Evidence of gene–environment interaction for the IRF6 gene and maternal multivitamin supplementation in controlling the risk of cleft lip with/without cleft palate

Although multiple genes have been identified as genetic risk factors for isolated, non-syndromic cleft lip with/without cleft palate (CL/P), a complex and heterogeneous birth defect, interferon regulatory factor 6 gene (IRF6) is one of the best documented genetic risk factors. In this study, we tested for association between markers in IRF6 and CL/P in 326 Chinese case–parent trios, considering gene–environment interaction for two common maternal exposures, and parent-of-origin effects. CL/P case–parent trios from three sites in mainland China and Taiwan were genotyped for 22 single nucleotide polymorphisms (SNPs) in IRF6. The transmission disequilibrium test was used to test for marginal effects of individual SNPs. We used PBAT to screen the SNPs and haplotypes for gene–environment (G × E) interaction and conditional logistic regression models to quantify effect sizes for SNP–environment interaction. After Bonferroni correction, 14 SNPs showed statistically significant association with CL/P. Evidence of G × E interaction was found for both maternal exposures, multivitamin supplementation and environmental tobacco smoke (ETS). Two SNPs showed evidence of interaction with multivitamin supplementation in conditional logistic regression models (rs2076153 nominal P = 0.019, rs17015218 nominal P = 0.012). In addition, rs1044516 yielded evidence for interaction with maternal ETS (nominal P = 0.041). Haplotype analysis using PBAT also suggested interaction between SNPs in IRF6 and both multivitamin supplementation and ETS. However, no evidence for maternal genotypic effects or significant parent-of-origin effects was seen in these data. These results suggest IRF6 gene may influence risk of CL/P through interaction with multivitamin supplementation and ETS in the Chinese population.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.     

Fipronil recognition by the FA1 site of human serum albumin

Fipronil is a broad‐spectrum pesticide widely used in agriculture, horticulture, and forestry. Because fipronil can cause a variety of toxic effects in animals and humans, its use is authorized as a pesticide in veterinary medicinal products for pets, but not for the treatment of livestock animals whose products are intended for consumption. Recently, however, the presence of fipronil residues has been detected in the eggs and meat of layer hens from farms located in different European countries. Given the relevance of fipronil toxicity for human health, it is important to gain information concerning its fate in the human body, including its binding mode to human serum albumin (HSA), the most abundant protein in plasma. Here, the inhibition of heme‐Fe(III) binding to the fatty acid site 1 (FA1) of HSA by fipronil is reported. Docking simulations support functional data, indicating that the FA1 site is the preferential cleft for fipronil recognition by HSA. The affinity of fipronil for HSA (K_f = 1.9 × 10^?6 M, at pH 7.3, and 20.0°C) may be relevant in vivo. Indeed, HSA could play a pivotal role in fipronil transport and scavenging, thus reducing the pesticide‐free plasmatic levels, with consequent reduced systemic toxicity. In turn, fipronil binding to the FA1 site of HSA could impair the recognition of endogenous and exogenous molecules.     

Thermal specialization across large geographical scales predicts the resilience of mangrove crab populations to global warming

The broad prediction that ectotherms will be more vulnerable to climate change in the tropics than in temperate regions includes assumptions about centre/edge population effects that can only be tested by within‐species comparisons across wide latitudinal gradients. Here, we investigated the thermal vulnerability of two mangrove crab species, comparing populations at the centre (Kenya) and edge (South Africa) of their distributions. At the same time, we investigated the role of respiratory mode (water‐ versus air‐breathing) in determining the thermal tolerance in amphibious organisms. To do this, we compared the vulnerability to acute temperature fluctuations of two sympatric species with two different lifestyle adaptations: the free living Perisesarma guttatum and the burrowing Uca urvillei, both pivotal to the ecosystem functioning of mangroves. The results revealed the air‐breathing U. urvillei to be a thermal generalist with much higher thermal tolerances than P. guttatum. Importantly, however, we found that, while U. urvillei showed little difference between edge and centre populations, P. guttatum showed adaptation to local conditions. Equatorial populations had elevated tolerances to acute heat stress and mechanisms of partial thermoregulation, which make them less vulnerable to global warming than temperate conspecifics. The results reveal both the importance of respiratory mode to thermal tolerance and the unexpected potential for low latitude populations/species to endure a warming climate. The results also contribute to a conceptual model on the latitudinal thermal tolerance of these key species. This highlights the need for an integrated population‐level approach to predict the consequences of climate change.     

Nusinersen treatment and cerebrospinal fluid neurofilaments: An explorative study on Spinal Muscular Atrophy type 3 patients

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response.     

Interpopulation variation in pollinators and floral scent of the lady’s-slipper orchid <Emphasis Type="Italic">Cypripedium calceolus</Emphasis> L.

Floral scent is a key mediator in many plant–pollinator interactions. It is known to vary not only among plant species, but also within species among populations. However, there is a big gap in our knowledge of whether such variability is the result of divergent selective pressures exerted by a variable pollinator climate or alternative scenarios (e.g., genetic drift). Cypripedium calceolus is a Eurasian deceptive lady’s-slipper orchid pollinated by bees. It is found from near sea level to altitudes of 2500 m. We asked whether pollinator climate and floral scents vary in a concerted manner among different altitudes. Floral scents of four populations in the Limestone Alps were collected by dynamic headspace and analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Flower visitors and pollinators (the subset of visitors with pollen loads) were collected and identified. Preliminary coupled gas chromatographic and electroantennographic measurements with floral scents and pollinators revealed biologically active components. More than 70 compounds were detected in the scent samples, mainly aliphatics, terpenoids, and aromatics. Although several compounds were found in all samples, and all samples were dominated by linalool and octyl acetate, scents differed among populations. Similarly, there were strong differences in flower visitor spectra among populations with most abundant flower visitors being bees and syrphid flies at low and high altitudes, respectively. Pollinator climate differed also among populations; however, independent of altitude, most pollinators were bees of Lasioglossum, Andrena, and Nomada. Only few syrphids acted as pollinators and this is the first record of flies as pollinators in C. calceolus. The electrophysiological tests showed that bees and syrphid flies sensed many of the compounds released by the flowers, among them linalool and octyl acetate. Overall, we found that both floral scent and visitor/pollinator climate differ among populations. We discuss whether interpopulation variation in scent is a result of pollinator-mediated selection.     

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.     

Functionalized pyrazoles and pyrazolo[3,4-d]pyridazinones: Synthesis and evaluation of their phosphodiesterase 4 inhibitory activity

A series of pyrazoles and pyrazolo[3,4-d]pyridazinones were synthesized and evaluated for their PDE4 inhibitory activity. All the pyrazoles were found devoid of activity, whereas some of the novel pyrazolo[3,4-d]pyridazinones showed good activity as PDE4 inhibitors. The most potent compounds in this series showed an IC₅₀ in the nanomolar range. The ability to inhibit TNF-α release in human PBMCs was determined for two representative compounds, finding values in the sub-micromolar range. SARs studies demonstrated that the best arranged groups around the heterocyclic core are 2-chloro-, 2-methyl- and 3-nitrophenyl at position 2, an ethyl ester at position 4 and a small alkyl group at position 6. Molecular modeling studies performed on a representative compound allowed to define its binding mode to the PDE4B isoform.