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PubMed1976

Do evolutionary changes in cytochrome c structure reflect functional adaptations?

Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.

Amino Acid Sequence↗

PubMedDate not supplied

[Dominant heredity in tardive congenital glaucoma with high insertion of the iris].

Seven cases of tardive congenital glaucoma determined by a high insertion of the iris are analyzed. Biomicroscopy revealed in all cases irian stromal atrophy and the complete or incomplete, total or partial insertion of the iris at the level of trabeculum was gonioscopically observed. With respect to the reducing of trabecular space exposed to aqueous humour, tension increase appeared between 11 and 20 years old at three patients, between 21 and 30 years old at three patients and at 34 years old at one patient. The authors consider the age of 35 to be an age limit in tardive congenital glaucoma. The transmission was dominant with variable expressivity. The systematic examination of family members allowed the discovering of other cases of tardive congenital glaucoma, which were unknown or known as open-angle glaucoma cases. The associations between tardive congenital glaucoma and myopia delayed the setting of diagnosis which lead to a more serious evolution. In the therapy with myotics was not sufficient, protected filtering interventions and especially trepanotrabeculectomy were recommended.

Adolescent↗

PubMed2004

Can fat explain the human brain's big bang evolution?-Horrobin's leads for comparative and functional genomics.

When David Horrobin suggested that phospholipid and fatty acid metabolism played a major role in human evolution, his 'fat utilization hypothesis' unified intriguing work from paleoanthropology, evolutionary biology, genetic and nervous system research in a novel and coherent lipid-related context. Interestingly, unlike most other evolutionary concepts, the hypothesis allows specific predictions which can be empirically tested in the near future. This paper summarizes some of Horrobin's intriguing propositions and suggests as to how approaches of comparative genomics published in Cell, Nature, Science and elsewhere since 1997 may be used to examine his evolutionary hypothesis. Indeed, systematic investigations of the genomic clock in the species' mitochondrial DNA, the Y and autosomal chromosomes as evidence of evolutionary relationships and distinctions can help to scrutinize associated predictions for their validity, namely that key mutations which differentiate us from Neanderthals and from great apes are in the genes coding for proteins which regulate fat metabolism, and particularly the phospholipid metabolism of the synapses of the brain. It is concluded that beyond clues to humans' relationships with living primates and to the Neanderthals' cognitive performance and their disappearance, the suggested molecular clock analyses may provide crucial insights into the biochemical evolution-and means of possible manipulation-of our brain.

Animals↗

PubMed2001

Ongoing evolution of strand composition in bacterial genomes.

We tried to identify the substitutions involved in the establishment of replication strand bias, which has been recognized as an important evolutionary factor in the evolution of bacterial genomes. First, we analyzed the composition asymmetry of 28 complete bacterial genomes and used it to test the possibility that asymmetric deamination of cytosine might be at the origin of the bias. The model showed significant correlation to the data but left unexplained a significant portion of the variance and indicated a systematic underestimation of GC skews in comparison with TA skews. Second, we analyzed the substitutions acting on the genes from five fully sequenced Chlamydia genomes that had not suffered strand switch since speciation. This analysis showed that substitutions were not at equilibrium in Chlamydia trachomatis or in C. muridarum and that strand bias is still an on-going process in these genes. Third, we identified substitutions involved in the adaptation of genes that had switched strands after speciation. These genes adapted quickly to the skewed composition of the new strand, mostly due to C-->T, A-->G, and C-->G asymmetric substitutions. This observation was reinforced by the analysis of genes that switched strands after divergence between Bacillus subtilis and B. halodurans. Finally, we propose a more extended model based on the analysis of the substitution asymmetries of CHLAMYDIA: This model fits well with the data provided by bacterial genomes presenting strong strand bias.

Bacteria↗

PubMed2005

The epidemiology of childhood recurrent abdominal pain in Western countries: a systematic review.

OBJECTIVE: Recurrent abdominal pain (RAP) of childhood is a common problem encountered by clinicians. The aim of this study was to systematically review published literature about the prevalence, incidence, natural history, and co-morbid conditions of childhood RAP in western countries. METHODS: A computer-assisted search of MEDLINE, EMBASE, and Current Contents/Science Edition databases was performed. Study selection criteria included: (1) United States and European population and school-based samples of children; (2) diagnostic criteria of RAP; and (3) published in full manuscript form in English. Data were extracted, tabulated, and presented in descriptive form. RESULT: The prevalence of RAP ranged from 0.3 to 19% (median 8.4; IQR 2.3-14.7). Published studies in children demonstrate a higher prevalence of RAP in females, with the highest prevalence of symptoms between 4 and 6 yr and early adolescence. Identified studies demonstrated associations between RAP and the child's familial and socioeconomic environment. In addition, childhood RAP was reported to be associated with psychological co-morbidity in childhood and adulthood. Population/school-based studies have not, however, established the incidence of this disorder, what features predict long-standing symptoms, or whether RAP is a risk factor for adult functional bowel disorders. CONCLUSION: RAP is a common complaint of childhood with associated familial, psychological, and co-morbid conditions. Epidemiologic studies of RAP in children may offer information on the evolution of functional bowel disorders through the lifespan.

