Intra- and inter-rater reliability in ultrasonographic measurements of coracohumeral distance.

BACKGROUND: Ultrasonography (US) is a widely used diagnostic tool in physical therapy. One of the US variables often utilized to quantify the dimensions of the subacromial space and its association with shoulder pathology is the coracohumeral distance (CHD), however, this measurement presents diverse evidence in terms of reliability. OBJECTIVES: To assess the intra- and inter-rater reliability of both expert and non-expert raters when measuring CHD through US in asymptomatic subjects. Additionally, we determined the effect of rater experience and measurement conditions on the recording of CHD. METHODS: CHD of 15 individuals were recorded from US images of the glenohumeral joint of both upper extremities in three different positions. An expert and a non-expert rater in US usage recorded three CHD measurements, after a randomization procedure. To determine intra- and inter-rater reliability, the interclass correlation coefficient (ICC) and a multivariate variance model for the effects of rater experience, joint position and time of measure were used. Standard Error of Measurement and Minimal Detectable Change was also estimated for CHD measurements. RESULTS: Intra-rater reliability ranged 0.970 to 0.998) and Inter-rater reliability ranged 0.48 to 0.876). Joint position (F2;55 = 38.308; p < 0.001; ηp2 = 0.582) and measurement time (F2;55 = 6.019; p = 0.004; ηp2 = 0.180) effect was observed on CHD. CONCLUSION: Excellent intra- and poor to moderate inter-rater reliability between expert and non-expert clinicians was determined, the latter being influenced by the position of the glenohumeral joint position at the time of US recording and the time of recording the measurement.

A new genus and species of Neotropical Hybosorinae Erichson, 1847 (Coleoptera: Hybosoridae).

Among the 26 Hybosorinae genera, seven are present exclusively in the Neotropical region. However, Hybosorinae from the New World have been less studied. One new genus and new species collected in Brazil is herein described to this subfamily. The genus is recognizable by the following combination of characters: labium with semicircular mentum; mandibles with lateral projection excavated, separated in the middle by longitudinal carina, inner side sheltering the labrum; antennae with nine antenomeres; scutellar shield with strong punctures at the proximal half; elytra with single rows formed by punctures; and protibia without well-defined denticles.

Liogenys Guerin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) of northern South America and Central America: taxonomic overview with four new species.

The biodiversity of northern South American and Central American Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) is reviewed. Four new species are described: L. clipeosetosa Cherman, new species; L. genieri Smith Cherman, new species; L. granadina Cherman, new species; and L. schneiderae Cherman, new species. The male of L. quadridens (Fabricius, 1798) and the female of L. quadridentata Blanchard, 1851 are described for the first time. Liogenys gebieni Moser, 1921 is a new junior subjective synonym of L. macropelma Bates, 1887. The northernmost record of Liogenys is emended to Trinidad and Tobago for L. granadina Cherman, new species and L. schneiderae new species. Diplotaxis puberea cuprascens (Bates, 1887) new combination, Diplotaxis puberea puberea (Bates, 1887) new combination, and Diplotaxis pubisternis (Bates, 1887) new combination are all transferred from Liogenys to Diplotaxis Kirby, 1837. Lectotypes are designated for Liogenys gebieni Moser, 1921; Melolontha quadridens Fabricius, 1798; and Liogenys quadridentatus Blanchard, 1851. An identification key to northern South American Liogenys is presented.

In vivo measurements of glenohumeral distraction technique performed in three different joint positions.

Introduction: Distraction techniques are an important part of the manual approach in the glenohumeral joint; however, there is controversy regarding the initial joint position to maximize separation of joint surfaces.Objective: To identify, through an in vivo exploration, the behavior of the coracohumeral distance (CHD) during the application of a short lever arm grade III distraction technique on the humeral head, executed in three different glenohumeral positions (zero position (P0), rest position (RP) and 90º abduction position with maximum external rotation (ABD+ER)).Methods: 15 participants were selected. A physical therapist executed grade III distraction techniques in described joint positions. Ultrasound was used to visualize the differences in CHD. Statistical analysis included interclass correlation coefficients (ICC) and repeated measurements of ANOVA.Results: The range of ICC values was 0.740 and 0.948. The differences in CHD were 5.74 ± 0.51 mm, 3.97 ± 0.24 mm, and 0.64 ± 0.02 mm, for P0, RP and ABD+ER during application of distraction technique, respectively. There were differences between P0 and RP with ABD+ER (P < 0.001), and no differences between P0 and RP.Conclusion: Initial joint position of application influences the separation of the articular surfaces, being wider in the zero and rest positions.

Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) from the Chacoan Province and its boundaries: taxonomic overview with four new species.

