The Trimeric Autotransporter Adhesin EmaA and Infective Endocarditis.

Infective endocarditis (IE), a disease of the endocardial surface of the heart, is usually of bacterial origin and disproportionally affects individuals with underlying structural heart disease. Although IE is typically associated with Gram-positive bacteria, a minority of cases are caused by a group of Gram-negative species referred to as the HACEK group. These species, classically associated with the oral cavity, consist of bacteria from the genera Haemophilus (excluding Haemophilus influenzae), Aggregatibacter, Cardiobacterium, Eikenella, and Kingella. Aggregatibacter actinomycetemcomitans, a bacterium of the Pasteurellaceae family, is classically associated with Aggressive Periodontitis and is also concomitant with the chronic form of the disease. Bacterial colonization of the oral cavity serves as a reservoir for infection at distal body sites via hematological spreading. A. actinomycetemcomitans adheres to and causes disease at multiple physiologic niches using a diverse array of bacterial cell surface structures, which include both fimbrial and nonfimbrial adhesins. The nonfimbrial adhesin EmaA (extracellular matrix binding protein adhesin A), which displays sequence heterogeneity dependent on the serotype of the bacterium, has been identified as a virulence determinant in the initiation of IE. In this chapter, we will discuss the known biochemical, molecular, and structural aspects of this protein, including its interactions with extracellular matrix components and how this multifunctional adhesin may contribute to the pathogenicity of A. actinomycetemcomitans.

CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Switching to an advanced hybrid closed-loop system in real-world practice improves hypoglycemia awareness and metabolic control in adults with type 1 diabetes, particularly in those with impaired perception of hypoglycemia symptoms.

AIMS: To evaluate the efficacy of an advance closed-loop (AHCL) system in restoring awareness of hypoglycemia in patients with type 1 diabetes (T1D). METHODS: We conducted a prospective study including 46 subjects with T1D flash glucose monitoring (FGM) or continuous glucose monitoring (CGM) switching to a Minimed 780G® system. Patients were classified in three groups according to the therapy used before switching to Minimed® 780G: multiple dose insulin (MDI) therapy + FGM (n = 6), continuous subcutaneous insulin infusion + FGM (n = 21), and sensor-augmented pump with predictive low-glucose suspend (n = 19). FGM/CGM data were analyzed at baseline, after 2 and 6 months on AHCL. Clarke's score of hypoglycemia awareness was compared at baseline and 6 months recordings. We also compared the efficacy of the AHCL system in improving A1c among patients with appropriate perception of symptoms of hypoglycemia compared to those presenting with impaired awareness of hypoglycemia (IAH). RESULTS: Participants had a mean age of 37 ± 15 and a diabetes duration of 20 ± 10 years. At baseline, 12 patients (27%) showed IAH as defined by a Clarke's score ≥ 3. Patients with IAH were older and had lower estimated glomerular filtration rate (eGFR) compared with those who did not have IAH; with no differences in baseline CGM metrics or A1c. An overall decrease in A1c was observed after 6 months on AHCL system (from 6.9 ± 0.5% to 6.7 ± 0.6%, P < 0.001), regardless of prior insulin therapy. The improvement in metabolic control was greater in patients with IAH, showing a reduction in A1c from 6.9 ± 0.5 to 6.4 ± 0.4% vs 6.9 ± 0.5 to 6.8 ± 0.6% (P = 0.003), showing a parallel increase in total daily boluses of insulin and automatic bolus correction administered by the AHCL system. In patients with IAH Clarke's score decreased from 3.6 ± 0.8 at baseline to 1.9 ± 1.6 after 6 months (P < 0.001). After 6 months on AHCL system, only 3 patients (7%) presented with a Clarke's score ≥ 3, resulting in an absolute risk reduction of 20% (95% confidence interval: 7-32) of having IAH. CONCLUSIONS: Switching from any type of insulin administration to AHCL system improves restoration of hypoglycemia awareness and metabolic control in patients with T1D, particularly in adults with impaired perception of hypoglycemia symptoms. TRIAL REGISTRATION: ClinicalTrial.gov ID NCT04900636.