Abdominal Pain↗

PubMed2006

A generalized discrete model linking rippling pattern formation and individual cell reversal statistics in colonies of myxobacteria.

Self-organization processes in multicellular aggregates of bacteria and amoebae offer fascinating insights into the evolution of cooperation and differentiation of cells. During myxobacterial development a variety of spatio-temporal patterns emerges such as counterpropagating waves of cell density that are known as rippling. Recently, several models have been introduced that qualitatively reproduce these patterns. All models include active motion and a collision-triggered reversal of individual bacteria. Here, we present a systematic study of a generalized discrete model that is based on similar assumptions as the continuous model by Igoshin et al (2001 Proc. Natl Acad. Sci. USA 98 14913). We find counterpropagating as well as unidirectional rippling waves in extended regions of the parameter space. If the interaction strength and the degree of cooperativity are large enough, rippling patterns appear even in the absence of a refractory period. We show for the first time that the experimentally observed double peak in the reversal statistics of bacteria in rippling colonies (Welch and Kaiser 2001 Proc. Natl Acad. Sci. USA 98 14907) can be reproduced in simulations of counterpropagating rippling waves which are dominant in experiments. In addition, the reversal statistics in the pre-rippling phase is correctly reproduced.

Biological Evolution↗

PubMed2001

Contrasting evolution of the human leukocyte N-formylpeptide receptor subtypes FPR and FPRL1R.

N-formylpeptides are phagocyte chemoattractants that act by binding to two structurally related receptors, FPR (formylpeptide receptor) and FPRL1R (FPR-like-1 receptor), which are encoded by the human genes FPR1 and FPRL1. Single nucleotide polymorphisms (SNPs) in the FPR coding region have been reported and two have been associated with the disease juvenile periodontitis; however, their frequency and linkage relationships are unknown. Here we systematically analyzed polymorphism in the open reading frames of FPR1 and FPRL1 by direct sequencing of cloned alleles from random blood donors from North America. For FPR1 we detected five non-synonymous SNPs and two synonymous SNPs in a sample of 26 chromosomes one each from 17 Caucasian and nine black random blood donors. Although all five non-synonymous SNPs were common in Caucasians, Blacks, and Asians, notable differences in allele frequency were found for each SNP in the different racial groups, suggesting differential selective pressures. We found that the FPR1 polymorphisms are linked in 15 common haplotypes. No polymorphisms were detected in FPRL1 after sampling 44 chromosomes from 36 random blood donors from the same three racial groups. Thus FPR1 and FPRL1, though they originated from a common gene, appear to have undergone markedly different evolutionary events.

Amino Acid Sequence↗

PubMed2026

Predicting food taste with bound-driven optimization.

The prediction of sensory attributes from ingredient-level formulations is an emerging challenge at the intersection of food science and artificial intelligence. We address the fundamental question of whether the taste of a food can be predicted from its ingredients by treating recipes as composite materials. We apply Hashin-Shtrikman (HS) and Reuss-Voigt (RV) bounds, techniques originally developed for elastic moduli, as a null-hypothesis additive baseline for five taste dimensions (sweetness, sourness, bitterness, umami, saltiness) on a curated dataset of 70 recipes decomposed into 115 distinct ingredients scored against a library of 209 ingredient-level taste references with trained-panel ground truth. This baseline systematically under-predicts perceived taste: 77% of actual taste values exceeded the HS upper bound, with the exceedance rate ranging from 26% (bitterness) to 97% (saltiness). We traced this gap to specific processing chemistry (Maillard reactions, caramelization, evaporative concentration, protein hydrolysis, and nucleotide synergy) and introduced a hybrid model that augments the HS baseline with eight chemistry-proxy features encoding these mechanisms. Our results show that our interpretable hybrid model eliminates the systematic bias and reduces mean absolute error by 27%-62% for sweetness, sourness, umami, and saltiness while using only 10 interpretable features, achieving performance comparable to a black-box Lasso regression on 115 per-ingredient features. We further demonstrate constrained inverse design via Differential Evolution, recovering ingredient formulations that match target taste profiles subject to compositional bounds. Our work demonstrates how key chemical processes during food preparation can inform and augment physics-based and machine learning models, providing a quantitative fingerprint of processing chemistry's contribution to taste perception and paving the way for model-driven food formulation with targeted sensory characteristics.