A taxonomic revision of the Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) from the Chacoan Biogeographical Province is presented. Liogenys now includes 92 species, including four new species described here: L. neoforcipata Cherman, new species; L. foveata Cherman, new species; L. isotarsis Cherman, new species; and L. truncata Cherman, new species; and the female of L. tarsalis Moser is described for the first time. Six new synonymies are proposed: L. denticulata Moser, 1918 is a new synonym of L. denticeps Blanchard, 1851; L. ophtalmica Frey, 1973 is a new synonym of L. bidenticeps Moser, 1919; L. mendozana incisa Frey, 1969 is a new synonym of L. mendozana Moser, 1918; L. flavicollis Blanchard, 1851 and L. fulvescens Blanchard, 1851 are new synonyms of L. pallens Blanchard, 1851; and L. densicollis Moser, 1921 is a new synonym of L. opacicollis Fairmaire, 1892. Liogenys cribricollis Moser, 1921 species status is revalidated from its synonymy with L. densicollis. A neotype is designated for Liogenys mendozana incisa Frey, 1969, as well as lectotypes for: L. bruchi Moser, 1924; L. cribricollis, L. denticulata, L. denticeps, L. fulvescens, L. latitarsis Moser, 1918; L. mendozana Moser, 1918; L. obscura Blanchard, 1851; L. opacicollis; and L. pallens. Redescriptions and/or diagnoses and updated geographical distributions are provided for 16 species. Six species previously known only from Argentina have their distribution expanded to Bolivia (L. mendozana; L. opacicollis; L. rectangula Frey, 1969), Paraguay (L. nigrofusca Moser, 1918; L. pallens), or to both of these countries (L. latitarsis).

Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae) from the southern South American Transition Zone and boundaries: taxonomic overview with four new species.

The biodiversity of Liogenys Guérin-Méneville, 1831 (Coleoptera: Scarabaeidae: Melolonthinae: Diplotaxini) from the southern South American Transition Zone and boundaries is presented. Four new species are described: L. brachyclypeata Cherman, new species; L. lucialmeidae Cherman, new species; L. martinezi Cherman, new species; and L. maxillaricuspis Cherman, new species. The synonymy of L. flaveola Moser, 1924 (= L. kadleci Frey, 1970) is proposed. Lectotypes are designated for L. flavida Moser, 1918; L. pallidicornis Blanchard, 1851 (currently L. xanthocera Harold, 1869); and L. rufoflava Moser, 1918. Redescriptions are provided for all the species mentioned above plus L. calcarata Frey, 1970 and L. kunzteni Moser, 1921, as well as an identification key and updated geographical distributions to all species in the region. All species are present in the Monte province, except of L. kuntzeni (Andean provinces of Chile). Liogenys flavida and L. rufoflava have the broadest distribution, the latter here expanded to Paraguay and Chile.

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation.

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Probing the conformation of a conserved glutamic acid within the Cl- pathway of a CLC H+/Cl- exchanger.

The CLC proteins form a broad family of anion-selective transport proteins that includes both channels and exchangers. Despite extensive structural, functional, and computational studies, the transport mechanism of the CLC exchangers remains poorly understood. Several transport models have been proposed but have failed to capture all the key features of these transporters. Multiple CLC crystal structures have suggested that a conserved glutamic acid, Gluex, can adopt three conformations and that the interconversion of its side chain between these states underlies H+/Cl- exchange. One of these states, in which Gluex occupies the central binding site (Scen) while Cl- ions fill the internal and external sites (Sint and Sext), has only been observed in one homologue, the eukaryotic cmCLC. The existence of such a state in other CLCs has not been demonstrated. In this study, we find that during transport, the prototypical prokaryotic CLC exchanger, CLC-ec1, adopts a conformation with functional characteristics that match those predicted for a cmCLC-like state, with Gluex trapped in Scen between two Cl- ions. Transport by CLC-ec1 is reduced when [Cl-] is symmetrically increased on both sides of the membrane and mutations that disrupt the hydrogen bonds stabilizing Gluex in Scen destabilize this trapped state. Furthermore, inhibition of transport by high [Cl-] is abolished in the E148A mutant, in which the Gluex side chain is removed. Collectively, our results suggest that, during the CLC transport cycle, Gluex can occupy Scen as well as the Sext position in which it has been captured crystallographically and that hydrogen bonds with the side chains of residues that coordinate ion binding to Scen play a role in determining the equilibrium between these two conformations.

A morphological reappraisal of the immature stages and life history of Elachista synethes Meyrick (Lepidoptera, Elachistidae), an Australian leaf miner alien to Chile

ABSTRACT Elachista synethes was recently recognized as an alien species in northern Chile, where its larvae mine the rescue grass Bromus catharticus (Poaceae). In order to provide the necessary information to allow field detection of E. synethes during early ontogeny, we conducted a morphological reappraisal of the immature stages of this leaf-miner moth, based on light and scanning electron microscopy, including the first descriptions of the egg and the first-instar larva. This is the first report of the existence of an apodal early larva for a species of Elachista Treitschke. The legs and prolegs are absent in the first two instars, but are well developed in the last two. Additional observations on the life history are also provided, including a description of the mine.

