The architecture and contraction time of intrinsic foot muscles

Although critical for effective human locomotion and posture, little data exists regarding the segmentation, architecture and contraction time of the human intrinsic foot muscles. To address this issue, the Abductor Hallucis (AH), Abductor Digiti Minimi (ADM), Flexor Digitorum Brevis (FDB) and Extensor Digitorum Brevis (EDB) were investigated utilizing a cadaveric dissection and a non-invasive whole muscle mechanomyographic (wMMG) technique. The segmental structure and architecture of formaldehyde-fixed foot specimens were determined in nine cadavers aged 60–80 years. The wMMG technique was used to determine the contraction time (Tc) of individual muscle segments, within each intrinsic foot muscle, in 12 volunteers of both genders aged between 19 and 24 years.While the pattern of segmentation and segmental –architecture (e.g. fibre length) and –Tc of individual muscle segments within the same muscle were similar, they varied between muscles. Also, the average whole muscle Tc of FDB was significantly (p < 0.05) shorter (faster) (Tc = 58 ms) than in all other foot muscles investigated (ADM Tc = 72 ms, EDB Tc = 72 ms and ABH Tc = 69 ms). The results suggest that the architecture and contraction time of the FDB reflect its unique direct contribution, through toe flexion, to postural stability and the rapid development of ground reaction forces during forceful activities such as running and jumping.     

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

Background  

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas.     

Evolution of Bacterial-Like Phosphoprotein Phosphatases in Photosynthetic Eukaryotes Features Ancestral Mitochondrial or Archaeal Origin and Possible Lateral Gene Transfer

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.Reversible protein phosphorylation is a posttranslational mechanism central to the proper function of living organisms (Brautigan, 2013). Governed by two large groups of enzymes, protein kinases and protein phosphatases, this mechanism has been suggested to regulate upwards of 70% of all eukaryotic proteins (Olsen et al., 2010). Protein phosphatases represent one-half of this dynamic regulatory system and have been shown to be highly regulated proteins themselves (Roy and Cyert, 2009; Shi, 2009; Uhrig et al., 2013). Classically, protein phosphatases have been placed into four families defined by a combination of their catalytic mechanisms, metal ion requirements, and phosphorylated amino acid targets (Kerk et al., 2008). These four families are the phosphoprotein phosphatases (PPPs), metallo-dependent protein phosphatases, protein Tyr phosphatases, and Asp-based phosphatases. The PPP protein phosphatases, best known to include PP1, PP2A, PP2B, and PP4 to PP7 (Kerk et al., 2008; Shi, 2009), have been found to regulate a diverse number of biological processes in plants ranging from cell signaling (Ahn et al., 2011; Di Rubbo et al., 2011; Tran et al., 2012) to metabolism (Heidari et al., 2011; Leivar et al., 2011) and hormone biosynthesis (Skottke et al., 2011). The classical PPP protein phosphatase family has been expanded to include three novel classes that show greatest similarity to PPP-like protein phosphatases of prokaryotic origin (Andreeva and Kutuzov, 2004; Uhrig and Moorhead, 2011a; Uhrig et al., 2013). These bacterial-like phosphatase classes were annotated as Shewanella-like (SLP) phosphatases, Rhizobiales-like (RLPH) phosphatases, and ApaH-like (ALPH) phosphatases based on their similarity to prokaryotic sequences from these respective sources (Andreeva and Kutuzov, 2004). Recent characterization of the SLP phosphatases from Arabidopsis (Arabidopsis thaliana) provided biochemical evidence of insensitivity to the classic PPP protein phosphatase inhibitors okadaic acid and microcystin in addition to revealing a lack of genetic redundancy across sequenced plant genomes (Uhrig and Moorhead, 2011a).The characterization of eukaryotic protein evolution can provide insight into individual protein or protein class conservation across the domains of life for biotechnological applications in addition to furthering our understanding of how multicellular life evolved. In particular, investigation into the evolution of key signaling proteins, such as protein kinases and phosphatases from plants, can have wide-ranging agribiotechnological and medical potential. This can include the development of healthier, disease- or stress-resistant crops in addition to treatments for parasitic organisms such as Plasmodium spp. (malaria; Patzewitz et al., 2013) and other chromoalveolates (Kutuzov and Andreeva, 2008; Uhrig and Moorhead, 2011b) that are derived from photosynthetic eukaryotes and maintain a remnant chloroplast (apicoplast; Le Corguillé et al., 2009; Janouskovec et al., 2010; Kalanon and McFadden, 2010; Walker et al., 2011). The existence of proteins that are conserved across diverse eukaryotic phyla but absent in metazoa, such as the majority of bacterial-like PPP protein phosphatases described here, presents unique research opportunities.Conventional understanding of the acquisition by eukaryotes of prokaryotic genes and proteins largely involves ancient endosymbiotic gene transfer events stemming from primary endosymbiosis of α-Proteobacteria and Cyanobacteria to form eukaryotic mitochondria and chloroplasts, respectively (Keeling and Palmer, 2008; Dorrell and Smith, 2011; Tirichine and Bowler, 2011). Over time, however, it has become apparent that alternative modes of eukaryotic gene and protein acquisition exist, such as independent horizontal or lateral gene transfer (LGT) events (Keeling and Palmer, 2008; Keeling, 2009). Targeted studies of protein evolution have seen a steady rise in documented LGT events across a wide variety of eukaryotic organisms, including photosynthetic eukaryotes (Derelle et al., 2006; Raymond and Kim, 2012; Schönknecht et al., 2013), nematodes (Mayer et al., 2011), arthropods (Acuña et al., 2012), fungi (Wenzl et al., 2005), amoebozoa (Clarke et al., 2013), and oomycetes (Belbahri et al., 2008). Each instance documents the integration of a bacterial gene(s) into a eukaryotic organism, seemingly resulting in an adaptive advantage(s) important to organism survival.Utilizing a number of in silico bioinformatic techniques and available sequenced genomes, the molecular evolution of three bacterial-like PPP classes found in eukaryotes is revealed to involve ancient mitochondrial or archaeal origin plus additional possible LGT events. A third, more ancient group of SLP phosphatases (SLP3 phosphatases) is defined in green algae. Subcellular localization predictions reveal distinctive subsets of bacterial-like PPPs, which may correlate with altered functions. In addition, the large sequence collections compiled here have allowed the elucidation of two highly conserved C-terminal domain motifs, which are specific to each bacterial-like PPP class and whose differences are particularly pronounced in photosynthetic eukaryotes. Together, these findings substantially expand our knowledge of the molecular evolution of the bacterial-like PPPs and point the way toward attractive future research avenues.     