Composite material bounds↗

PubMed2002

[Impact of medical counselling on giving up smoking during pregnancy].

OBJECTIVE: Women s tobacco consumption has increased in Spain in recent years, especially among women of reproductive age. This study aims to evaluate the impact of medical counselling integrated into pre-natal care on tobacco consumption during pregnancy and the period after delivery. DESIGN: Quasi-experimental intervention study.Setting. Hospital del Mar, Barcelona. PATIENTS: 219 patients who attended the Hospital del Mar for delivery during 1996 (control group) and 169 patients seen during their pregnancies at the same hospital in 1997 (intervention group). INTERVENTIONS: The control group patients had received normal care. The pregnant women in the intervention group received systematic structured counselling on giving up smoking, backed up by a special brochure composed for this purpose. MEASUREMENTS AND RESULTS: The intervention and control groups showed no statistically significant differences either in their social or demographic variables or in their tobacco consumption. In both groups the evolution of their smoking during pregnancy was determined during their pre-natal visits and six months after delivery through a telephone interview. 44 of the women in the control group (20.1%) gave up smoking before their first pre-natal visit, and 11 (5%) gave up during pregnancy. In the intervention group 26 (17.7%) had given up spontaneously and 16 (10.9%) gave up during pregnancy. Of those who gave up completely during pregnancy, 36.4% of women in the control group and 64.3% in the intervention group remained abstinent at six months (P=.002). CONCLUSIONS: Counselling at pre-natal check-ups to give up smoking lightly increases the number of women who give up and reduces significantly the number of post-delivery backsliders.

Adult↗

PubMedDate not supplied

[Depression].

The Portuguese Medical Association, through its Colleges of Specialistes, has decided to establish working groups to establish Therapeutic Guidelines in pathologies of great interest and current relevance in order to improve and rationalize health care. The group in charge of establishing the Therapeutic Recommendations in Depression, whose work I had the responsability and pleasure of coordinating during part of 1998 and 1999, is comprised of colleagues selected by the Portuguese Medical Association (Ordem dos Médicos). In the last few years, there has been rapidly increasing clinical and scientific interest in the study and treatment of depression throughout the life cycle, in different contexts and levels of medical practice. The rapid progress made in the neurosciences and psychopharmacology, new research in the field of psychotherapies and improved knowledge of relevant psychosocial aspects has not only broadened our knowledge of the aetiology and pathogenesis of depression, but also improved our criteria of diagnosis and classification. This has allowed the development of new therapeutic approaches and new drugs of proven efficacy. These Recommendations are aimed at systematizing and disseminating a consensus on interventions in depression, supported by evidence and the most recent scientific developments, in a way that will optimise therapeutic treatment. Due to some delay in the publication of these Recommendations (through no fault of the working group), and the rapid evolution of knowledge in this area, some of the contents may shortly need to be revised and updated. The writers and publishers of this document are fully aware of this and in accordance.

Clinical Protocols↗

PubMed1984

Xiphophorus as an in vivo model for studies on oncogenes.

The capacity of Xiphophorus to develop neoplasia can be formally assigned to a "tumor gene" (Tu), which appears to be a normal part of the genome of all individuals. The wild fish have evolved population-specific and cell type-specific systems of regulatory genes (R) for Tu that protect the fish from neoplasia. Hybridization of members of different wild populations in the laboratory followed by treatment of the hybrids with carcinogens led to disintegration of the R systems permitting excessive expression of Tu and thus resulting in neoplasia. Certain hybrids developed neoplasia even spontaneously. Observations on the genuine phenotypic effect of the derepressed Tu in the early embryo indicated an essential normal function of this oncogene in cell differentiation, proliferation and cell-cell communication. Tu appeared to be indispensable in the genome but may also be present in accessory copies. Recently, c-src, the cellular homolog of the Rous sarcoma virus oncogene v-src, was detected in Xiphophorus. The protein product of c-src, pp60c-src, was identified and then examined by its associated kinase activity. This pp60c-src was found in all individuals tested, but, depending on the genotype, its kinase activity was different. The genetic characters of c-src, such as linkage relations, dosage relations, expression, etc., correspond to those of Tu. From a systematic study which showed that pp60c-src was present in all metazoa tested ranging from mammals down to sponges, we concluded that c-src has evolved with the multicellular organization of animals. Neoplasia of animals and humans is a characteristic closely related to this evolution. Our data showed that small aquarium fish, besides being used successfully because they are time-, space-, and money-saving systems for carcinogenicity testing, are also highly suitable for basic studies on neoplasia at the populational, morphological, developmental, cell biological, and molecular levels.

