On the origin, homologies and evolution of primate facial muscles, with a particular focus on hominoids and a suggested unifying nomenclature for the facial muscles of the Mammalia

The mammalian facial muscles are a subgroup of hyoid muscles (i.e. muscles innervated by cranial nerve VII). They are usually attached to freely movable skin and are responsible for facial expressions. In this study we provide an account of the origin, homologies and evolution of the primate facial muscles, based on dissections of various primate and non‐primate taxa and a review of the literature. We provide data not previously reported, including photographs showing in detail the facial muscles of primates such as gibbons and orangutans. We show that the facial muscles usually present in strepsirhines are basically the same muscles that are present in non‐primate mammals such as tree‐shrews. The exceptions are that strepsirhines often have a muscle that is usually not differentiated in tree‐shrews, the depressor supercilii, and lack two muscles that are usually differentiated in these mammals, the zygomatico‐orbicularis and sphincter colli superficialis. Monkeys such as macaques usually lack two muscles that are often present in strepsirhines, the sphincter colli profundus and mandibulo‐auricularis, but have some muscles that are usually absent as distinct structures in non‐anthropoid primates, e.g. the levator labii superioris alaeque nasi, levator labii superioris, nasalis, depressor septi nasi, depressor anguli oris and depressor labii inferioris. In turn, macaques typically lack a risorius, auricularis anterior and temporoparietalis, which are found in hominoids such as humans, but have muscles that are usually not differentiated in members of some hominoid taxa, e.g. the platysma cervicale (usually not differentiated in orangutans, panins and humans) and auricularis posterior (usually not differentiated in orangutans). Based on our observations, comparisons and review of the literature, we propose a unifying, coherent nomenclature for the facial muscles of the Mammalia as a whole and provide a list of more than 300 synonyms that have been used in the literature to designate the facial muscles of primates and other mammals. A main advantage of this nomenclature is that it combines, and thus creates a bridge between, those names used by human anatomists and the names often employed in the literature dealing with non‐human primates and non‐primate mammals.     

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi–Rhodnius prolixus interaction using confocal microscopy

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi–R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.     

First reported cases: renal denervation with secondgeneration multi-electrode catheter via brachial and radial access

Renal denervation is a minimally invasive procedure that aims to reduce brain–kidney sympathetic cross-talk. Despite the negative results of the recent SYMPLICITY HTN-3 trial, the procedure is considered safe and has been associated with many beneficial effects, including the reversal of hypertensive heart disease substrate and the prevention of cardiac arrhythmia. The first-generation radiofrequency catheter system featured a monopolar catheter that required sequential singlepoint energy application, followed by rotation, partial withdrawal of the catheter and re-application of energy. The latest generation device features four electrodes configured in a helical arrangement that can simultaneously ablate in four quadrants of the vessel circumference. Renal denervation via brachial or radial arterial access with the second-generation system has not been described before.     

Inter- and intra-rater reliability of diffusion tensor imaging parameters in the normal pediatric spinal cord

AIM: To assess inter- and intra-rater reliability (agreement) between two region of interest (ROI) methods in pediatric spinal cord diffusion tensor imaging (DTI).METHODS: Inner-Field-of-View DTI data previously acquired from ten pediatric healthy subjects (mean age = 12.10 years) was used to assess for reliability. ROIs were drawn by two neuroradiologists on each subject data twice within a 3-mo interval. ROIs were placed on axial B₀ maps along the cervical spine using free-hand and fixed-size ROIs. Agreement analyses for fractional anisotropy (FA), axial diffusivity, radial diffusivity and mean diffusivity were performed using intra-class-correlation (ICC) and Cronbach’s alpha statistical methods.RESULTS: Inter- and intra-rater agreement between the two ROI methods showed moderate (ICC = 0.5) to strong (ICC = 0.84). There were significant differences between raters in the number of pixels selected using free-hand ROIs (P < 0.05). However, no significant differences were observed in DTI parameter values. FA showed highest variability in ICC values (0.10-0.87). Cronbach’s alpha showed moderate-high values for raters and ROI methods.CONCLUSION: The study showed that high reproducibility in spinal cord DTI can be achieved, and demonstrated the importance of setting detailed methodology for post-processing DTI data, specifically the placement of ROIs.     

Mitochondrial involvement in memory impairment induced by scopolamine in rats

Objective: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action.

Methods: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function.

Results: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H₂O₂ generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly.

Conclusion: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.     

Distribution of virulence markers in clinical and environmental Vibrio cholerae non-O1/non-O139 strains isolated in Brazil from 1991 to 2000

One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed.     

