PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Antisaccades and memory-guided saccades in genetic generalized epilepsy and temporal lobe epilepsy.

OBJECTIVE: Oculomotor tasks can be used to measure volitional control of behavior sensitive to frontal dysfunction. This study aimed to examine the saccadic eye movement in Genetic Generalized Epilepsy (GGE) which could correlate with the abnormality of the frontal lobe or the thalamo-frontal network. METHODS: Twenty-one patients with GGE were compared with 22 patients with Temporal Lobe Epilepsy (TLE) and 39 healthy controls. Visual-guided saccades, Antisaccades, and Memory-guided saccades as oculomotor tasks were performed using a novel gaze-tracker designed for clinical practice use. RESULTS: Patients with epilepsy (either GEE or TLE) had similar latency, accuracy, and velocity in visual-guided saccades and memory-guided saccades. Patients with epilepsy had similar latencies and correct antisaccade number. However, healthy volunteers, matched by age, had faster responses and more accurate results than patients with epilepsy. CONCLUSIONS: Our investigations did not reveal differences between TLE and GGE patients' groups in visually guided saccades, antisaccades, and memory-guided saccades, thus suggesting that the frontal cortical mechanisms responsible for them are not explicitly impaired in patients with GGE.

Clinical, pathological and genetic spectrum in 89 cases of mitochondrial progressive external ophthalmoplegia.

BACKGROUND: Mitochondrial progressive external ophthalmoplegia (PEO) encompasses a broad spectrum of clinical and genetic disorders. We describe the phenotypic subtypes of PEO and its correlation with molecular defects and propose a diagnostic algorithm. METHODS: Retrospective analysis of the clinical, pathological and genetic features of 89 cases. RESULTS: Three main phenotypes were found: 'pure PEO' (42%), consisting of isolated palpebral ptosis with ophthalmoparesis; Kearns-Sayre syndrome (10%); and 'PEO plus', which associates extraocular symptoms, distinguishing the following subtypes: : myopathic (33%), bulbar (12%) and others (3%). Muscle biopsy was the most accurate test, showing mitochondrial changes in 95%. Genetic diagnosis was achieved in 96% of the patients. Single large-scale mitochondrial DNA (mtDNA) deletion was the most frequent finding (63%), followed by multiple mtDNA deletions (26%) due to mutations in TWNK (n=8), POLG (n=7), TK2 (n=6) or RRM2B (n=2) genes, and point mtDNA mutations (7%). Three new likely pathogenic mutations were identified in the TWNK and MT-TN genes. CONCLUSIONS: Phenotype-genotype correlations cannot be brought in mitochondrial PEO. Muscle biopsy should be the first step in the diagnostic flow of PEO when mitochondrial aetiology is suspected since it also enables the study of mtDNA rearrangements. If no mtDNA deletions are identified, whole mtDNA sequencing should be performed.

Hypophonia as a sign of thalamus lesion: a case report.

Hypophonia is a neurological sign usually seen after brainstem or peripheral damage, either at the recurrent laryngeal nerve or vocal cord level. However, it has been described as a sign of supratentorial strokes in a few studies, specifically when anterior and ventral thalamic involvement is reported. In addition, it is a prominent sign of other neurological disorders such as Parkinson disease and other extrapyramidal conditions. We describe a case of hypophonia secondary to a left thalamic hemorrhage, after a careful search of other potential causes of this deficit, and we discuss the underlying neuroanatomical circuits.

Prognosis of Tandem Occlusions versus Isolated Intracranial Occlusions.

BACKGROUND: Both acute single intracranial and tandem occlusions are managed with intravascular thrombectomy with success, but little evidence exists about the differences in their mid-term outcome. We aim to compare the outcome at 3 months after tandem (extracranial internal carotid and/or middle cerebral artery) and single intracranial (M1 division) occlusions, and to identify the factors, which determine such prognosis. METHODS: A total of 66 patients (33 with tandem and 33 with singleM1 occlusions) who underwent emergent intravascular therapy in our center between November of 2013 and November of 2016 were collected. Patients' medical histories were reviewed for clinical and radiological variables. A modified Rankin Scale of 3 or more was considered as bad outcome. An interobserver concordance analysis evaluated the quality of collaterals in the initial computed tomography through the Maas, Miteff, and CGS (collateral grading scale) scales. RESULTS: No differences were found in theprognosis of tandem versus single M1 occlusions (P = .30). The kappa index for the Maas scale was .77 (95% confidence interval [CI] .59-.94) and bad collaterals were defined by a score of 1 or 2. The factors independently associated with a worse prognosis were the presence of bad collaterals (adjusted odds ratio [OR] 6.03, 95% CI 1.01-35.9, P = .048) and an incomplete revascularization (adjusted OR 6.01, 95% CI 1.01-35.7, p = .049). CONCLUSIONS: The outcome of patients with acute stroke secondary to tandem or M1 occlusions has not been found to depend on their localization. The bad quality of collaterals is the main factor related to an unfavorable prognosis.

