Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing

Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS

Bcl11b—A Critical Neurodevelopmental Transcription Factor—Roles in Health and Disease

B cell leukemia 11b (Bcl11b) is a zinc finger protein transcription factor with a multiplicity of functions. It works as both a genetic suppressor and activator, acting directly, attaching to promoter regions, as well as indirectly, attaching to promoter-bound transcription factors. Bcl11b is a fundamental transcription factor in fetal development, with important roles for the differentiation and development of various neuronal subtypes in the central nervous system (CNS). It has been used as a specific marker of layer V subcerebral projection neurons as well as striatal interneurons. Bcl11b also has critical developmental functions in the immune, integumentary and cardiac systems, to the extent that Bcl11b knockout mice are incompatible with extra-uterine life. Bcl11b has been implicated in a number of disease states including Huntington’s disease, Alzheimer’s disease, HIV and T-Cell malignancy, amongst others. Bcl11b is a fascinating protein whose critical roles in the CNS and other parts of the body are yet to be fully explicated. This review summarizes the current literature on Bcl11b and its functions in development, health, and disease as well as future directions for research

The use of a smartwatch as a prompting device for people with acquired brain injury : a single case experimental design study

Prompting-based memory compensation is a potential application for smartwatches. This study investigated the usability and efficacy of a Moto360 smartwatch as a memory aid. Four community dwelling adults with memory difficulties following acquired brain injury (ABI) were included in an A-B-A single case experimental design study. Performance of everyday memory tasks was tested over six weeks with the smartwatch and software provided during weeks three and four. Participants were asked to use their usual memory aids and strategies during the control phases (weeks 1–2, 5–6). Three participants successfully used the smartwatch throughout the intervention weeks and gave positive usability ratings. A fourth participant experienced a seizure and subsequently left the study before the intervention phase. Three participants showed improved memory performance when using the smartwatch. Nonoverlap of all pairs (NAP) analysis showed a non-significant small increase in memory performance between baseline and intervention phases (mean NAP = 0.1, p = .84). There was a larger, significant decline between the intervention and return to baseline (mean NAP = 0.58, p < .01). The use of an off-the-shelf smartwatch device and software was feasible for people with ABI in the community. It was effective compared to practice as usual, although this was only apparent on withdrawal of the device

The use of a smartwatch as a prompting device for people with acquired brain injury: A single case experimental design study

Prompting-based memory compensation is a potential application for smartwatches. This study investigated the usability and efficacy of a Moto360 smartwatch as a memory aid. Four community dwelling adults with memory difficulties following acquired brain injury (ABI) were included in an A-B-A single case experimental design study. Performance of everyday memory tasks was tested over six weeks with the smartwatch and software provided during weeks three and four. Participants were asked to use their usual memory aids and strategies during the control phases (weeks 1-2, 5-6). Three participants successfully used the smartwatch throughout the intervention weeks and gave positive usability ratings. A fourth participant experienced a seizure and subsequently left the study before the intervention phase. Three participants showed improved memory performance when using the smartwatch. Nonoverlap of all pairs (NAP) analysis showed a non-significant small increase in memory performance between baseline and intervention phases (mean NAP = 0.1, p = .84). There was a larger, significant decline between the intervention and return to baseline (mean NAP = 0.58, p < .01). The use of an off-the-shelf smartwatch device and software was feasible for people with ABI in the community. It was effective compared to practice as usual, although this was only apparent on withdrawal of the device

A FUSE Survey of Interstellar Molecular Hydrogen in the Small and Large Magellanic Clouds

We describe a moderate-resolution FUSE survey of H2 along 70 sight lines to the Small and Large Magellanic Clouds, using hot stars as background sources. FUSE spectra of 67% of observed Magellanic Cloud sources (52% of LMC and 92% of SMC) exhibit absorption lines from the H2 Lyman and Werner bands between 912 and 1120 A. Our survey is sensitive to N(H2) >= 10^14 cm^-2; the highest column densities are log N(H2) = 19.9 in the LMC and 20.6 in the SMC. We find reduced H2 abundances in the Magellanic Clouds relative to the Milky Way, with average molecular fractions = 0.010 (+0.005, -0.002) for the SMC and = 0.012 (+0.006, -0.003) for the LMC, compared with = 0.095 for the Galactic disk over a similar range of reddening. The dominant uncertainty in this measurement results from the systematic differences between 21 cm radio emission and Lya in pencil-beam sight lines as measures of N(HI). These results imply that the diffuse H2 masses of the LMC and SMC are 8 x 10^6 Msun and 2 x 10^6 Msun, respectively, 2% and 0.5% of the H I masses derived from 21 cm emission measurements. The LMC and SMC abundance patterns can be reproduced in ensembles of model clouds with a reduced H2 formation rate coefficient, R ~ 3 x 10^-18 cm^3 s^-1, and incident radiation fields ranging from 10 - 100 times the Galactic mean value. We find that these high-radiation, low-formation-rate models can also explain the enhanced N(4)/N(2) and N(5)/N(3) rotational excitation ratios in the Clouds. We use H2 column densities in low rotational states (J = 0 and 1) to derive a mean kinetic and/or rotational temperature = 82 +/- 21 K for clouds with N(H2) >= 10^16 cm^-2, similar to Galactic gas. We discuss the implications of this work for theories of star formation in low-metallicity environments. [Abstract abridged]Comment: 30 pages emulateapj, 14 figures (7 color), 7 tables, accepted for publication in the Astrophysical Journal, figures 11 and 12 compressed at slight loss of quality, see http://casa.colorado.edu/~tumlinso/h2/ for full version

Next-generation sequencing for HLA typing of class I loci

<p>Abstract</p> <p>Background</p> <p>Comprehensive sequence characterization across the MHC is important for successful organ transplantation and genetic association studies. To this end, we have developed an automated sample preparation, molecular barcoding and multiplexing protocol for the amplification and sequence-determination of class I HLA loci. We have coupled this process to a novel HLA calling algorithm to determine the most likely pair of alleles at each locus.</p> <p>Results</p> <p>We have benchmarked our protocol with 270 HapMap individuals from four worldwide populations with 96.4% accuracy at 4-digit resolution. A variation of this initial protocol, more suitable for large sample sizes, in which molecular barcodes are added during PCR rather than library construction, was tested on 95 HapMap individuals with 98.6% accuracy at 4-digit resolution.</p> <p>Conclusions</p> <p>Next-generation sequencing on the 454 FLX Titanium platform is a reliable, efficient, and scalable technology for HLA typing.</p

ER-stress and basement membrane defects combine to cause glomerular and tubular renal disease caused by Col4a1 mutations

Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies

Whole Genome Pyrosequencing of Rare Hepatitis C Virus Genotypes Enhances Subtype Classification and Identification of Naturally Occurring Drug Resistance Variants

Background. Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. Methods. Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. Results. Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. Conclusions. These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistanc

Endurance, Refuge, and Reemergence of Dengue Virus Type 2, Puerto Rico, 1986–2007

To study the evolution of dengue virus (DENV) serotype 2 in Puerto Rico, we examined the genetic composition and diversity of 160 DENV-2 genomes obtained through 22 consecutive years of sampling. A clade replacement took place in 1994–1997 during a period of high incidence of autochthonous DENV-2 and frequent, short-lived reintroductions of foreign DENV-2. This unique clade replacement was complete just before DENV-3 emerged. By temporally and geographically defining DENV-2 lineages, we describe a refuge of this virus through 4 years of low genome diversity. Our analyses may explain the long-term endurance of DENV-2 despite great epidemiologic changes in disease incidence and serotype distribution