Animals↗

PubMed2005

First analysis of the proteome in two nematomorph species, Paragordius tricuspidatus (Chordodidae) and Spinochordodes tellinii (Spinochordodidae).

The proteome of most parasite species is currently unknown. Hairworms (Nematomorpha), 300 species distributed around the world, are parasitic in arthropods (mainly terrestrial species) when juveniles, but they are free-living in aquatic environments when adult. Most aspects of their systematics and biology are currently unknown. The aim of this paper was (i) to report a novel and reproducible protocol for the analysis of the proteome of hairworms using two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (matrix laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)) and (ii) to determine the level of proteomic divergence between two sympatric but taxonomically unrelated nematomorph species in the adult stage, Paragordius tricuspidatus Dufour (Nematomorpha, Gordiidae) and Spinochordodes tellinii Camerano (Nematomorpha, Gordiidae). In total, 689 protein spots were observed for P. tricuspidatus, 575 for S. tellinii. Only 36.2% spots were shared between the two species. Quantitative analysis of the proteins which are common to both parasite species reveals substantial differences in the pattern of protein expression. These results suggest a rapid evolutionary divergence between these two nematomorph families. Also, to test the value of our MALDI-TOF protocol, we used Actin-2 (Act-2), a protein highly conserved in the course of evolution. Peptide mass fingerprint (PMF) data obtained for Act-2 of P. tricuspidatus and S. tellinii suggest a very high homology with Act-2 of different worms species belonging to the Bilateria phylum (Annelida and Nematoda) and more specifically to Lumbricus terrestris (Annelida, Lumbricidae) and Caenorhabditis elegans (Nematoda, Rhabditidae). We discuss our results in relationship with current ideas concerning the use of proteomics in systematics.

Animals↗

PubMed2005

Structural and functional evolution of three cardiac natriuretic peptides.

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.

Amino Acid Sequence↗

PubMed1995

The low temperature organic chemistry of Titan's geofluid.

Organic chemistry on Titan and prebiotic chemistry on Earth involve the same N-containing organics: nitriles and their oligomers. Couplings of their chemistry in the three parts of Titan's geofluid (atmosphere, aerosols and surface) seem to play a key role in the organic chemical evolution of the planet. If liquid water was present on Titan, then a prebiotic chemistry, involving eutectics, similar to that of the early Earth, may have occurred. However, liquid water is currently absent and a prebiotic chemistry based only on N-organics may be evolving now on Titan. The other consequence of the low temperatures of Titan is the possible formation of organics unstable at room temperature and very reactive. So far, these compounds have not been systematically searched for in experimental studies of Titan's organic chemistry. C4N2 has already been detected on Titan. Powerful reactants in organic chemistry, CH2N2, and CH3N3, may be also present. They exhibit spectral signatures in the mid-IR strong enough to allow their detection at the 10-100 ppb level. They may be detectable on future IR spectra (ISO and Cassini) of Titan.

Aerosols↗

PubMed2005

An empirical assessment of long-branch attraction artefacts in deep eukaryotic phylogenomics.

In the context of exponential growing molecular databases, it becomes increasingly easy to assemble large multigene data sets for phylogenomic studies. The expected increase of resolution due to the reduction of the sampling (stochastic) error is becoming a reality. However, the impact of systematic biases will also become more apparent or even dominant. We have chosen to study the case of the long-branch attraction artefact (LBA) using real instead of simulated sequences. Two fast-evolving eukaryotic lineages, whose evolutionary positions are well established, microsporidia and the nucleomorph of cryptophytes, were chosen as model species. A large data set was assembled (44 species, 133 genes, and 24,294 amino acid positions) and the resulting rooted eukaryotic phylogeny (using a distant archaeal outgroup) is positively misled by an LBA artefact despite the use of a maximum likelihood-based tree reconstruction method with a complex model of sequence evolution. When the fastest evolving proteins from the fast lineages are progressively removed (up to 90%), the bootstrap support for the apparently artefactual basal placement decreases to virtually 0%, and conversely only the expected placement, among all the possible locations of the fast-evolving species, receives increasing support that eventually converges to 100%. The percentage of removal of the fastest evolving proteins constitutes a reliable estimate of the sensitivity of phylogenetic inference to LBA. This protocol confirms that both a rich species sampling (especially the presence of a species that is closely related to the fast-evolving lineage) and a probabilistic method with a complex model are important to overcome the LBA artefact. Finally, we observed that phylogenetic inference methods perform strikingly better with simulated as opposed to real data, and suggest that testing the reliability of phylogenetic inference methods with simulated data leads to overconfidence in their performance. Although phylogenomic studies can be affected by systematic biases, the possibility of discarding a large amount of data containing most of the nonphylogenetic signal allows recovering a phylogeny that is less affected by systematic biases, while maintaining a high statistical support.

