Target Organs of Metals Released from Metal-on-Polyethylene Knee and Hip Arthroplasty Implants: Implications for Tissue Metal Profiles.

Metals are used in orthopedic implants. The wear of arthroplasty implant can lead to the release of arthroprosthetic metals, both locally and systemically, after migration into the organs. While the toxicity of metal-on-metal arthroplasty implants is well-known and monitored, the toxicity associated with metal-on-polyethylene (MoP) ones is not as comprehensively understood. This study aimed to investigate the release of metals from MoP arthroplasty implants and their impact on the tissue metal profile in autopsied individuals, comparing them to deceased controls without prostheses. High-resolution ICP-MS was employed to analyze 39 metals in the blood, urine, hair, organs, and periprosthetic tissue of 25 deceased individuals with arthroplasty implants and 20 control subjects (Prometox study, protocol ID: APHP180539, NCT03812627). Eight metals (beryllium, chromium, cobalt, lanthanum, molybdenum, nickel, tellurium, titanium) exhibited significant impacts in arthroplasty implant wearers across various organs. Increased concentrations of La and Be were observed, the origin of which could not be precisely defined within the scope of this study. Notably, the lungs emerged as the primary target organ for metallic ions contained in implants. This study suggests that MoP arthroplasty implants, even when functional and not visibly worn, release arthroprosthetic metals into the body, potentially causing disturbances. Furthermore, considering the presence of an arthroplasty implant in autopsy reports may be relevant, as the released metals could influence the tissue metal profile.

CRISPR/Cas9-Targeted Mutagenesis of CiGAS and CiGAO to Reduce Bitterness in Chicory (Cichorium intybus L.).

BACKGROUND: Chicory (Cichorium intybus L.), a member of the Asteraceae family, is known for its numerous health benefits, including its prebiotic, digestive, antioxidant or anti-inflammatory effects. Used as a coffee substitute, chicory roots is also appreciated for its bitterness, which can prove to be a disadvantage for other uses in food. The bitterness of chicory is largely linked to the presence of sesquiterpene lactones (STLs) in the roots. METHODS: In order to create less bitter industrial chicory varieties, CRISPR/Cas9 technology was used to inhibit the first two genes of the STL biosynthetic pathway: germacrene A synthase (CiGAS), short form, and germacrene A oxidase (CiGAO). To determine the impact of these reductions on the perception of bitterness, a sensory analysis of 13 field-grown chicories genotypes, contrasting for their STL composition, allowed the construction of obtain a bitterness scale by correlating STL content with perceived bitterness. The edited chicories were positioned on this scale according to their STL content. RESULTS: Biallelic mutations in two of the copies of CiGAS-short form or in the CiGAO gene led to a reduction in STL content of edited chicories and a reduction in bitterness, or even an absence of perception, was obtained for some mutants. CONCLUSIONS: The use of the CRISPR/Cas9 tool as well as the choice of targets therefore makes it possible to modulate the bitterness of chicory.

Multivariate analysis of chemical and genetic diversity of wild Humulus lupulus L. (hop) collected in situ in northern France.

The hop plant (Humulus lupulus L.) has been exploited for a long time for both its brewing and medicinal uses, due in particular to its specific chemical composition. These last years, hop cultivation that was in decline has been experiencing a renewal for several reasons, such as a craze for strongly hopped aromatic beers. In this context, the present work aims at investigating the genetic and chemical diversity of fifty wild hops collected from different locations in Northern France. These wild hops were compared to ten commercial varieties and three heirloom varieties cultivated in the same sampled geographical area. Genetic analysis relying on genome fingerprinting using 11 microsatellite markers showed a high level of diversity. A total of 56 alleles were determined with an average of 10.9 alleles per locus and assessed a significant population structure (mean pairwise FST = 0.29). Phytochemical characterization of hops was based on volatile compound analysis by HS-SPME GC-MS, quantification of the main prenylated phenolic compounds by UHPLC-UV as well as untargeted metabolomics by UHPLC-HRMS and revealed a high level of chemical diversity among the assessed wild accessions. In particular, analysis of volatile compounds revealed the presence of some minor but original compounds, such as aromadendrene, allo-aromadendrene, isoledene, ß-guaiene, α-ylangene and ß-pinene in some wild accessions; while analysis of phenolic compounds showed high content of ß-acids in these wild accessions, up to 2.37% of colupulone. Genetic diversity of wild hops previously observed was hence supported by their chemical diversity. Sample soil analysis was also performed to get a pedological classification of these different collection sites. Results of the multivariate statistical analysis suggest that wild hops constitute a huge pool of chemical and genetic diversity of this species.

A recurrent de novo splice site variant involving DNM1 exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism.

