (17). The microtubule tip region is chemically different from the microtubule lattice region, due to delayed hydrolysis of guanosine triphosphate (GTP) that is associated with tubulin subunits that incorporate into growing microtubules. This region is recognized by microtubule end-binding (EB) proteins (21–23) as well as EB partners such as TipAct. However, the processivity of the actin transport mechanism and its dependency on relevant parameters such as actin filament length and microtubule growth velocity, as well as the magnitude of the generated forces, are currently unresolved.Here, we investigate the conditions necessary for microtubule-mediated actin transport and elucidate the cross-linker–dependent mechanism. We combine biochemical reconstitution experiments with coarse-grained computer simulations and a minimal theoretical model and show that in the presence of passive cross-linkers, microtubules can transport actin filaments over large (micrometer-range) distances during periods that last up to several minutes. We propose and test a mechanism to explain this movement, in which actin transport is the result of a competition between a forward force that tends to maximize the overlap of the actin filament with the plus end of a growing microtubule and a backward force caused by the friction between the actin and microtubule lattice. These two antagonistic forces are both caused by the binding of cross-linkers. The preferential binding of cross-linkers at the interface between two objects (such as filaments) creates a driving force that tends to maximize the overlap region. This type of force, which, following earlier work (6, 7), we call condensation force, can drive the movement of intracellular cargos (2, 24–26) and the contraction of filament bundles (5, 6, 27, 28) in various biological contexts. However, the combination of a processive transport of cargo by a cross-linker–based condensation force toward a chemically distinct region that is simultaneously hindered by a friction force caused by the same cross-linkers is specific to filament transport and has to our knowledge not been studied before. The active transport of filaments by growing microtubule plus ends thus provides a minimal mechanism by which two cytoskeletal systems can interact, which is distinct from cytoskeletal crosstalk that is driven by motor proteins (11, 29–32). We expect this mechanism to be especially relevant in migrating cells, where growing microtubules nucleate actin filaments and encounter membrane-bound actin arrays.     

A multi-element analysis of language facilitation games with educationally deprived Mexican migrant workers

A behavior analysis was conducted to identify simple ways to promote language use among non-English-speaking migrant workers during relatively unstructured periods of an Adult Basic Education class. The context for the analysis was a language facilitation game which involved a modified version of “Go Fish” requiring the migrants to match English words printed on cards. This game and two variations involving a simple alteration of the stimulus on the cards and changes in the game's rules were examined. A multi-element analysis revealed that more English and less Spanish was spoken as a function of the complexity of both the antecedent and consequent stimuli involved in the games.