The shell matrix of the european thorny oyster, Spondylus gaederopus: microstructural and molecular characterization.

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids - fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

Wounding of Arabidopsis halleri leaves enhances cadmium accumulation that acts as a defense against herbivory.

Approximately 0.2% of all angiosperms are classified as metal hyperaccumulators based on their extraordinarily high leaf metal contents, for example >1% zinc, >0.1% nickel or >0.01% cadmium (Cd) in dry biomass. So far, metal hyperaccumulation has been considered to be a taxon-wide, constitutively expressed trait, the extent of which depends solely on available metal concentrations in the soil. Here we show that in the facultative metallophyte Arabidopsis halleri, both insect herbivory and mechanical wounding of leaves trigger an increase specifically in leaf Cd accumulation. Moreover, the Cd concentrations accumulated in leaves can serve as an elemental defense against herbivory by larvae of the Brassicaceae specialist small white (Pieris rapae), thus allowing the plant to take advantage of this non-essential trace element and toxin. Metal homeostasis genes are overrepresented in the systemic transcriptional response of roots to the wounding of leaves in A. halleri, supporting that leaf Cd accumulation is preceded by systemic signaling events. A similar, but quantitatively less pronounced transcriptional response was observed in A. thaliana, suggesting that the systemically regulated modulation of metal homeostasis in response to leaf wounding also occurs in non-hyperaccumulator plants. This is the first report of an environmental stimulus influencing metal hyperaccumulation.

The genome of the fire ant Solenopsis invicta.

Ants have evolved very complex societies and are key ecosystem members. Some ants, such as the fire ant Solenopsis invicta, are also major pests. Here, we present a draft genome of S. invicta, assembled from Roche 454 and Illumina sequencing reads obtained from a focal haploid male and his brothers. We used comparative genomic methods to obtain insight into the unique features of the S. invicta genome. For example, we found that this genome harbors four adjacent copies of vitellogenin. A phylogenetic analysis revealed that an ancestral vitellogenin gene first underwent a duplication that was followed by possibly independent duplications of each of the daughter vitellogenins. The vitellogenin genes have undergone subfunctionalization with queen- and worker-specific expression, possibly reflecting differential selection acting on the queen and worker castes. Additionally, we identified more than 400 putative olfactory receptors of which at least 297 are intact. This represents the largest repertoire reported so far in insects. S. invicta also harbors an expansion of a specific family of lipid-processing genes, two putative orthologs to the transformer/feminizer sex differentiation gene, a functional DNA methylation system, and a single putative telomerase ortholog. EST data indicate that this S. invicta telomerase ortholog has at least four spliceforms that differ in their use of two sets of mutually exclusive exons. Some of these and other unique aspects of the fire ant genome are likely linked to the complex social behavior of this species.

Diverting the Use of Hand-Operated Tablet Press Machines to Bioassays: A Novel Protocol to Test 'Waste' Insoluble Shell Matrices.

To mineralize their shells, molluscs secrete a complex cocktail of proteins-collectively defined as the calcifying shell matrix-that remains occluded in the exoskeleton. Nowadays, protein extracts from shells are recognized as a potential source of bioactive substances, among which signalling molecules, bactericides or protease inhibitors offer the most tangible perspectives in applied sciences, health, and aquaculture. However, one technical obstacle in testing the activity of shell extracts lies in their high insolubility. In this paper, we present a protocol that circumvents this impediment. After an adapted shell protein extraction and the production of two organic fractions-one soluble, one insoluble-we employ a hand-operated tablet press machine to generate well-calibrated tablets composed of 100% insoluble shell matrix. FT-IR monitoring of the quality of the tablets shows that the pressure used in the press machine does not impair the molecular properties of the insoluble extracts. The produced tablets can be directly tested in different biological assays, such as the bactericidal inhibition zone assay in Petri dish, as illustrated here. Diverting the use of the hand-operated tablet press opens new perspectives in the analysis of insoluble shell matrices, for discovering novel bioactive components.

Management of pulmonary hypertension associated with valvular heart disease with angiotensin-receptor neprilysin inhibitor.

Pulmonary hypertension secondary to left-sided valvular disease (VHD-PH) is associated with high morbidity and mortality. Angiotensin-receptor neprilysin inhibitor (ARNI) is a novel pharmacotherapy, which reduces afterload with natriuresis and peripheral vasodilation. Our cases demonstrate that ARNI may also have a role in the treatment of combined pre- and postcapillary pulmonary hypertension that is independent of its effect on pulmonary capillary wedge pressure and cardiac output. Future prospective trials are needed to evaluate role of ARNIs in treatment of VHD-PH.

Molecular characterization of accripin11, a soluble shell protein with an acidic C-terminus, identified in the prismatic layer of the Mediterranean fan mussel Pinna nobilis (Bivalvia, Pteriomorphia).

