Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle

Background

The phenotype of large diameter sensory afferent neurons changes in several models of neuropathic pain. We asked if similar changes also occur in “functional” pain syndromes.

Methodology/Principal Findings

Acidic saline (AS, pH 4.0) injections into the masseter muscle were used to induce persistent myalgia. Controls received saline at pH 7.2. Nocifensive responses of Experimental rats to applications of Von Frey Filaments to the masseters were above control levels 1–38 days post-injection. This effect was bilateral. Expression of c-Fos in the Trigeminal Mesencephalic Nucleus (NVmes), which contains the somata of masseter muscle spindle afferents (MSA), was above baseline levels 1 and 4 days after AS. The resting membrane potentials of neurons exposed to AS (n = 167) were hyperpolarized when compared to their control counterparts (n = 141), as were their thresholds for firing, high frequency membrane oscillations (HFMO), bursting, inward and outward rectification. The amplitude of HFMO was increased and spontaneous ectopic firing occurred in 10% of acid-exposed neurons, but never in Controls. These changes appeared within the same time frame as the observed nocifensive behaviour. Ectopic action potentials can travel centrally, but also antidromically to the peripheral terminals of MSA where they could cause neurotransmitter release and activation of adjacent fibre terminals. Using immunohistochemistry, we confirmed that annulospiral endings of masseter MSA express the glutamate vesicular transporter VGLUT1, indicating that they can release glutamate. Many capsules also contained fine fibers that were labelled by markers associated with nociceptors (calcitonin gene-related peptide, Substance P, P2X3 receptors and TRPV1 receptors) and that expressed the metabotropic glutamate receptor, mGluR5. Antagonists of glutamatergic receptors given together with the 2^nd injection of AS prevented the hypersensitivity observed bilaterally but were ineffective if given contralaterally.

Conclusions/Significance

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some “functional” pain syndromes.     

Viral haemorrhagic septicaemia (VHS) outbreaks in Finnish rainbow trout farms

In Finland, viral haemorrhagic septicaemia virus (VHSV) was diagnosed for the first time in 2000 from 4 rainbow trout farms in brackish water. Since then the infection has spread and, by the end of 2004, VHSV had been isolated from 24 farms in 3 separate locations: 2 in the Baltic Sea and 1 in the Gulf of Finland. The pathogenicity of 3 of these isolates from 2 separate locations was analysed in infection experiments with rainbow trout fry. The cumulative mortalities induced by waterborne and intraperitoneal challenge were approximately 40 and 90 %, respectively. Pair-wise comparisons of the G and NV gene regions of Finnish VHSV isolates collected between 2000 and 2004 revealed that all isolates were closely related, with 99.3 to 100% nucleotide identity, which suggests the same origin of infection. Phylogenetic analysis revealed that they were closely related to the old freshwater isolates from rainbow trout in Denmark and to one old marine isolate from cod in the Baltic Sea, and that they were located close to the presumed ancestral source. As the Finnish isolates induce lower mortality than freshwater VHSV isolates in infection experiments, they could represent an intermediate stage of marine isolates evolving towards pathogenicity in rainbow trout.     

Genetic and maternal determinants of effective dispersal: the effect of sire genotype and size at birth in side-blotched lizards

We assessed genetic factors on progeny dispersal due to sire color morph genotypes in a field pedigree and lab crosses, and we measured maternal effects by studying both natural and experimentally induced egg size variation. Progeny were released into nature upon hatching, but we recorded dispersal distance at maturity, which reflects effective dispersal after viability selection has run its course. Progeny dispersal was significantly affected by sire genotype. Progeny from orange sires dispersed the farthest. Progeny from blue sires dispersed intermediate distances. Progeny from yellow sires were the most philopatric. Sire genotype effects interacted with egg size. In particular, enlarged progeny from orange sires dispersed farther, while enlarged progeny from yellow sires were more philopatric. Progeny from blue sires were unaffected by egg size manipulations. Egg manipulations and natural variation generally had concordant effects indicative of causation. However, asymmetry of gigantization and miniaturization on progeny dispersal from some sire genotypes suggest the involvement of maternal factors besides egg size. Results of laboratory crosses with progeny released into nature confirmed key sire genotype effects and identified additional maternal effects that modulated dispersal as a function of progeny gender. We discuss the adaptive implications of progeny dispersal in the context of male (rock-paper-scissors) and female strategies (r- and K-density cycle) that are associated with color morphs.     

