Identification of hidden associations among eukaryotic genes through statistical analysis of coevolutionary transitions.

Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of (S)-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes.

A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.

Cysteine Enrichment Mediates Co-Option of Uricase in Reptilian Skin and Transition to Uricotelism.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with ß-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

Functional characterization and transcriptional repression by Lacticaseibacillus paracasei DinJ-YafQ.

DinJ-YafQ is a bacterial type II TA system formed by the toxin RNase YafQ and the antitoxin protein DinJ. The activity of YafQ and DinJ has been rigorously studied in Escherichia coli, but little has been reported about orthologous systems identified in different microorganisms. In this work, we report an in vitro and in vivo functional characterization of YafQ and DinJ identified in two different strains of Lacticaseibacillus paracasei and isolated as recombinant proteins. While DinJ is identical in both strains, the two YafQ orthologs differ only for the D72G substitution in the catalytic site. Both YafQ orthologs digest ribosomal RNA, albeit with different catalytic efficiencies, and their RNase activity is neutralized by DinJ. We further show that DinJ alone or in complex with YafQ can bind cooperatively to a 28-nt inverted repeat overlapping the -35 element of the TA operon promoter. Atomic force microscopy imaging of DinJ-YafQ in complex with DNA harboring the cognate site reveals the formation of different oligomeric states that prevent the binding of RNA polymerase to the promoter. A single amino acid substitution (R13A) within the RHH DNA-binding motif of DinJ is sufficient to abolish DinJ and DinJ-YafQ DNA binding in vitro. In vivo experiments confirm the negative regulation of the TA promoter by DinJ and DinJ-YafQ and unveil an unexpected high expression-related toxicity of the gfp reporter gene. A model for the binding of two YafQ-(DinJ)2-YafQ tetramers to the promoter inverted repeat showing the absence of protein-protein steric clash is also presented. KEY POINTS: • The RNase activity of L. paracasei YafQ toxin is neutralized by DinJ antitoxin. • DinJ and DinJ-YafQ bind to an inverted repeat to repress their own promoter. • The R13A mutation of DinJ abolishes DNA binding of both DinJ and DinJ-YafQ.

A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249 -encoded transmembrane domain containing protein, CATSPERθ, is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper transmembrane subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might acts as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.

Origin and significance of the human DNase repertoire.

The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics and transcriptomics data to infer the evolutionary history of DNases and investigate their biological significance. Both DNase1 and DNase2 families have expanded in vertebrates since ~ 650 million years ago before the divergence of jawless and jawed vertebrates. DNase1, DNase1L1, and DNase1L3 co-existed in jawless fish, whereas DNase1L2 originated in amniotes by tandem duplication of DNase1. Among the non-human DNases, DNase1L4 and newly identified DNase1L5 derived from early duplications that were lost in terrestrial vertebrates. The ancestral gene of the DNase2 family, DNase2b, has been conserved in synteny with the Uox gene across 700 million years of animal evolution,while DNase2 originated in jawless fish. DNase1L1 acquired a GPI-anchor for plasma membrane attachment in bony fishes, and DNase1L3 acquired a C-terminal basic peptide for the degradation of microparticle DNA in jawed vertebrates. The appearance of DNase1L2, with a distinct low pH optimum and skin localization, is among the amniote adaptations to life on land. The expansion of the DNase repertoire in vertebrates meets the diversified demand for DNA debris removal in complex multicellular organisms.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

Birth of a pathway for sulfur metabolism in early amniote evolution.

Among amniotes, reptiles and mammals are differently adapted to terrestrial life. It is generally appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo, but rather repurposing of existing ones. Here, by comparing the inventory of pyridoxal 5'-phosphate-dependent enzymes in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase contains haem and pyridoxal 5'-phosphate co-factors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide, respectively. A specific cysteic acid decarboxylase produces taurine, while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase. This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by cystathionine beta-synthase duplication, cysteine lyase neofunctionalization and cysteic acid decarboxylase co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways, and reveal the molecular mechanisms by which such innovations arose in amniote evolution.

Application of The Sepsis-3 Consensus Criteria in a Geriatric Acute Care Unit: A Prospective Study.