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO3 , while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization.

In situ mapping of biomineral skeletal proteins by molecular recognition imaging with antibody-functionalized AFM tips.

Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine.

The priming agent ß-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.

Level I trauma center internal campaign to increase safe driving by staff.

A 3-month internal campaign at our trauma center was conducted to improve employee motor vehicle safety and have employees serve as community role models. The injury-prevention coordinator and trauma program manager developed and implemented a campaign of high-visibility events. Effectiveness was evaluated through pre- and postcampaign surveys and seat belt-use observation and observation at 1 year. Although survey responses did not change, observed seat belt use increased significantly from 79% to 87%, with maintenance at 1 year (89%). No direct causal effect can be proven, but the campaign appears to have increased employee seat belt use.

Impact of normalization on miRNA microarray expression profiling.

Profiling miRNA levels in cells with miRNA microarrays is becoming a widely used technique. Although normalization methods for mRNA gene expression arrays are well established, miRNA array normalization has so far not been investigated in detail. In this study we investigate the impact of normalization on data generated with the Agilent miRNA array platform. We have developed a method to select nonchanging miRNAs (invariants) and use them to compute linear regression normalization coefficients or variance stabilizing normalization (VSN) parameters. We compared the invariants normalization to normalization by scaling, quantile, and VSN with default parameters as well as to no normalization using samples with strong differential expression of miRNAs (heart-brain comparison) and samples where only a few miRNAs are affected (by p53 overexpression in squamous carcinoma cells versus control). All normalization methods performed better than no normalization. Normalization procedures based on the set of invariants and quantile were the most robust over all experimental conditions tested. Our method of invariant selection and normalization is not limited to Agilent miRNA arrays and can be applied to other data sets including those from one color miRNA microarray platforms, focused gene expression arrays, and gene expression analysis using quantitative PCR.

Impact of 6-Month Exposure to Aerosols From Potential Modified Risk Tobacco Products Relative to Cigarette Smoke on the Rodent Gastrointestinal Tract.

Cigarette smoking causes adverse health effects that might occur shortly after smoking initiation and lead to the development of inflammation and cardiorespiratory disease. Emerging studies have demonstrated the role of the intestinal microbiome in disease pathogenesis. The intestinal microbiome is susceptible to the influence of environmental factors such as smoking, and recent studies have indicated microbiome changes in smokers. Candidate modified risk tobacco products (CMRTP) are being developed to provide substitute products to lower smoking-related health risks in smokers who are unable or unwilling to quit. In this study, the ApoE-/- mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F and aerosols from two CMRTPs based on the heat-not-burn principle [carbon-heated tobacco product 1.2 (CHTP 1.2) and tobacco heating system 2.2 (THS 2.2)] on the intestinal microbiome over a 6-month period. The effect of cessation or switching to CHTP 1.2 after 3 months of CS exposure was also assessed. Next-generation sequencing was used to evaluate the impact of CMRTP aerosols in comparison to CS on microbiome composition and gene expression in the digestive tract of mice. Our analyses highlighted significant gene dysregulation in response to 3R4F exposure at 4 and 6 months. The findings showed an increase in the abundance of Akkermansiaceae upon CS exposure, which was reversed upon cessation. Cessation resulted in a significant decrease in Akkemansiaceae abundance, whereas switching to CHTP 1.2 resulted in an increase in Lactobacillaceae abundance. These microbial changes could be important for understanding the effect of CS on gut function and its relevance to disease pathogenesis via the microbiome.

Comprehensive analysis of Arabidopsis expression level polymorphisms with simple inheritance.

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5' regulatory sequence variation in the corresponding genes is indeed increased. However, approximately 42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a >4-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL.

Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins.

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo- and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicity-related host adaptation mechanisms of these human pathogenic fungi.

Tobacco Heating System 2.2 has a limited impact on DNA methylation of candidate enhancers in mouse lung compared with cigarette smoke.

Cigarette smoke (CS) exposure has been shown to correlate with changes in DNA methylation levels, however, the impact of CS on DNA methylation at genome-wide scale is missing. Here, we used whole-genome bisulfite sequencing to assess the effects of CS extract and aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product, on DNA methylation in lung and liver tissues from apolipoprotein E-deficient mice during an eight-month period of exposure. We found that in lung tissue, CS mainly induced hypermethylation of candidate enhancers at late time points, while promoters were less affected. This effect was strongly reduced upon cessation or switching to THS 2.2. By contrast, chronic exposure to THS 2.2 had a limited effect on DNA methylation at both promoters and enhancers. We also identified members of the Ets and Fox families of transcription factors as potential players in the epigenetic response to CS exposure in lung tissue. In contrast to the lung, DNA methylation in the liver was largely insensitive to all investigated exposures. In summary, our investigations indicate that CS-related DNA methylation alterations are tissue-specific, occur mainly at enhancers and are strongly reduced upon smoking cessation or switching to THS2.2.