INTRALOCUS SEXUAL CONFLICT OVER IMMUNE DEFENSE,GENDER LOAD,AND SEX‐SPECIFIC SIGNALING IN A NATURAL LIZARD POPULATION

In species with separate sexes, antagonistic selection on males and females (intralocus sexual conflict) can result in a gender load that can be resolved through the evolution of sexual dimorphism. We present data on intralocus sexual conflict over immune defense in a natural population of free‐ranging lizards (Uta stansburiana) and discuss the resolution of this conflict. Intralocus sexual conflict arises from correlational selection between immune defense and orange throat coloration in these lizards. Males with orange throats and high antibody responses had enhanced survival, but the same trait combination reduced female fitness. This sexual antagonism persisted across the life cycle and was concordant between the juvenile and adult life stages. The opposing selective pressure on males and females is ameliorated by a negative intersexual genetic correlation (r_m,f=?0.86) for immune defense. Throat coloration was also genetically correlated with immune defense, but the sign of this genetic correlation differed between the sexes. This resulted in sex‐specific signaling of immunological condition. We also found evidence for a sex‐specific maternal effect on sons with potential to additionally reduce the gender load. These results have implications for signaling evolution, genetic integration between adaptive traits, sex allocation, and mutual mate choice for indirect fitness benefits.     

Mechanism of Initiation Site Selection Promoted by the Human Rhinovirus 2 Internal Ribosome Entry Site

Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5′ side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by ∼70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.Until the recent discovery of animal picornaviruses with internal ribosome entry sites (IRESs) resembling that of hepatitis C virus, most picornavirus IRESs have been classified into two groups (1, 17): type 1 (exemplified by entero- and rhinoviruses) and type 2 (cardio- and aphthoviruses). Primary sequences and especially secondary structures are strongly conserved within each group but there is very little similarity between the two groups apart from an AUG triplet at the 3′ end of the IRES (as defined by deletion analysis), which is preceded by a ∼25 nucleotide (nt) pyrimidine-rich tract (17). In type 2 IRESs, notably encephalomyocarditis virus (EMCV), this AUG triplet is the authentic initiation codon for viral polyprotein synthesis, and the totality of the evidence indicates that all ribosomes bind at, or very close to, this AUG and that all initiate translation at this site (18, 19). The foot-and-mouth disease virus (FMDV), although a type 2 IRES, is not quite so straightforward in that a minority of initiation events occur at the AUG immediately downstream of the oligopyrimidine tract, and the rest occur at the next AUG, 84 nt downstream (3, 45).In contrast, initiation on type 1 IRESs seems much more complicated and rather puzzling. The first puzzling feature is that there is very little, if any, initiation at the AUG just downstream of the oligopyrimidine tract, at nt 586 in poliovirus type 1 (PV-1) (39), and the initiation site for polyprotein synthesis is the next AUG further downstream, at a distance of ∼160 nt in enteroviruses and ∼35 nt in rhinoviruses (17). Nevertheless, AUG-586 is important for efficient initiation at the authentic polyprotein initiation site. Mutation of AUG-586 in a PV-1 infectious clone was found to be quasi-infectious (42), while mutation of the equivalent site in PV-2 conferred a small-plaque phenotype and reduced initiation at the polyprotein initiation site by ∼70% in both in vitro assays and in transfection assays (32, 33, 37).This observation has led to the idea that ribosomes first bind at AUG-586, but instead of initiating at this site, virtually all of them get transferred to the polyprotein initiation site (17). This raises questions as to the nature of the transfer process. Because insertion of an AUG codon between PV-1 nt 586 and the authentic initiation site conferred a small-plaque phenotype and because all large-plaque pseudo-revertants had lost the inserted AUG either by deletion or point mutation (25, 26), linear scanning is likely to be important. However, as the insertion resulted in a small-plaque phenotype rather than lethality, there remains the possibility that some ribosomes were transferred directly without scanning the whole distance. This has also been suggested on the grounds that insertion of AUGs or a hairpin loop between nt 586 and the authentic initiation site of PV-1 did not seem to reduce polyprotein synthesis in vitro as much as might be expected if the authentic initiation site is accessed by strictly linear scanning (8).The final puzzle is that AUG-586 is located in a stem-loop structure, domain VI (Fig. ​(Fig.1A),1A), which is conserved in all entero- and rhinoviruses apart from bovine enterovirus. If the initiating 40S subunits do inspect AUG-586 in some way, albeit an unproductive way, this stem-loop would need to open at least partly, if not completely. This need for domain VI to be opened might be considered an impediment to efficient initiation, and yet its strong conservation suggests the opposite, namely, that it might have a positive effect. Precise deletion of the spacer downstream of AUG-586 in PV-1(Mahoney), so that polyprotein synthesis now started at 586, reduced virus yield by ∼10-fold (39), and in an independent study a deletion that brought the polyprotein initiation site to nt 586 or 580 caused a very similar growth defect in PV-1(Sabin) although the defect was considerably less in a Mahoney background (13, 27). On the other hand, two smaller deletions in PV-1(Sabin) that retained just the whole base-paired domain VI or only its 5′ side, placing the polyprotein initiation site 52 or 31 nt, respectively, downstream of AUG-586, did not confer any significant negative phenotype (13, 27). Taken together, these results would seem to imply that the base pairing in domain VI is neutral to initiation efficiency, but the primary sequence of its 5′ side may confer a moderate positive effect. In this respect it is interesting that bovine enterovirus retains most of the sequence of the 5′ side of domain VI but lacks the complementary sequence of the 3′ side.Open in a separate windowFIG. 1.(A) Sequence and base pairing of IRES domain VI of HRV-2 and PV-1(Mahoney), numbered with respect to the viral genome sequence. (B) Hypothetical model for the opening of HRV-2 domain VI in two stages, showing that in the intermediate state AUG-576 and AUG-611 are both exposed.We have reexamined these issues but in the context of human rhinovirus 2 (HRV-2), mainly because the close proximity of the polyprotein initiation site (at nt 611) to the AUG (at nt 576) just downstream of the oligopyrimidine tract makes the interpretation of results less ambiguous than is the case with enteroviruses. A recent comprehensive sequence comparison of 106 different HRV strains plus 10 field isolates shows that HRV-2 domain VI is typical of the 106 serotypes and the one field isolate that differs in domain VI from its parent strain (35). In 95% of these sequences, the number of residues between the two AUG codons is in the range of 28 to 34 nt (median, 31 nt), with five outliers at 20 or 22 nt. The two AUGs are invariably base paired in a back-to-back configuration (Fig. ​(Fig.1A),1A), and the intervening residues fold into a base-paired structure, usually with a single mismatch (Fig. ​(Fig.1A)1A) or at least one G-U codon at around the mid-point and an apical loop of 3 to 6 residues (depending on the strain). The base-paired stem of enteroviruses is considerably shorter (usually without a mismatch), and the extra length in HRV domain VI generally consists of A-U and U-A pairs (often alternating) in the apical part (Fig. ​(Fig.1A).1A). In 23% of these 107 HRV domain VI sequences, the two AUGs are in the same reading frame, and in 17 (approximately two-thirds) of these there is no in-frame stop codon between them so that any initiation at the upstream AUG would result in synthesis of a VP0 protein (and, hence, also VP4) with an N-terminal extension.We first asked whether AUG-576 in HRV-2 is similar to AUG-586 in PV-1 in that there is very little initiation at this site, and yet AUG-576 is important for efficient initiation at the downstream polyprotein initiation site. We then looked for evidence that the domain VI stem-loop opens and whether all ribosomes access the authentic initiation site (AUG-611) by strictly linear scanning from some upstream site. We conclude that most ribosomes do access AUG-611 in this way, but a significant minority may take a shortcut, which could be facilitated if the apical part of this domain remains closed and base paired, with the single mismatch in the domain VI stem possibly causing the opening of this domain to occur in two stages (Fig. ​(Fig.1B1B).     

Dynamics of haplogroup frequencies and survival rates in a contact zone of two mtDNA lineages of the lizard Lacerta vivipara

The analysis of contact zones between lineages that were previously isolated in allopatry can lead to important insights on evolutionary processes such as selection and adaptation. In this paper we conducted a comparative demographic study of two mitochondrial DNA (mtDNA) lineages of the lizard Lacerta vivipara in the western Pyrénées to provide detail on the dynamics of their contact zone. By surveying haplogroup frequency across the contact area, we revealed the existence of a stable and very narrow contact zone between two parapatric lineages, which we infer to demonstrate a role for selection in the maintenance of this contact zone. We suggest these two lineages evolved in allopatry after retreating to different refugia during the Pleistocene glaciations, and subsequently came into secondary contact after the last glacial maximum. Although haplogroup frequencies were stable over time, we found significant age and environment (temperature) dependent survival differences between mtDNA haplogroups in one contact population sampled yearly from 2002 to 2009. Therefore, temperature‐induced demographic differences between the two mtDNA lineages may be responsible for the stability of this narrow contact zone. This is one of the first demographic studies conducted under natural conditions indicating the possibility of selection on mtDNA.     