Estrogen Receptor Beta Agonist Influences Presynaptic NMDA Receptor Distribution in the Paraventricular Hypothalamic Nucleus Following Hypertension in a Mouse Model of Perimenopause.

Women become susceptible to hypertension as they transition to menopause (i.e., perimenopause); however, the underlying mechanisms are unclear. Animal studies using an accelerated ovarian failure (AOF) model of peri-menopause (peri-AOF) demonstrate that peri-AOF hypertension is associated with increased postsynaptic NMDA receptor plasticity in the paraventricular hypothalamic nucleus (PVN), a brain area critical for blood pressure regulation. However, recent evidence indicates that presynaptic NMDA receptors also play a role in neural plasticity. Here, using immuno-electron microscopy, we examine the influence of peri-AOF hypertension on the subcellular distribution of the essential NMDA GluN1 receptor subunit in PVN axon terminals in peri-AOF and in male mice. Hypertension was produced by 14-day slow-pressor angiotensin II (AngII) infusion. The involvement of estrogen signaling was investigated by co-administering an estrogen receptor beta (ERß) agonist. Although AngII induced hypertension in both peri-AOF and male mice, peri-AOF females showed higher cytoplasmic GluN1 levels. In peri-AOF females, activation of ERß blocked hypertension and increased plasmalemmal GluN1 in axon terminals. In contrast, stimulation of ERß did not inhibit hypertension or influence presynaptic GluN1 localization in males. These results indicate that sex-dependent recruitment of presynaptic NMDA receptors in the PVN is influenced by ERß signaling in mice during early ovarian failure.

Pathogenic Mis-splicing of CPEB4 in Schizophrenia.

BACKGROUND: Schizophrenia (SCZ) is caused by an interplay of polygenic risk and environmental factors, which may alter regulators of gene expression leading to pathogenic misexpression of SCZ risk genes. The CPEB family of RNA-binding proteins (CPEB1-4) regulates translation of target RNAs (approximately 40% of overall genes). We previously identified CPEB4 as a key dysregulated translational regulator in autism spectrum disorder (ASD) because its neuronal-specific microexon (exon 4) is mis-spliced in ASD brains, causing underexpression of numerous ASD risk genes. The genetic factors and pathogenic mechanisms shared between SCZ and ASD led us to hypothesize CPEB4 mis-splicing in SCZ leading to underexpression of multiple SCZ-related genes. METHODS: We performed MAGMA-enrichment analysis on Psychiatric Genomics Consortium genome-wide association study data and analyzed RNA sequencing data from the PsychENCODE Consortium. Reverse transcriptase polymerase chain reaction and Western blot were performed on postmortem brain tissue, and the presence/absence of antipsychotics was assessed through toxicological analysis. Finally, mice with mild overexpression of exon 4-lacking CPEB4 (CPEB4Δ4) were generated and analyzed biochemically and behaviorally. RESULTS: First, we found enrichment of SCZ-associated genes for CPEB4-binder transcripts. We also found decreased usage of CPEB4 microexon in SCZ probands, which was correlated with decreased protein levels of CPEB4-target SCZ-associated genes only in antipsychotic-free individuals. Interestingly, differentially expressed genes fit those reported for SCZ, specifically in the SCZ probands with decreased CPEB4-microexon inclusion. Finally, we demonstrated that mice with mild overexpression of CPEB4Δ4 showed decreased protein levels of CPEB4-target SCZ genes and SCZ-linked behaviors. CONCLUSIONS: We identified aberrant CPEB4 splicing and downstream misexpression of SCZ risk genes as a novel etiological mechanism in SCZ.

Lipidomic profiling of bioactive lipids during spontaneous fermentations of fine-flavor cocoa.