The prognostic value of quick Sepsis-related Organ Failure Assessment (qSOFA) score in geriatric patients is uncertain. We aimed to compare qSOFA vs. Systemic Inflammatory Response Syndrome (SIRS) criteria for mortality prediction in older multimorbid subjects, admitted for suspected sepsis in a geriatric ward. We prospectively enrolled 272 patients (aged 83.7 ± 7.4). At admission, qSOFA and SIRS scores were calculated. Mortality was assessed during hospital stay and three months after discharge. The predictive capacity of qSOFA and SIRS was assessed by calculating the Area Under the Receiver Operating Characteristic Curve (AUROC), through pairwise AUROC comparison, and multivariable logistic regression analysis. Both qSOFA and SIRS exhibited a poor prognostic performance (AUROCs 0.676, 95% CI 0.609⁻0.738, and 0.626, 95% CI 0.558⁻0.691 for in-hospital mortality; 0.684, 95% CI 0.614⁻0.748, and 0.596, 95% CI 0.558⁻0.691 for pooled three-month mortality, respectively). The predictive capacity of qSOFA showed no difference to that of SIRS for in-hospital mortality (difference between AUROCs 0.05, 95% CI -0.05 to 0.14, p = 0.31), but was superior for pooled three-month mortality (difference between AUROCs 0.09, 95% CI 0.01⁻0.17, p = 0.029). Multivariable logistic regression analysis, accounting for possible confounders, including frailty, showed that both scores were not associated with in-hospital mortality, although qSOFA, unlike SIRS, was associated with pooled three-month mortality. In conclusion, neither qSOFA nor SIRS at admission were strong predictors of mortality in a geriatric acute-care setting. Traditional geriatric measures of frailty may be more useful for predicting adverse outcomes in this setting.

Safety evaluations and lipid-lowering activity of an Arthrospira platensis enriched diet: A 1-month study in rats.

Arthrospira platensis (A. platensis) is worldwide consumed as dietary supplement, but its use in the form of whole biomass for food purposes may raise toxicity concerns. The aim of this study was to preliminarily evaluate the safety of an A. platensis F&M-C256-enriched diet (20% (weight/weight) corresponding to 12g/kg body weight/day), administered to rats for 1month. A. platensis F&M-C256-enriched diet was well tolerated: behavior, body weight, food consumption and growth curves were not affected; no discomfort, no deaths and no physical signs related to the treatment were observed during the administration period; food daily consumption and apparent digestibility were comparable to those of the standard laboratory AIN-76 control diet. Daily water consumption and urine excretion were, on the contrary, significantly higher (27.18±1.24 vs 21.53±1.68ml and 12.63±0.99 vs 7.00±1.29ml respectively), probably because of a slight increase in sodium intake in rats fed A. platensis F&M-C256-enriched diet. Biochemical markers of kidney and liver function were not varied but a significant increase in cholesterol-HDL and a decreased plasma triglycerides level was observed in rats fed A. platensis F&M-C256-enriched diet. These last changes were associated with an increased fecal lipids excretion and liver PPAR-α gene expression. These results indicate that A. platensis F&M-C256 is likely safe and well tolerated even at a high dosage in rodents and suggest that it may represent a promising functional food for preventing or even for managing dyslipidemias.

[Recent advances in urate metabolism]. / Nuovi avanzamenti nelle conoscenze del metabolismo dell'urato.

In the last fifteen years, genomics and other -omics sciences have revolutionized our understanding of biological processes at the molecular level. An illustrative example is urate metabolism. Before the publication of the complete human genome, in 2003 it was believed that a single enzyme (urate oxidase) was responsible for uricolysis that is the conversion of urate into the more soluble allantoin. Now we know with great detail that this process requires the consecutive action of three enzymes that have been lost by gene inactivation in our hominoid ancestor. Similarly, a single urate transporter (URAT1) was known at that time. Now we have evidence that urate homeostasis depends on a complex set of transporters located on the epithelial cells of the kidney and the intestine. In this review article, we give an account of the recent discoveries on urate metabolism and how these discoveries can be applied to the development of novel drugs to treat hyperuricemia, tumor lysis syndrome and the Lesch-Nyhan disease.

Heme binding and peroxidase activity of a secreted minicatalase.