Description and life history of a new cecidogenous species of Palaeomystella Fletcher (Lepidoptera, Momphidae) from Brazil

ABSTRACTMale, female, pupa, and last-instar larva of Palaeomystella beckeri (Moreira and Basilio) a new species from the Atlantic forest, southern Brazil, are described and illustrated with the aid of optical and scanning electron microscopy. Larvae induce galls on apical branches of Tibouchina trichopoda (DC.) Baill. (Melastomataceae) within which pupation occurs. Gall description and preliminary data on life history are also provided.

Mechanism of potassium ion uptake by the Na(+)/K(+)-ATPase.

The Na(+)/K(+)-ATPase restores sodium (Na(+)) and potassium (K(+)) electrochemical gradients dissipated by action potentials and ion-coupled transport processes. As ions are transported, they become transiently trapped between intracellular and extracellular gates. Once the external gate opens, three Na(+) ions are released, followed by the binding and occlusion of two K(+) ions. While the mechanisms of Na(+) release have been well characterized by the study of transient Na(+) currents, smaller and faster transient currents mediated by external K(+) have been more difficult to study. Here we show that external K(+) ions travelling to their binding sites sense only a small fraction of the electric field as they rapidly and simultaneously become occluded. Consistent with these results, molecular dynamics simulations of a pump model show a wide water-filled access channel connecting the binding site to the external solution. These results suggest a mechanism of K(+) gating different from that of Na(+) occlusion.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters.

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl(-) channels or transporters is described. The assay is based on the measure of the efflux rate of Cl(-) from proteoliposomes following the addition of the K(+) ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H(+)/Cl(-) exchanger, as a model system. The efflux assay can be utilized to study any Cl(-) conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A new cecidogenous species of Eugnosta Hübner (Lepidoptera: Tortricidae) associated with Baccharis salicifolia (Asteraceae) in the northern Chilean Atacama Desert: Life-history description and phylogenetic inferences.

Eugnosta Hübner, 1825 (Lepidoptera, Tortricidae, Tortricinae, Cochylini, Cochylina) is reported for the first time in Chile. Male and female adults, the pupa, the last-instar larva, and galls of Eugnosta azapaensis Vargas & Moreira, sp. n., are described and illustrated from the Azapa Valley in the northern Atacama Desert. The larvae induce fusiform galls on shoots of the shrub Baccharis salicifolia (Ruiz & Pav.) Pers. (Asteraceae). An assessment of phylogenetic relationships of E. azapaensis with two congeneric species based on mitochondrial DNA is provided.

Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter.

CLC-type exchangers mediate transmembrane Cl(-) transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood. In conventional models, two gates alternatively expose substrates to the intra- or extracellular solutions. A glutamate was identified as the only gate in the CLCs, suggesting that CLCs function by a nonconventional mechanism. Here we show that transport in CLC-ec1, a prokaryotic homolog, is inhibited by cross-links constraining movement of helix O far from the transport pathway. Cross-linked CLC-ec1 adopts a wild-type-like structure, indicating stabilization of a native conformation. Movements of helix O are transduced to the ion pathway via a direct contact between its C terminus and a tyrosine that is a constitutive element of the second gate of CLC transporters. Therefore, the CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway.

The role of gap junction channels during physiologic and pathologic conditions of the human central nervous system.

Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP(3), and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.

A kinetic analysis of protein transport through the anthrax toxin channel.

Anthrax toxin is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal factor (LF) and edema factor (EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel can be driven by voltage on a timescale of seconds. A characteristic of the translocation of LF(N), the N-terminal 263 residues of LF, is its S-shaped kinetics. Because all of the translocation experiments reported in the literature have been performed with more than one LF(N) molecule bound to most of the channels, it is not clear whether the S-shaped kinetics are an intrinsic characteristic of translocation kinetics or are merely a consequence of the translocation in tandem of two or three LF(N)s. In this paper, we show both in macroscopic and single-channel experiments that even with only one LF(N) bound to the channel, the translocation kinetics are S shaped. As expected, the translocation rate is slower with more than one LF(N) bound. We also present a simple electrodiffusion model of translocation in which LF(N) is represented as a charged rod that moves subject to both Brownian motion and an applied electric field. The cumulative distribution of first-passage times of the rod past the end of the channel displays S-shaped kinetics with a voltage dependence in agreement with experimental data.

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins.

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA(63))(7), in the host cell's endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA(63))(7) incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å-long, 15 Å-wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF(N) (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF(N) adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF(N) in the normal direction but also can, at least partially, translocate LF(N) in the opposite direction.

Evidence for a proton-protein symport mechanism in the anthrax toxin channel.

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

Connexin-based gap junction hemichannels: gating mechanisms.

Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions.