Cardiac looping and the vertebrate left-right axis: antagonism of left-sided Vg1 activity by a right-sided ALK2-dependent BMP pathway.

The rightward looping of the primary heart tube is dependent upon upstream patterning events that establish the vertebrate left-right axis. In Xenopus, a left-sided Vg1 signaling pathway has been implicated in instructing cells to adopt a 'left-sided identity'; however, it is not known whether 'right-sided identity' is acquired by a default pathway or by antagonism of Vg1 signaling. Here, we propose that an antagonistic, BMP/ALK2/Smad-mediated signaling pathway is active on the right side of the Xenopus embryo. Truncated ALK2 receptor expression on the right side of the blastula elicits heart reversals and altered nodal expression. Consistent with these findings, constitutively active ALK2 (CA-ALK2) receptor expression on the left side of the blastula also elicits heart reversals and altered nodal expression. Coexpression of CA-ALK2 with mature Vg1 ligand results in predominantly left-sided nodal expression patterns and normal heart looping, demonstrating that the ALK2 pathway can 'rescue' left-right reversals that otherwise occur following right-sided misexpression of mature Vg1 ligand alone. Results with chimeric precursor proteins indicate that the mature domain of BMP ligands can mimic the ability of the ALK2 signaling pathway to antagonize the Vg1 pathway. Consistent with the observed antagonism between BMP and Vg1 ligands, left-sided ectopic expression of Xolloid results in heart reversals. Moreover, ectopic expression of Smad1 or Smad7 identified two downstream modulators of the BMP/ALK2 signaling pathway that also can regulate cardiac orientation. Collectively, these results define a BMP/ALK2-mediated pathway on the right side of the Xenopus embryo and, moreover, suggest that left-right patterning preceding cardiac morphogenesis involves the activation of two distinct and antagonistic, left- and right-sided TGF(beta)-related signaling pathways.     

Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.

Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M(3) muscarinic receptors. Acetylcholine release is limited by inhibitory M(2) muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M(2) receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M(2) dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M(2) dysfunction. M(2) receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M(2) function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M(2) receptor function after ozone is due to release of eosinophil MBP.     

Evidence for panmixia despite barriers to gene flow in the southern African endemic, <Emphasis Type="Italic">Caffrogobius caffer</Emphasis> (Teleostei: Gobiidae)

Background  

Oceanography and life-history characteristics are known to influence the genetic structure of marine species, however the relative role that these factors play in shaping phylogeographic patterns remains unresolved. The population genetic structure of the endemic, rocky shore dwelling Caffrogobius caffer was investigated across a known major oceanographic barrier, Cape Agulhas, which has previously been shown to strongly influence genetic structuring of South African rocky shore and intertidal marine organisms. Given the variable and dynamic oceanographical features of the region, we further sought to test how the pattern of gene flow between C. caffer populations is affected by the dominant Agulhas and Benguela current systems of the southern oceans.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP) signaling to inhibit steroidogenesis in human granulosa cells

Background  

Obesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells.     

Diverse initiation in a conserved left-right pathway?

Preceding stereotypical left-right asymmetric morphogenesis, asymmetric gene expression patterns of nodal and pitx2 are very similar in major groups of vertebrates. I propose that these conserved expression patterns are indicative of 'left-right' phylotypic stages' of development. It is not known whether these patterns are initiated by conserved or divergent developmental mechanisms.     

Opportunities and challenges with transitioning to non-lethal sampling of wild fish for microbiome research

The microbial communities of fish are considered an integral part of maintaining the overall health and fitness of their host. Research has shown that resident microbes reside on various mucosal surfaces, such as the gills, skin, and gastrointestinal tract, and play a key role in various host functions, including digestion, immunity, and disease resistance. A second, more transient group of microbes reside in the digesta, or feces, and are primarily influenced by environmental factors such as the host diet. The vast majority of fish microbiome research currently uses lethal sampling to analyse any one of these mucosal and/or digesta microbial communities. The present paper discusses the various opportunities that non-lethal microbiome sampling offers, as well as some inherent challenges, with the ultimate goal of creating a sound argument for future researchers to transition to non-lethal sampling of wild fish in microbiome research. Doing so will reduce animal welfare and population impacts on fish while creating novel opportunities to link host microbial communities to an individual's behavior and survival across space and time (e.g., life-stages, seasons). Current lethal sampling efforts constrain our ability to understand the mechanistic ecological consequences of variation in microbiome communities in the wild. Transitioning to non-lethal sampling will open new frontiers in ecological and microbial research.     

Long-Term Weight-Loss in Gastric Bypass Patients Carrying Melanocortin 4 Receptor Variants

Background

The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects.

Subjects/Methods

In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy.

Results

While carrying a rare MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype.

Conclusions

These data suggest that subtle differences in receptor signaling conferred by rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.     

Markers of genetic variation among the Waorani Indians of the Ecuadorian Amazon headwaters

Until recently, the Waorani Indians of Ecuador's Amazon headwaters maintained a fierce resistance to all intruders into their territory, and as a result of their actions and reputations a population of 600 people controlled a very large territory (about 8,000 square miles). The isolation of the Waorani has resulted in a large linguistic and genetic distance from their neighbors. Our survey of red cell enzymes, immunoglobulin allotypes, and dermatoglyphics demonstrates that the Waorani are a highly inbred and homogeneous population. Of 18 red cell enzymes studied, the Waorani have a limited polymorphism for only 6. Only two Gm haplotypes (Gm1,2,17,21, Gm1,17,21) were found and 60% of those tested were homozygous for the Gm1,17,21 haplotype. All individuals were A2m (1) and 95% of these were homozygous. The Waorani's dermatoglyphic traits fell within the wide range found among other South American Indians with close affinity to the Ecuadorian Jivaro group. Despite the limitations of these genetic systems, they demonstrate that the Waorani share limited genetic traits with the neighboring Jivaro Indians and are isolated from other tribal populations in South America.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.