Discordant Brucella melitensis Antigens Yield Cognate CD8+ T Cells In Vivo

Brucella spp. are intracellular bacteria that cause the most frequent zoonosis in the world. Although recent work has advanced the field of Brucella vaccine development, there remains no safe human vaccine. In order to produce a safe and effective human vaccine, the immune response to Brucella spp. requires greater understanding. Induction of Brucella-specific CD8⁺ T cells is considered an important aspect of the host response; however, the CD8⁺ T-cell response is not clearly defined. Discovering the epitope containing antigens recognized by Brucella-specific CD8⁺ T cells and correlating them with microarray data will aid in determining proteins critical for vaccine development that cover a kinetic continuum during infection. Developing tools to take advantage of the BALB/c mouse model of Brucella melitensis infection will help to clarify the correlates of immunity and improve the efficacy of this model. Two H-2^d CD8⁺ T-cell epitopes have been characterized, and a group of immunogenic proteins have provoked gamma interferon production by CD8⁺ T cells. RYCINSASL and NGSSSMATV induced cognate CD8⁺ T cells after peptide immunization that showed specific killing in vivo. Importantly, we found by microarray analysis that the genes encoding these epitopes are differentially expressed following macrophage infection, further emphasizing that these discordant genes may play an important role in the pathogenesis of B. melitensis infection.Brucellosis is the world''s most common zoonosis, with more than half a million new human infections each year (44). Brucellosis has been endemic to the Mediterranean and Middle East since ancient times, since carbonized cheese and skeletal remains in Pompeii show evidence of Brucella spp. (8). Evidence of brucellosis also exists in the skeleton of a 2.4- to 2.8-million-year-old hominid (16). In areas of endemicity, domestic animal brucellosis severely affects regional economies, and vaccination campaigns cannot always reach nomadic herders. Human infections occur in these regions mainly from the ingestion of infected animal products, including unpasteurized milk and fresh cheeses (14). Antibiotic treatment exists but is costly and prolonged, lasting at least 6 weeks in moderate cases, and it may extend for years depending on complications that arise. Even after treatment, PCR data have revealed that low levels of bacteria are detectable years after the resolution of symptoms, and relapses occur in 5 to 30% of cases (20, 30, 55, 62). In areas where brucellosis is endemic, prevention of infection via vaccine would be far more cost-effective than the regimen of antibiotics suggested by the World Health Organization (WHO). Unfortunately, this disease flies below the radar of many of the major world health agencies, and the problem is compounded by frequent misdiagnosis and under-reporting (15, 20).Although brucellosis is eradicated from food sources here, in the post-Gulf War United States, awareness was raised to fund vaccine research concerning potential biological weapons. Brucella melitensis, B. abortus, and B. suis are considered category B select agents because of the ease of aerosolization, diverse symptoms, and chronic persistence. The spectrum of disease that results from Brucella infection suggests that Brucella spp. could be a biological weapon in the current absence of any human vaccine (43). Human symptoms begin with a general malaise and fever, followed by organ-specific “hot spots” of infection, for instance, endocarditis and orchitis. In the United States, infections are due to accidental infection with a live animal vaccine by veterinarians and laboratory workers. In fact, brucellosis is one of the most common laboratory-acquired infections, and the lack of a human vaccine discourages work with the agent (20, 37, 40).Three vaccines are currently recommended by the WHO for livestock, and all of them are live-attenuated Brucella strains: B. abortus S-19 and RB-51 for bovine brucellosis and B. melitensis Rev-1 for goat and sheep brucellosis. These vaccine constructs are not completely effective and pose safety risks, including abortifacient effects and residual virulence, making them unsuitable for human application (33). Heat-killed Brucella does not induce detectable interleukin-12 (IL-12) in vivo, and killed bacteria actively suppress IL-12 production in response to challenge with live bacteria by unknown mechanisms (24). Studies conducted in our laboratory, and confirmed by others, have shown that subunit vaccines can confer a degree of short-term protection but have not elicited long-term effective immunity (3, 39). Only live bacteria appear to induce cell-mediated immunity, whereas dead bacteria induce a nonprotective humoral response (31, 36).CD4⁺ T cells induce the production of IgG2 antibodies from B cells during the course of murine and ovine B. melitensis infections (9, 56). There is evidence that this humoral response is an indispensable aspect of the host defenses in that opsonization may be required for successful uptake by macrophages, although a humoral response is not protective (7, 18, 31). In addition, although opsonization may result in increased bacterial uptake by macrophages, bacterial survival is unchanged (18). Previous studies have shown that host protection can be mediated by gamma interferon (IFN-γ) produced by CD4⁺ T cells, although data have also shown that treatment of macrophages with optimal concentrations of IFN-γ still allows some intracellular Brucella to survive (19, 26, 57, 63). Brucella can escape complement-mediated killing and thrive inside the acidified phagosomes of macrophages, using the common bactericidal host mechanisms to its own advantage (11, 13, 28a). In addition, major histocompatibility complex (MHC) class II antigen presentation can be disrupted by Brucella lipopolysaccharide that has incorporated into the host cell membrane (28). In our lab and others, evidence supports that protection in animal models is engendered by CD8⁺ T cells (10, 12, 22, 27, 38, 42, 64). Therefore, we chose to investigate the Brucella antigens that are recognized by CD8⁺ T cells in the context of MHC class I molecules.In the United States, most select agent work is confined to biosafety level 3 and above, the logistics of which largely dictate the use of small-animal models in Brucella research. Mice are not a natural host of B. melitensis, making the optimization of this model a high priority. By exploring the CD8⁺ T-cell component of the BALB/c mouse response to B. melitensis infection, we are further refining the mouse as a valuable tool in Brucella research and vaccine development.Determining the epitopes recognized by Brucella-specific CD8⁺ T cells and the Brucella genes encoding the proteins containing these epitopes will help establish proteins critical for vaccine development (47, 48, 51, 52, 60). Epitopes were predicted from the Brucella genome using an algorithm based on allele-specific binding motifs and cleavage sites (49, 50). Select peptides were then tested for their capacity to bind their respective MHC alleles in vitro (54). Peptides subsequently deemed epitopes displayed a combination of immunogenicity, natural processing, and functional avidity, while eliciting CD8⁺ T cells that kill in vivo. Peptide immunogenicity was evaluated using peptide pools in adjuvant, whereas natural processing and functional avidity tests used nonreplicating but metabolically active whole B. melitensis to immunize mice. Our approach has identified the first B. melitensis-specific MHC class I CD8⁺ T-cell epitopes that are recognized in H-2^d mice and generate CD8⁺ T cells that kill in vivo. These present findings offer insight regarding the debate concerning Brucella correlates of immunity and provide guidance in designing a safe and viable human vaccine.