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) as a result of a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied with a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in 39 pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, c.1197-8G>A (GenBank: NM_001288739.1), which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of enlarged synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.Arg399Trp and p.Gly401Asp, had a similar DEE phenotype. In contrast, one individual with a missense variant affecting exon 10b, p.Pro405Leu, which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants as well as the possibility of splice site variants acting through a dominant-negative mechanism.

The Ogival Palate: A New Risk Marker of Sudden Unexpected Death in Infancy?

Objective: Ogival palate (i.e., a narrow and high-arched palate) is usually described in obstructive breath disorder but has been found in infants unexpectedly deceased. We studied the association between ogival palate and sudden unexpected death in infancy (SUDI) on the basis of a computed tomography (CT) evaluation. Methods: We conducted a monocentric case-control study of children under 2 years of age who died of SUDI, for which a head CT scan and an autopsy were performed between 2011 and 2018. Each case was matched by sex and age (± 30 days) to two controls selected among living children in the same center who benefited from a cranio-encephalic CT scan. Four parameters of the hard palate were measured by CT: height, width, length, and sagittal angle; the height/width ratio was calculated. The presence of an ogival palate was also subjectively evaluated by the radiologists, independently from the measurements. Standardized odds ratios (OR) were calculated using conditional logistic regression models, all expressed for +1 standard deviation (SD). Results: Thirty-two deceased children were matched to 64 living control children. Mean ages were 5.0 and 5.3 months, respectively. Twenty-eight cases were considered to have died as a result of SIDS. The mean heights of the hard palate were significantly higher in the deceased children [4.1 (± 0.7) millimeters (mm)] than in the living children [3.2 (± 0.6) mm], with OR (+1SD) = 4.30 (95% confidence interval [CI], 2.04-9.06, P = 0.0001). The mean widths of the hard palate were 21.0 (± 1.9) mm and 23.2 (± 2.1) mm, respectively, with OR = 0.15 (95% CI, 0.06-0.40, P = 0.0001). The mean sagittal angles were significantly more acute in deceased children [134.5° (± 9.3)] than in living children [142.9° (± 8.1)], with OR = 0.28 (95% CI, 0.14-0.56, P = 0.0003). The mean height/width ratios were 19.8 (± 3.7) and 14.1 (± 3.3), respectively, with OR = 6.10 (95% CI, 2.50-14.9, P = 0.0001). The hard palate was subjectively considered as ogival in 59.4% (19/32) of the cases versus 12.5% (8/64) of the controls. Conclusion: Radiological features of the ogival palate were strongly associated with SUDI. This observation still needs to be confirmed and the corresponding clinical features must be identified.

Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Chicory: Understanding the Effects and Effectors of This Functional Food.

Industrial chicory has been the subject of numerous studies, most of which provide clinical observations on its health effects. Whether it is the roasted root, the flour obtained from the roots or the different classes of molecules that enter into the composition of this plant, understanding the molecular mechanisms of action on the human organism remains incomplete. In this study, we were interested in three molecules or classes of molecules present in chicory root: fructose, chlorogenic acids, and sesquiterpene lactones. We conducted experiments on the murine model and performed a nutrigenomic analysis, a metabolic hormone assay and a gut microbiota analysis, associated with in vitro observations for different responses. We have highlighted a large number of effects of all these classes of molecules that suggest a pro-apoptotic activity, an anti-inflammatory, antimicrobial, antioxidant, hypolipidemic and hypoglycemic effect and also an important role in appetite regulation. A significant prebiotic activity was also identified. Fructose seems to be the most involved in these activities, contributing to approximately 83% of recorded responses, but the other classes of tested molecules have shown a specific role for these different effects, with an estimated contribution of 23-24%.

Is bridging vein rupture/thrombosis associated with subdural hematoma at birth?

BACKGROUND: The combination of bridging vein rupture/thrombosis and subdural hematoma in infants has recently gained attention as highly suggestive of abusive head trauma. While subdural hematomas are frequently observed at birth, there are no previous studies of bridging vein rupture/thrombosis prevalence in that context. OBJECTIVE: To evaluate the prevalence of bridging vein rupture/thrombosis in newborns with and without subdural hematoma. MATERIALS AND METHODS: This bicentric retrospective study (2012-2019) looked at all brain MRIs performed in neonates. We noted delivery method, demographic data and intracranial injuries and analyzed any clots at the vertex as potential markers of bridging vein rupture/thrombosis. RESULTS: We analyzed 412 MRIs in 412 neonates. Age was (mean ± standard deviation [SD]) 5.4±2.2 days and 312 (76%) infants were full term (38.3±2.9 weeks from last menstrual period). The delivery method was vaginal birth for 42% (n=174), cesarean section for 43% (n=179), and unknown for 14% (n=59). Subdural hematoma was present in 281 MRIs (68.0%, [95% confidence interval = 63.3-72.5]). Six MRIs showed at least one clot at the vertex, assumed to be possible bridging vein rupture/thrombosis (1.5%, [0.5-3.1%]). Only one MRI showed more than two clots at the vertex, in a context of maternal infection. There was no significant difference in terms of gestational age at birth, delivery method or the presence of subdural hematoma or parenchymal injuries between those 6 infants and the 406 others. CONCLUSION: Bridging vein rupture/thrombosis at birth is very rare and unlikely to be related to subdural hematoma.