The impact of cocoa lipid content on chocolate quality has been extensively described. Nevertheless, few studies have elucidated the cocoa lipid composition and their bioactive properties, focusing only on specific lipids. In the present study the lipidome of fine-flavor cocoa fermentation was analyzed using LC-MS-QTOF and a Machine Learning model to assess potential bioactivity was developed. Our results revealed that the cocoa lipidome, comprised mainly of fatty acyls and glycerophospholipids, remains stable during fine-flavor cocoa fermentations. Also, several Machine Learning algorithms were trained to explore potential biological activity among the identified lipids. We found that K-Nearest Neighbors had the best performance. This model was used to classify the identified lipids as bioactive or non-bioactive, nominating 28 molecules as potential bioactive lipids. None of these compounds have been previously reported as bioactive. Our work is the first untargeted lipidomic study and systematic effort to investigate potential bioactivity in fine-flavor cocoa lipids.

Dissecting fine-flavor cocoa bean fermentation through metabolomics analysis to break down the current metabolic paradigm.

Cocoa fermentation plays a crucial role in producing flavor and bioactive compounds of high demand for food and nutraceutical industries. Such fermentations are frequently described as a succession of three main groups of microorganisms (i.e., yeast, lactic acid, and acetic acid bacteria), each producing a relevant metabolite (i.e., ethanol, lactic acid, and acetic acid). Nevertheless, this view of fermentation overlooks two critical observations: the role of minor groups of microorganisms to produce valuable compounds and the influence of environmental factors (other than oxygen availability) on their biosynthesis. Dissecting the metabolome during spontaneous cocoa fermentation is a current challenge for the rational design of controlled fermentations. This study evaluates variations in the metabolic fingerprint during spontaneous fermentation of fine flavor cocoa through a multiplatform metabolomics approach. Our data suggested the presence of two phases of differential metabolic activity that correlate with the observed variations on temperature over fermentations: an exothermic and an isothermic phase. We observed a continuous increase in temperature from day 0 to day 4 of fermentation and a significant variation in flavonoids and peptides between phases. While the second phase, from day four on, was characterized for lower metabolic activity, concomitant with small upward and downward fluctuations in temperature. Our work is the first to reveal two phases of metabolic activity concomitant with two temperature phases during spontaneous cocoa fermentation. Here, we proposed a new paradigm of cocoa fermentation that considers the changes in the global metabolic activity over fermentation, thus changing the current paradigm based only on three main groups of microorganism and their primary metabolic products.

Dissecting industrial fermentations of fine flavour cocoa through metagenomic analysis.

The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.

Bethlem myopathy: a series of 16 patients and description of seven new associated mutations.

BACKGROUND: Bethlem myopathy represents the milder phenotype of collagen type VI-related myopathies. However, clinical manifestations are highly variable among patients and no phenotype-genotype correlation has been described. We aim to analyse the clinical, pathological and genetic features of a series of patients with Bethlem myopathy, and we describe seven new mutations. METHODS: A series of 16 patients with the diagnosis of Bethlem myopathy were analyzed retrospectively from their medical records for clinical, creatine kinase (CK), muscle biopsy, and muscle magnetic resonance (MRI) data. Genetic testing was performed through next-generation sequencing of custom amplicon-based targeted genes panel of myopathies. Mutations were confirmed by Sanger sequencing. RESULTS: The most frequent phenotype consisted of proximal limb weakness associated with interphalangeal and wrists contractures. However, cases with isolated contractures or isolated myopathy were found. CK levels did not correlate with severity of the disease. The most frequent mutation was the COL6A3 variant c.7447A>G, p.Lys2486Glu, with either an homozygous or compound heterozygous presentation. Five new mutations were found in COL6A1 gene and other two in COL6A3 gene, all of them with a dominant heritability pattern. From these, a new COL6A1 mutation (c.1657G>A, p.Glu553Arg) was related to an oligosymptomatic phenotype with predominating contractures in the absence of weakness and a normal muscle MRI. Finally, the most common COL6A1 mutation reported to date that leads to an Ullrich phenotype (c. 868G>A, p.Gly290Arg), has been found here as Bethlem presentation. CONCLUSIONS: Manifestations of Bethlem myopathy are quite variable, so either contractures or weakness may be lacking, and no phenotype-genotype associations can be brought.

A new MR radiological sign in HaNDL syndrome. A case report.

BACKGROUND: One of the classical diagnostic criteria of the syndrome of transient headache and neurologic deficits with cerebrospinal fluid lymphocytosis (HaNDL syndrome) is the normality of imaging studies except from some reversible alterations as leptomeningeal enhancement or focal hypoperfusion. CASE: We present a 41 year-old man who abruptly started with a set of right parietal symptoms, meeting the diagnostic criteria for HaNDL syndrome. An electroencephalographic record showed a slowing of the right hemisphere. MR susceptibility weighted sequences demonstrated a reduced venous signal in the symptomatic hemisphere, unlike other transient disorders as migraine aura where an opposite pattern with prominence of the venous structures in the symptomatic hemisphere has been reported. This sign could reflect a decrease in metabolic demands or a fail in oxygen employment by the affected tissue. CONCLUSION: To our knowledge, this finding has not been described yet, and it may provide a new insight on the pathogenesis of HaNDL syndrome.