Assembly and annotation of a draft genome sequence for Glycine latifolia,a perennial wild relative of soybean

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939‐Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked‐reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome‐scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91‐bp centromere‐specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92‐bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein‐coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine‐specific orthologous gene families. A total of 304 putative nucleotide‐binding site (NBS)‐leucine‐rich‐repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR‐NBS‐LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR‐receptor‐like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost‐effectiveness of the application of Chromium linked‐reads in diploid plant genome de novo assembly.     

A mutant lymphoma cell line with a defectiveThy-1 glycoprotein gene

We characterized a mutant T -cell lymphoma line selected for the inability to express the Thy-1 glycoprotein. This cell line is a member of the D complementation class of Thy-1^– somatic cell mutants, and it lacks detectable cell-surface Thy-1.1 glycoprotein and detectable cytoplasmic Thy-1 mRNA. Southern blot analysis using a number of probes isolated from the clonedThy-1.2 gene demonstrated that, in the mutant, one copy of theThy-1 gene is absent from the genome and the other has undergone rearrangement. This rearrangement results from a deletion of the 5 portion of the gene removing the first two alternate exons and promoters and a portion of the second intron. The deletion breakpoint within the mutantThy-1 gene was localized to within 400 nucleotides by Southern blot analysis. The breakpoint is near two classes of mouse repetitive elements-a mouse B1-family repetitive element and a simple repetitive sequence-suggesting a mechanism of rearrangement leading to the mutation. Southern blot analysis demonstrated that two closely linked molecular markers on chromosome 9 are unaltered, demonstrating that the deletion in this mutant cell line is subchromosomal.     

Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Adenovirus (Ad) endocytosis via α_v integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

Uptake of saline groundwater by plants: An analytical model for semi-arid and arid areas

An analytical model, based on unsaturated zone water and solute balances, was developed to describe the uptake of saline groundwater by plants in dry regions. It was assumed that: i. initially, the profile had low water and salt contents to some depth; ii. both water and solutes move upwards from the water table by piston flow due only to plant water extraction; iii. the uptake of water concentrates solutes in the soil solution until some threshold salinity is reached, above which plants can no longer extract water due to osmotic effects; iv. uptake of the groundwater does not affect the water table level; and v. uptake of groundwater is only limited by transmission of groundwater through the soil. Model predictions were compared with measurements of groundwater uptake made over 15 months at five sites in aEucalyptus forest in a semi-arid area, using independently measured model parameters. Depth and salinity of groundwater, and soil type varied greatly between sites. Predicted groundwater uptake rates were close to measured values, generally being within ∼ 0.1 mm day^-1. Sensitivity analysis showed that groundwater depth and salinity were the main controls on uptake of groundwater, while soil properties appeared to have a lesser effect. The model showed that uptake of groundwater would result in complete salinisation of the soil profile within 4 to 30 yr at the sites studied, unless salts were leached from the soil by rainfall or flood waters. However, a relatively small amount of annual leaching may be sufficient to allow groundwater uptake to continue. Thus groundwaters, even when saline, may be important sources of water to plants in arid and semi-arid areas.     

Effect of temperature and the F27W mutation on the Ca2+ activated structural transition of trout cardiac troponin C

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.     

Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones

We investigated hair bundle mechanoreceptors in sea anemones for a homolog of cadherin 23. A candidate sequence was identified from the database for Nematostella vectensis that has a shared lineage with vertebrate cadherin 23s. This cadherin 23-like protein comprises 6,074 residues. It is an integral protein that features three transmembrane alpha-helices and a large extracellular loop with 44 contiguous, cadherin (CAD) domains. In the second half of the polypeptide, the CAD domains occur in a quadruple repeat pattern. Members of the same repeat group (i.e., CAD 18, 22, 26, and so on) share nearly identical amino acid sequences. An affinity-purified antibody was generated to a peptide from the C-terminus of the cadherin 23-like polypeptide. The peptide is expected to lie on the exoplasmic side of the plasma membrane. In LM, the immunolabel produced punctate fluorescence in hair bundles. In TEM, immunogold particles were observed medially and distally on stereocilia of hair bundles. Dilute solutions of the antibody disrupted vibration sensitivity in anemones. We conclude that the cadherin 23-like polypeptide likely contributes to the mechanotransduction apparatus of hair bundle mechanoreceptors of anemones.     

Attenuation of Vesicular Stomatitis Virus Encephalitis through MicroRNA Targeting