Microbial pathogens often require efficient and robust H2 O2 scavenger activities to survive in the presence of reactive oxygen species generated by inflammatory responses. In addition to catalases and peroxidases, enzymes known to scavenge H2 O2 , a novel class of secreted minicatalases is found in diderm bacteria. Here, we characterize the Helicobacter pylori (Hp) minicatalase: a monomeric hemoprotein with catalase core homology. Overexpression of Hp minicatalase rescued a catalase/peroxidase-deficient Escherichia coli phenotype under aerobic conditions and limited H2 O2 stress. The purified enzyme lacks catalase activity, but has strong (kcat > 100 s-1 ) H2 O2 -dependent peroxidase activity toward a variety of organic substrates. Our investigations into heme binding revealed that the heme cofactor is assembled in the periplasm to form the functional holoprotein. Furthermore, we observed the presence of a disulfide bond near the heme cavity of Hp minicatalase, which is conserved in secreted minicatalases and, therefore, may play a role in heme binding.

Lung ultrasound and chest x-ray for detecting pneumonia in an acute geriatric ward.

BACKGROUND: Our aim was to compare the accuracy of lung ultrasound (LUS) and standard chest x-ray (CXR) for diagnosing pneumonia in older patients with acute respiratory symptoms (dyspnea, cough, hemoptysis, and atypical chest pain) admitted to an acute-care geriatric ward. METHODS: We enrolled 169 (80 M, 89 F) multimorbid patients aged 83.0 ±â€Š9.2 years from January 1 to October 31, 2015. Each participant underwent CXR and bedside LUS within 6 hours from ward admission. LUS was performed by skilled clinicians, blinded to CXR results and clinical history. The final diagnosis (pneumonia vs no-pneumonia) was established by another clinician reviewing clinical and laboratory data independent of LUS results and possibly prescribing chest contrast-enhanced CT. Diagnostic parameters of CXR and LUS were compared with McNemar test on the whole cohort and after stratification for Rockwood Clinical Frailty Scale. RESULTS: Diagnostic accuracy for pneumonia (96 patients) was significantly higher in LUS (0.90, 95% confidence interval [CI] 0.83-0.96) compared with CXR (0.67, 95%CI 0.60-0.74, P < 0.001). LUS had a better sensitivity (0.92, 95%CI 0.86-0.97 vs 0.47, 95%CI 0.37-0.57) and negative predictive value (0.95, 95% CI 0.83-0.96 vs 0.57, 95%CI 0.48-0.56). In those patients with frailty (n = 87 with Rockwood Clinical Frailty Scale ≥5), LUS maintained a high diagnostic accuracy, but CXR did not (P = 0.0003). Interobserver agreement for LUS, calculated in a subsample of 29 patients, was high (k = 0.90). CONCLUSIONS: In multimorbid patients admitted to an acute geriatric ward, LUS was more accurate than CXR for the diagnosis of pneumonia, particularly in those with frailty. A wider use of LUS should be implemented in this setting.

Evolution of the Selenoproteome in Helicobacter pylori and Epsilonproteobacteria.

By competing for the acquisition of essential nutrients, Helicobacter pylori has the unique ability to persist in the human stomach, also causing nutritional insufficiencies in the host. Although the H. pylori genome apparently encodes selenocysteine synthase (SelA, HP1513), a key pyridoxal phosphate (PLP)-dependent enzyme for the incorporation of selenium into bacterial proteins, nothing is known about the use of this essential element in protein synthesis by this pathogen. We analyzed the evolution of the complete machinery for incorporation of selenium into proteins and the selenoproteome of several H. pylori strains and related Epsilonproteobacteria. Our searches identified the presence of selenoproteins-including the previously unknown DUF466 family-in various Epsilonproteobacteria, but not in H. pylori. We found that a complete system for selenocysteine incorporation was present in the Helicobacteriaceae ancestor and has been recently lost before the split of Helicobacter acinonychis and H. pylori. Our results indicate that H. pylori, at variance with other gastric and enterohepatic Helicobacter, does not use selenocysteine in protein synthesis and does not use selenium for tRNA wobble base modification. However, selA has survived as a functional gene, having lost the domain for the binding of selenocysteine tRNA, but maintaining the ability to bind the PLP cofactor. The evolutionary modifications described for the SelA protein of H. pylori find parallels in other bacterial and archaeal species, suggesting that an alternative enzymatic function is hidden in many proteins annotated as selenocysteinyl-tRNA synthase.