Animals↗

PubMed1982

Ochotona princeps (pika) myoglobin: an appraisal of lagomorph phylogeny.

Myoglobin was purified from skeletal muscle of the pika (Ochotona princeps) and its primary structure was determined. This sequence was added to the set of 64 already known vertebrate myoglobin sequences and used to evaluate the phylogenetic position of the pika among other mammals, using a computerized search procedure based on maximum parsimony criteria. The pika is clearly related to the rabbit (Oryctolagus cuniculus), with which it is traditionally associated as a member of the order Lagomorpha. A monophyletic group composed of Lagomorpha, Scandentia, and Carnivora is a consistent feature of the dendrograms produced. An association of Carnivora and Lagomorpha casts doubt on the results of those investigations using rabbit antisera in systematic studies and depending on carnivores as an outside reference group for cladogram construction.

Amino Acid Sequence↗

PubMed2002

Evolutionary adaptations to pre-eclampsia/eclampsia in humans: low fecundability rate, loss of oestrus, prohibitions of incest and systematic polyandry.

Gestational-hypertension/pre-eclampsia occurs in approximately 10% of human pregnancies. This persistent complication of pregnancy has been reported to occur more frequently in couples conceiving very shortly after the beginning of their sexual relationship and/or after a change in paternity. Primipaternity may be the leading cause of pre-eclampsia in women under 30 years of age when genetic susceptibility to cardio-vascular disease has not yet been expressed, especially in women before their twenties, who for the last 40,000 years have perhaps comprised the age group when the majority of parturients classified as Homo sapiens sapiens initiated their reproductive life. In terms of evolution, the prevalence of pre-eclampsia represents a distinct reproductive disadvantage in humans as compared with other mammals. Indeed, pre-eclampsia is a consequence of the defect of the normal human-specific deep endovascular invasion of the trophoblast. The large size of the human fetal brain imposing this deep trophoblastic invasion induced the need for major immunogenetic compromises in terms of paternal-maternal tissue tolerance. The price that mankind has had to pay to adapt to the pre-eclampsia risk is a low fecundability rate and therefore loss of oestrus, possibly a step in the deviation between apes and hominids. Further, pre-eclampsia risk may be a contributing factor leading to the rejection of systematic polyandry in human societies and have influenced prohibition of incest.

Adaptation, Biological↗

PubMed1976

[Frequency of hepatitis after extra-corporeal circulation].

In cardio-vascular surgery the risk that the hepatitis-B (H.B.) virus be transmitted was measured in 3 groups of patients (Professor Ch. Dubost), before and since systematic screening for HBS antigen in donors' blood. The same questionnaire was sent to each patient in each group 6 months after extra-corporeal-circulation (ECC), in order to determine the efficiency of HB virus screening tests. The present study gives the following results: --in the first group (no screening for HBS antigen in donors' blood), the frequency of icteric hepatitis was 6.7% (26 cases of jaundice, probably due to hepatitis viruses, out of 386 ECC); --in the second group (screening for HBS antigen in donors' blood by immuno-diffusion (often made after transfusion of fresh blood) the frequency of icteric hepatitis after transfusion of HBS antigen positive blood); --in the third groups, icteric hepatitis was less frequent (4.6%) since systematic screening for HBS antigen by counter-electrophoresis and complement fixation was performed (29 cas out of 629 ECC). Additionally, viral hepatitis appeared more frequently in men than in women, and among older patients than younger ones. Recovery was generally complete in 2 months; however, the evolution of the disease was severe in 3 cases and fatal in one case. In sum, after ECC, hepatitis still remains a frequent side-effect, compelling blood centers to use th most sensitive screening tests now available for HBS screening: radio-immuno-assay and reverse passive haemagglutination tests. As a consequence of the present study, these 2 techniques are being applied on a daily basis in our laboratory for HBS antigen screening tests are ineffective in preventing the transmission of other strains of viral hepatitis by blood transfusion.

Age Factors↗