Vesicular stomatitis virus (VSV) has long been regarded as a promising recombinant vaccine platform and oncolytic agent but has not yet been tested in humans because it causes encephalomyelitis in rodents and primates. Recent studies have shown that specific tropisms of several viruses could be eliminated by engineering microRNA target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. We therefore sought to determine whether microRNA targets could be engineered into VSV to ameliorate its neuropathogenicity. Using a panel of recombinant VSVs incorporating microRNA target sequences corresponding to neuron-specific or control microRNAs (in forward and reverse orientations), we tested viral replication kinetics in cell lines treated with microRNA mimics, neurotoxicity after direct intracerebral inoculation in mice, and antitumor efficacy. Compared to picornaviruses and adenoviruses, the engineered VSVs were relatively resistant to microRNA-mediated inhibition, but neurotoxicity could nevertheless be ameliorated significantly using this approach, without compromise to antitumor efficacy. Neurotoxicity was most profoundly reduced in a virus carrying four tandem copies of a neuronal mir125 target sequence inserted in the 3′-untranslated region of the viral polymerase (L) gene.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand rhabdovirus widely used as a vaccine platform as well as an anticancer therapeutic. While VSV is predominantly a pathogen of livestock (34), it has a very broad species tropism. The cellular tropism of VSV is determined predominantly at postentry steps, since the G glycoprotein of the virus mediates entry into most tissues in nearly all animal species (10).Though viral entry can take place in nearly all cell types, in vivo models of VSV infection have revealed that the virus is highly sensitive to the innate immune response, limiting its pathogenesis (4). VSV is intensively responsive to type I interferon (IFN), as the double-stranded RNA (dsRNA)-dependent PKR (2), the downstream effector of pattern recognition receptors MyD88 (32), and other molecules mediate shutdown of viral translation and allow the adaptive immune response to clear the virus. The vulnerability of the virus to the type I IFN response, typically defective in many cancers, has been exploited to generate tumor-selective replication (49), such that the virus is now poised to enter phase I trials. However, the virus remains potently neurotoxic, causing lethal encephalitis not only in rodent models (7, 22, 53) but also in nonhuman primates (25).VSV very often infiltrates the central nervous system (CNS) through infection of the olfactory nerves (41). When administered intranasally, the virus replicates rapidly in the nasal epithelium and is transmitted to olfactory neurons, from which it then moves retrograde axonally to the brain and replicates robustly, causing neuropathogenesis. While intranasal inoculation does cause neuropathy in mice, neurotoxicity following viral administration also occurs when the virus is delivered intravascularly (47), intraperitoneally (42), and (not surprisingly) intracranially (13). Previously, other groups have modified the VSV genome to be more sensitive to cellular IFNs (49) and have actually encoded IFN in the virus (36). However, the former can result in attenuation of the virus, such that it has reduced anticancer potential, while the latter still results in lethal encephalitis (unpublished results). In order to mitigate the effects of VSV infection on the brain without perturbing the potent oncolytic activity of the virus, we utilized a microRNA (miRNA) targeting paradigm, whereby viral replication is restricted in the brain without altering the tropism of the virus for other tissues.To redirect the tissue tropism of anticancer therapeutics, we (26) and others (11, 14, 55) have previously exploited the tissue-specific expression of cellular miRNAs. miRNAs are ∼22-nucleotide (nt) regulatory RNAs that regulate a diverse and expansive array of cellular activities. Through recognition of sequence-complementary target elements, miRNAs can either translationally suppress or catalytically degrade both cellular (6) and viral (50) RNAs. We have determined that cellular miRNAs can potentially regulate numerous steps of a virus life cycle and that this regulation of the virus by endogenous miRNAs can then abrogate toxicities of replication-competent viruses (27; E. J. Kelly et al., unpublished data).miRNAs are known to be highly upregulated in many different tissues, including (but not limited to) muscle (40), lung (44), liver (15, 44), spleen (44, 46), and kidney (51). In addition, the brain has a number of upregulated miRNAs, with each different subtype of cell having a unique miRNA profile. miR-125 is highly upregulated in all cells in the brain (neurons, astrocytes, and glia cells), while miR-124 is found predominantly in neuronal cells (48). Glial cells and glioblastomas are thought to have decreased expression of miR-128 compared to neurons (17), while miR-134 is particularly abundant in dendrites of neurons in the hippocampus (43). In addition to these miRNAs, the tumor suppressor miRNA let-7 and miRs 9, 26, and 29 (51) are also found to be enriched in the brain, with expression varying not only between different cell types and regions of the brain but also temporally (48).MicroRNAs have previously been exploited to modulate the tissue tropism of nonreplicating lentiviral vectors (8, 9), as well as curbing known toxicities of replication-competent picornaviruses (5, 26), adenoviruses (11), herpes simplex virus 1 (33), and influenza A virus (39). In addition, a recombinant VSV encoding a tumor suppressor target was found to be responsive to sequence-complementary miRNAs in vitro, possibly by affecting expression of the matrix (M) protein (14), and evidence from Dicer-deficient mice suggests that endogenously expressed microRNA targets within the P and L genes of VSV could restrict enhanced pathogenicity of the virus (37). However, in vivo protection from neuropathogenesis by this means has not been demonstrated for VSV.Here we evaluate the efficiencies of different brain-specific miRNAs for shutting down gene expression and extensively characterize the ability of miRNA targeting to attenuate the neurotoxicity of vesicular stomatitis virus in vivo. We constructed and evaluated recombinant VSVs with miRNA target (miRT) insertions at different regions of the viral genome, with special focus upon those affecting viral L expression. In addition, we looked at the regulatory efficiency of different brain-specific miRNAs and the impact of miRT orientation on VSV replication and determined the impact of the virus on oncolytic activity in vivo.