Pure alexia: two cases and a new neuroanatomical classification.

Pure alexia without visual or language accompanying deficits (isolated pure alexia), represents an infrequent finding in clinical practice. It has been linked to lesions involving the splenium of the callosal corpus in classical descriptions; however, it has also been reported after occipito-temporal cortex damage in the absence of white matter implication. In this regard, a functional region called the visual word form area has been recently related to the posterior aspect of the occipitotemporal gyrus. We report two new cases of cortical hematomas leading to this rare condition and we discuss the neuroanatomical evolution of this syndrome. Finally, we propose a new classification of pure alexia based on the neuroanatomical location of the lesion, namely: (1) disconnection alexia, after posterior and dorsal lesions involving the splenium of the callosal corpus or the paraventricular white matter, often associated with visual deficits, and (2) cortical alexia, after more anterior and ventral lesions in the occipito-temporal cortex with damage of the visual word form area, that usually manifests as isolated pure alexia.

The Mesoaccumbens Pathway: A Retrograde Labeling and Single-Cell Axon Tracing Analysis in the Mouse.

Neurons in the ventral tegmental area (VTA) that innervate the nucleus accumbens (Acb) constitute the so-called mesoaccumbens system. Increased activity by these neurons is correlated with the expectation and achievement of reward. The mesoaccumbens projection neurons are regarded as a central node in the brain networks that regulate drive and hedonic experience, and their dysregulation is a common pathophysiological step in addictive behaviors as well as major depression. Despite previous anatomical studies that have analyzed the origin of the mesoaccumbens axons within the VTA, regarded as a unit, the exact contributions of the various cytoarchitectural subdivisions of the VTA to this innervation is still unexplored; understanding these contributions would help further our understanding of their precise anatomical organization. With the aim of deciphering the contribution of the various VTA subdivisions to accumbal innervation, the present study has used retrograde tracer microinjections in the Acb to map the location within the various VTA subdivisions of neurons targeting either the shell or core compartments of the Acb in mice. Furthermore, the dopaminergic nature of these projections has also been analyzed using tyrosine-hydroxylase immunohistochemistry. We demonstrate here that small territories of the Acb core and shell are innervated simultaneously by many VTA subdivisions, contributing dopaminergic as well as non-dopaminergic axons to the accumbal innervation. In fact, single VTA subdivisions harbor both dopaminergic and non-dopaminergic neurons that project to the same accumbal territory. The most medial VTA subnuclei, like the caudal linear nucleus, project abundantly to medial aspects of the Acb core, whereas more lateral territories of the Acb are preferentially targeted by neurons located in the parabrachial pigmented and paranigral nuclei. Overall, about half of the mesoaccumbens neurons are putatively dopaminergic in mice. Anterograde single-cell labeling (Sindbis-pal-eGFP vector) of a limited sample of neurons revealed that mesoaccumbens neurons form profuse terminal arborizations to cover large volumes of either the Acb core or shell, and, unlike other VTA projection neuron populations, they do not branch to other striatal or extrastriatal structures. These anatomical observations are consistent with reports of an intense response in many Acb neurons after stimulation of very few VTA cells.

AISLAMIENTO E IDENTIFICACIÓN DE Lactobacillus spp. (LACTOBACILLACEAE) RESISTENTES A Cd(II) Y As(III) RECUPERADOS DE FERMENTO DE CACAO / Isolation and identification of Lactobacillus spp. (Lactobacillaceae) resistant to Cd(II) and As(III) from fermented cocoa

RESUMEN El objetivo de este estudio fue aislar e identificar a partir de cacao fermentado en Caldas Colombia, bacterias con potencial de aplicación en procesos biotecnológicos, como la detoxificación de cadmio (Cd(II)) y arsénico (As(III)) en el organismo humano. En total se recuperaron 36 aislados de los cuales se recuperaron 11 en presencia de 1,0 mg/L de Cd(II) y 25 en presencia de 0,1 mg/L de As(III). Su identificación molecular determinó que la mayoría de los aislados son del género Lactobacillus. Los ensayos de crecimiento en presencia de diferentes concentraciones de los elementos evaluados permitió determinar que gran parte de los aislamientos presentan resistencia a mayores concentraciones de As(III) (300 mg/L) que de Cd(II) (10 mg/L). En ensayos de tolerancia a la acidez (pH 2,5) se encontró que la cepa tipo Lactobacillus plantarum JCM 1055, junto con los aislamientos nativos L. plantarum A19, A26 y C16, mostraron la mayor tolerancia, por lo que se seleccionaron para evaluar su tolerancia a condiciones de salinidad. Las bacterias evaluadas mostraron crecimiento en concentraciones de hasta 4 g/L de sales biliares. Se concluye que los L. plantarum evaluados en este trabajo tienen un gran potencial para futuros ensayos en los que se busque demostrar la disminución de la bioaccesibilidad de Cd(II) y As(III) en condiciones in vitro del sistema digestivo humano debido a su resistencia a altas concentraciones de estos elementos y su tolerancia a condiciones de acidez y salinidad. Esto, junto con el reconocido potencial probiótico que tienen estos microorganismos, permitirá a futuro su uso en procesos biológicos de mitigación de Cd(II) y As(III).

ABSTRACT The aim of this study was to isolate and identify from fermented cocoa in Caldas Colombia, bacteria with potential application in biotechnological processes such as detoxification of cadmium (Cd(II)) and arsenic (As(III)) in the human organism. In total, 36 isolates were obtained, from which 11 were recovered in the presence of 1.0 mg/L of Cd(II) and 25 in presence of 0.1 mg/L of As(III). Molecular identification showed most isolates belong to the genera Lactobacillus. Minimum inhibitory concentration assays, in presence of different concentrations of the elements, allowed to determine that the majority of isolates have resistance to higher concentration of As(III) (300 mg/L) than Cd(II) (10 mg/L). Acidity tolerance assays at pH 2.5 showed that type strain Lactobacillus plantarum JCM 1055, and native isolates L. plantarum A19, A26, and C16, presented the highest tolerance, thus they were selected to evaluate their tolerance to salinity conditions. The evaluated bacteria could grow in bile salts up to 4 g/L. It is concluded that the evaluated L. plantarum have great potential to be used in assays in which bioaccessibility of Cd(II) and As(III) is diminished under in vitro conditions of the human digestive system, due to its resistance to high concentrations of the elements and tolerance to acidic and high bile salt conditions. These facts, together with the recognized probiotic potential of these microorganisms, may allow their future use in biological processes to mitigate Cd(II) and As(III).

Long-range projection neurons of the mouse ventral tegmental area: a single-cell axon tracing analysis.

Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four "subtypes" could be distinguished among the FPN cells based on their projection targets: (1) "Mesocorticolimbic" FPN projecting to both neocortex and basal forebrain; (2) "Mesocortical" FPN innervating the neocortex almost exclusively; (3) "Mesolimbic" FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) "Mesostriatal" FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and brainstem structures.

Earlobe crease shapes and cardiovascular events.

Earlobe crease (ELC) has been linked to coronary artery disease; however, systematic evaluations of the earlobe and its relation to ischemic stroke are lacking. The objectives were to define the ELC using a single-blind approach and to determine through multivariate analysis its association with cardiovascular events (CVEs) comprising coronary, ischemic cerebrovascular, and peripheral vascular diseases. A single-blind cross-sectional study was performed in 2 phases: (1) an initial study (n = 300) to define ELC classification criteria and (2) a confirmation stage (n = 1,000) to analyze ELC association with CVEs. Each of the participants' pinnae were photographed and classified blindly by joint decision according to ELC's inclination, length, depth, and bilateralism. Patients' medical histories were reviewed for age, cardiovascular risk factors, and CVEs. The concordance rate after the classification of all photographs was 89.6%. The first phase did not find any correlation between the different depth degrees or vertical creases and CVEs. The second stage concluded that diagonal bilateral ELC prevalence in patients with CVEs was 43% compared with 29% in the control patients (p <0.001). The multivariate analysis showed an association between ELC and CVEs (odds ratio 1.45, 95% confidence interval [CI] 1.08 to 1.93, p = 0.012), with a sensitivity and specificity of 43% and 70%, respectively. Ischemic stroke alone was also associated with diagonal bilateral ELC (odds ratio 1.67, 95% confidence interval 1.1 to 2.51, p = 0.015). In conclusion, diagonal bilateral ELC is independently associated with CVEs in the hospitalized population. An independent association with ischemic stroke has also been demonstrated for the first time.