CMC-modified cellulose biointerface for antibody conjugation

In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulose-based biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir-Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.     

Survival of the currently fittest: genetics of rainbow trout survival across time and space

As a fitness trait, survival is assumed to exhibit low heritability due to strong selection eroding genetic variation and/or spatio-temporal variation in mortality agents reducing genetic and increasing residual variation. The latter phenomenon in particular may contribute to low heritability in multigeneration data, even if certain cohorts exhibit significant genetic variation. Analysis of survival data from 10 year classes of rainbow trout reared at three test stations showed that treating survival as a single trait across all generations resulted in low heritability (h(2) = 0.08-0.17). However, when heritabilities were estimated from homogeneous generation and test station-specific cohorts, a wide range of heritability values was revealed (h(2) = 0.04-0.71). Of 64 genetic correlations between different cohorts, 20 were positive, but 16 were significantly negative, confirming that genetic architecture of survival is not stable across generations and environments. These results reveal the existence of hidden genetic variation for survival and demonstrate that treating survival as one trait over several generations may not reveal its true genetic architecture. Negative genetic correlations between cohorts indicate that overall survival has limited potential to predict general resistance, and care should be taken when using it as selection criterion.     

The role of pleiotropy vs signaller-receiver gene epistasis in life history trade-offs: dissecting the genomic architecture of organismal design in social systems

Traditional life history theory ignores trade-offs due to social interactions, yet social systems expand the set of possible trade-offs affecting a species evolution--by introducing asymmetric interactions between the sexes, age classes and invasion of alternative strategies. We outline principles for understanding gene epistasis due to signaller-receiver dynamics, gene interactions between individuals, and impacts on life history trade-offs. Signaller-receiver epistases create trade-offs among multiple correlated traits that affect fitness, and generate multiple fitness optima conditional on frequency of alternative strategies. In such cases, fitness epistasis generated by selection can maintain linkage disequilibrium, even among physically unlinked loci. In reviewing genetic methods for studying life history trade-offs, we conclude that current artificial selection or gene manipulation experiments focus on pleiotropy. Multi-trait selection experiments, multi-gene engineering methods or multiple endocrine manipulations can test for epistasis and circumvent these limitations. In nature, gene mapping in field pedigrees is required to study social gene epistases and associated trade-offs. Moreover, analyses of correlational selection and frequency-dependent selection are necessary to study epistatic social system trade-offs, which can be achieved with group-structured versions of Price's (1970) equation.     

Applicability of the co-inoculation technique using Agrobacterium tumefaciens shooty-tumour strain 82.139 in silver birch

In the co-inoculation technique, genetic transformation is performed using a mixture of Agrobacterium strains – shoot regeneration is induced by the wild-type strain 82.139, while the transferable genes are provided in a binary plasmid by another, non-oncogenic Agrobacterium strain. The aim of the present work was to study the applicability of co-inoculation under both in vitro and greenhouse conditions for in planta transformation in silver birch (Betula pendula Roth). In addition to the original method, several modifications of the technique including an genetically engineered 82.139 strain harbouring the binary pGUSINT were tested. The co-inoculations resulted in a gall formation frequency of 52–94% with greenhouse seedlings, and 4–63% with tissue-cultured plantlets, the shoot induction percentage varying by 0–13 in the greenhouse and 42–75 in vitro. PCR analysis verified that the majority of the regenerated material was non-transgenic, with a few individuals showing integration of the oncogenic T-DNA. According to the histochemical tests, however, some of the numerous differentiating buds and small shoots on gall tissues were transgenic, and contained the GUS reporter gene. The results show that it would have been necessary to apply selection pressure during differentiation in order to recover shoots transformed with the desired genes from the binary plasmid. The morphology and growth of all the regenerated plantlets was normal, suggesting that the oncogenic T-DNA was not expressed even though it was present. In conclusion, it was possible to obtain transgenic silver birch plantlets using the A. tumefaciens strain 82.139, but the co-inoculation method is not directly applicable as in planta transformation protocol.