Fulminant Myocardial Involvement in Neonatal Echovirus-induced Sepsis. Two Autopsy Cases.

Group-B Enteroviruses, such as Echoviruses, are a common cause of infections in neonates but fatal myocarditis during Echovirus-induced sepsis have been rarely reported. We report on 2 cases of neonatal Echovirus-related sepsis with myocarditis. Fatal cardiorespiratory failure occurred in both cases. Autopsies and thorough histologic and microbiologic investigations evidenced Echoviruses 5- and 11-induced myocarditis as the cause of death.

CC17 group B Streptococcus exploits integrins for neonatal meningitis development.

Group B Streptococcus (GBS) is the major cause of human neonatal infections. A single clone, designated CC17-GBS, accounts for more than 80% of meningitis cases, the most severe form of the infection. However, the events allowing blood-borne GBS to penetrate the brain remain largely elusive. In this study, we identified the host transmembrane receptors α5ß1 and αvß3 integrins as the ligands of Srr2, a major CC17-GBS-specific adhesin. Two motifs located in the binding region of Srr2 were responsible for the interaction between CC17-GBS and these integrins. We demonstrated in a blood-brain-barrier cellular model that both integrins contributed to the adhesion and internalization of CC17-GBS. Strikingly, both integrins were overexpressed during the postnatal period in the brain vessels of the blood-brain barrier and blood-cerebrospinal fluid barrier and contributed to juvenile susceptibility to CC17 meningitis. Finally, blocking these integrins decreased the ability of CC17-GBS to cross into the CNS of juvenile mice in an in vivo model of meningitis. Our study demonstrated that CC17-GBS exploits integrins in order to cross the brain vessels, leading to meningitis. Importantly, it provides host molecular insights into neonate's susceptibility to CC17-GBS meningitis, thereby opening new perspectives for therapeutic and prevention strategies of GBS-elicited meningitis.

Complement activation is a crucial driver of acute kidney injury in rhabdomyolysis.

Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury is lacking and the mechanisms of the injury are unclear. To clarify this, we studied intra-kidney complement activation (C3d and C5b-9 deposits) in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. The lectin complement pathway was found to be activated in the kidney, likely via an abnormal pattern of Fut2-dependent cell fucosylation, recognized by the pattern recognition molecule collectin-11 and this proceeded in a C4-independent, bypass manner. Concomitantly, myoglobin-derived heme activated the alternative pathway. Complement deposition and acute kidney injury were attenuated by pre-treatment with the heme scavenger hemopexin. This indicates that complement was activated in a unique double-trigger mechanism, via the alternative and lectin pathways. The direct pathological role of complement was demonstrated by the preservation of kidney function in C3 knockout mice after the induction of rhabdomyolysis. The transcriptomic signature for rhabdomyolysis-induced acute kidney injury included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury. Hence, heme scavenging and complement inhibition represent promising therapeutic strategies.

Clinical impact of post-mortem genetic testing in cardiac death and cardiomyopathy.

Post-mortem genetic analyses may help to elucidate the cause of cardiac death. The added value is however unclear when a cardiac disease is already suspected or affirmed. Our aim was to study the feasibility and medical impact of post-mortem genetic analyses in suspected cardiomyopathy. We studied 35 patients with cardiac death and suspected cardiomyopathy based on autopsy or clinical data. After targeted sequencing, we identified 15 causal variants in 15 patients (yield 43%) in sarcomeric (n = 8), desmosomal (n = 3), lamin A/C (n = 3) and transthyretin (n = 1) genes. The results had various impacts on families, i.e. allowed predictive genetic testing in relatives (15 families), planned early therapeutics based on the specific underlying gene (5 families), rectified the suspected cardiomyopathy subtype (2 families), assessed the genetic origin of cardiomyopathy that usually has an acquired cause (1 family), assessed the diagnosis in a patient with uncertain borderline cardiomyopathy (1 family), reassured the siblings because of a de novo mutation (2 families) and allowed prenatal testing (1 family). Our findings suggest that post-mortem molecular testing should be included in the strategy of family care after cardiac death and suspected cardiomyopathy, since genetic findings provide additional information useful for relatives, which are beyond conventional autopsy.

MeJA Elicitation of Chicory Hairy Roots Promotes Efficient Increase of 3,5-diCQA Accumulation, a Potent Antioxidant and Antibacterial Molecule.

Cichorium intybus L. (Asteraceae) is an important industrial crop, as well as a medicinal plant which produces some bioactive compounds implicated in various biological effects with potential applications in human health. Particularly, roots produce hydroxycinnamic acids like 5-caffeoyquinic acid and 3,5-dicaffeoylquinic acid (di-CQA). The present investigation relates to the use of methyl jasmonate for enhancing phenolic compounds accumulation and production in hairy root cultures of C. intybus. Elicitated hairy root growth rate increased 13.3 times compared with the initial inoculum in a period of 14 days and di-CQA production represented about 12% of DW. The elicitation has also promoted the production of tricaffeoylquinic acid never described in the chicory roots and identified as 3,4,5-tricaffeoyquinic acid by means of nuclear magnetic resonance. Our study confirmed the strong anti-oxidant effect of di-CQA. Our results also confirmed globally a selectivity of action of di-CQA against Gram-positive bacteria, in particular against some strains of Staphylococcus and Streptococcus. However, a non-negligible antibacterial activity of di-CQA against Pseudomonas aeruginosa was also underlined (MIC = 0.156 mg.mL-1 against some P. aeruginosa strains). The influence of di-CQA has been explored to evaluate its impact on the physiology of P. aeruginosa. Di-CQA showed no effect on the biofilm formation and the production of extracellular pyocyanin. However, it demonstrated an effect on virulence through the production of pyoverdine with a dose-dependent manner by more than 7-fold when treated at a concentration of 128 µg·mL-1, thus suggesting a link between di-CQA and iron sequestration. This study shows that elicitated hairy root cultures of chicory can be developed for the production of di-CQA, a secondary metabolite with high antibacterial potential.

FLNC pathogenic variants in patients with cardiomyopathies: Prevalence and genotype-phenotype correlations.

Pathogenic variants in FLNC encoding filamin C have been firstly reported to cause myopathies, and were recently linked to isolated cardiac phenotypes. Our aim was to estimate the prevalence of FLNC pathogenic variants in subtypes of cardiomyopathies and to study the relations between phenotype and genotype. DNAs from a cohort of 1150 unrelated index-patients with isolated cardiomyopathy (700 hypertrophic, 300 dilated, 50 restrictive cardiomyopathies, and 100 left ventricle non-compactions) have been sequenced on a custom panel of 51 cardiomyopathy disease-causing genes. An FLNC pathogenic variant was identified in 28 patients corresponding to a prevalence ranging from 1% to 8% depending on the cardiomyopathy subtype. Truncating variants were always identified in patients with dilated cardiomyopathy, while missense or in-frame indel variants were found in other phenotypes. A personal or family history of sudden cardiac death (SCD) was significantly higher in patients with truncating variants than in patients carrying missense variants (P = .01). This work reported the first observation of a left ventricular non-compaction associated with a unique probably causal variant in FLNC which highlights the role of FLNC in cardiomyopathies. A correlation between the nature of the variant and the cardiomyopathy subtype was observed as well as with SCD risk.

Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

A BAHD neofunctionalization promotes tetrahydroxycinnamoyl spermine accumulation in the pollen coat of the Asteraceae family.

In eudicotyledons, accumulation of trihydroxycinnamoyl spermidine that is restricted to the pollen wall constitutes an evolutionary conserved trait. However, the role of this compound, which is synthetized by the BAHD enzyme spermidine hydroxycinnamoyl transferase (SHT), is still a matter of debate. Here, we show that this particular phenolamide is replaced by tetrahydroxycinnamoyl spermine in the pollen coat of the Asteraceae. Phylogenetic analyses combined with quantitative RT-PCR experiments allowed the identification of two homologous genes from Cichorium intybus (chicory) putatively involved in its metabolism. In vitro biochemical characterization of the two enzymes, named CiSHT1 and CiSHT2, confirmed the capability of recombinant proteins to synthesize spermine as well as spermidine derivatives. The wild-type metabolic phenotype was partially restored in an Arabidopsis sht mutant expressing CiSHT2. Strikingly, the transgenic plants also accumulated spermine derivatives that were absent in the wild-type. Overexpression of CiSHT2 in chicory hairy roots led to the accumulation of spermine derivatives, confirming its in vivo function. Complementary sequence analyses revealed the presence of an amino acid motif typical of the SHTs among the BAHD enzyme family. Our results highlight a recent neofunctionalization among the SHTs that has promoted the emergence of new phenolamides in the Asteraceae, which could potentially have contributed to the evolutionary success of this family.