'Palaeoshellomics' reveals the use of freshwater mother-of-pearl in prehistory.

The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including 'palaeoshellomics', the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of 'double-buttons'. This craft is found throughout Europe between 4200-3800 BCE, highlighting the ornament-makers' profound knowledge of the biogeosphere and the existence of cross-cultural traditions.

Affymetrix Whole-Transcript Human Gene 1.0 ST array is highly concordant with standard 3' expression arrays.

The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3' based arrays: (i) it interrogates the entire mRNA transcript, and (ii) it uses DNA targets. To assess the impact of these differences on array performance, we performed a series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both RNA and DNA targets were hybridized on HG-U133 Plus 2.0 arrays. The results show that the overall reproducibility of the Gene 1.0 ST array is best. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. Agreements are best between the two labeling protocols using HG-U133 Plus 2.0 array. The Gene 1.0 ST array is most concordant with the HG-U133 array hybridized with cDNA targets. This may reflect the impact of the target type. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array.

Comparison of bare metal stents versus drug-eluting stents on clinical decision making in patients with previous percutaneous coronary intervention admitted for suspected acute coronary syndrome.

To determine whether the decreased rate of restenosis observed with drug-eluting stents (DES) has changed the treatment of patients with recurrent symptoms after stent placement, we compared patients hospitalized with presumed cardiac symptoms within 1 year after placement of either a DES or a bare metal stent (BMS). In this retrospective, single-center study, cases were identified from consecutive patients who received a DES from March 2003 to July 2004 or a BMS from August 2001 to June 2002. No differences were noted in the rate of hospitalization, hospitalization for presumed cardiac symptoms, use of coronary angiography in patients hospitalized for presumed cardiac symptoms, or average interval to hospitalization. In contrast, restenosis and the need for additional revascularization procedures were higher in the BMS group. The primary indication for additional revascularization was restenosis in the BMS group and progression of coronary artery disease in the DES group. In the DES group, the need for revascularization was significantly higher in patients with multi- versus single-vessel coronary artery disease (26% vs 7%, p < 0.05). In conclusion, the rate of hospitalization and use of coronary angiography in patients with recurrent symptoms were similar in patients who received a BMS or DES, despite the decreased rates of restenosis and additional revascularization procedures observed with DESs.

The prevalence of cognitive impairment among African-American patients with congestive heart failure.

This cross-sectional study sought to determine the prevalence of cognitive impairment among African-American patients with congestive heart failure (CHF). We studied 100 African-American CHF patients (aged 55-87 years) in New York Heart Association classes II-IV, who are enrolled in an ongoing, randomized, controlled trial, evaluating the effectiveness of a telemonitoring intervention to improve access to ambulatory care for heart failure patients. These CHF patients were recruited from an inner-city practice, rural physician practices and an urban physician practice in Atlanta. The Mini-Mental Status Examination (MMSE) was used to measure cognition. Cognitive impairment was defined as a MMSE score of less than 24. The crude prevalence of cognitive impairment was 10% in this population of African Americans with CHF. The results of multivariate logistic regression analysis indicated an increase in odds of cognitive impairment with increasing age [odds ratio (OR) = 1.10 and 95% confidence interval, 1.00-1.20; p=0.042]. There was no significant relationship between cognitive impairment and gender, education status, depression and severity of CHF. This study indicates that cognitive impairment is relatively prevalent among African Americans with CHF, but lower than previously reported among Caucasians with CHF.

Protection from HIV-1 infection of primary CD4 T cells by CCR5 silencing is effective for the full spectrum of CCR5 expression.

Stable gene silencing by RNA interference (RNAi) can be achieved by expression of small hairpin RNAs (shRNAs) from RNA polymerase III promoters. We have tested lentiviral vectors expressing shRNAs targetting CCR5 in primary CD4 T cells from donors representing various CCR5 and CCR2 genetic backgrounds covering the full spectrum of CCR5 expression levels and permissiveness for HIV-1 infection. A linear decrease in CCR5 expression resulted in a logarithmic decrease in cellular infection, giving up to three logs protection from HIV-1 infection in vitro. Protection was maintained at very high multiplicity of infection. This and other recent reports on RNAi should open a debate about the use of RNAi gene therapy for HIV infection.

Substantial deletion overlap among divergent Arabidopsis genomes revealed by intersection of short reads and tiling arrays.

Identification of small polymorphisms from next generation sequencing short read data is relatively easy, but detection of larger deletions is less straightforward. Here, we analyzed four divergent Arabidopsis accessions and found that intersection of absent short read coverage with weak tiling array hybridization signal reliably flags deletions. Interestingly, individual deletions were frequently observed in two or more of the accessions examined, suggesting that variation in gene content partly reflects a common history of deletion events.