Manejo conservador de pseudoaneurisma de vena cava iatrogénico / Conservative management of iatrogenic vena cava pseudoaneurysm

Introducción: los pseudoaneurismas de vena cava infrarrenal (VCI) son una patología infrecuente, sin tratamiento estandarizado; la mayoría, secundarios a traumatismos abdominales. Presentan una mortalidad del 20-57 %. Su manejo debe ser individualizado, con opciones como conservador, quirúrgico o endovascular. Caso clínico: varón de 23 años con cardiopatía congénita compleja que ingresa por síncope extrahospitalario con posterior aleteo auricular e inestabilidad hemodinámica. Durante el procedimiento de ablación presenta shock hemorrágico. Precisa drogas vasoactivas y transfusión masiva. Tras su estabilización, se realiza angio TAC abdominal en el que se visualiza hematoma retroperitoneal dependiente de VCI sin hemorragia activa. Dada la comorbilidad del paciente y la estabilidad hemodinámica, se decide tratamiento conservador y control radiológico. En el angio TAC a los 15 días se visualiza pseudoaneurisma de VCI. Decide mantenerse actitud conservadora, retirar la anticoagulación y realizar revisiones periódicas. Se mantiene estable y se decide el alta, con vigilancia estrecha. En el control a los dos meses se objetiva completa resolución del pseudoaneurisma. Discusión: dada la complejidad de la patología, la estabilidad hemodinámica y las comorbilidades del paciente, se optó por manejo conservador, sin descartar otras opciones terapéuticas si presentaba empeoramiento clínico o radiológico. El tratamiento del pseudoaneurisma de VCI debe individualizarse, priorizando la clínica y la estabilidad del paciente y vigilando la evolución de la lesión con control radiológico estrecho.(AU)

Background: infrarenal cava vein (ICV) pseudoaneurysms are an infrequent pathology, without standardized treatment. Most secondary to abdominal trauma and may associate arterial injuries. Presenting a mortality of 20-57 %, which has not been reduced, despite advances in treatment. Iatrogenic IVC injuries can develop retroperitoneal hematomas and pseudoaneurysms. Its management must be individualized, with options such as conservative, surgical or endovascular. Case report: a 23-year-old male with complex congenital heart disease was admitted due to out-of-hospital syncope with subsequent atrial flutter and hemodynamic instability. During the ablation procedure, he presented hemorrhagic shock requiring doses of vasoactive drugs and massive transfusion. After stabilizing the patient, an abdominal angio-CT was performed, visualizing an IVC-dependent retroperitoneal hematoma with no signs of active bleeding. Given the patient's comorbidity and hemodynamic stability, conservative treatment and radiological control were implemented. CT angiography at 15 days shows IVC pseudoaneurysm. It was decided to maintain a conservative attitude, withdraw anticoagulation and periodic check-ups. Remaining stable, discharge is decided, with close monitoring. At the two months check-up, complete resolution of the pseudoaneurysm is observed. Discussion: given the complexity of the pathology, the patient's hemodynamic stability and comorbidities, conservative management was chosen, without ruling out other therapeutic options if presented with clinical or radiological worsening. The treatment of IVC pseudoaneurysm must be individualized, prioritizing the patient's symptoms and stability and monitoring the evolution of the lesion with close radiological control.(AU)

Serotype-Specific Sugars Impact Structure but Not Functions of the Trimeric Autotransporter Adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors, including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with O-polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202-kDa protein was expressed in a non-serotype b, emaA mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by two-dimensional (2D) electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were posttranslationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII, and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes, however, did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. IMPORTANCE The human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for colonization by this pathobiont in vivo. This adhesin is modified with sugars associated with the O-polysaccharide (O-PS), and the modification is mediated using the enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the posttranslational modification of this adhesin is exchangeable without compromising functional activities.

Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence.

Cell senescence is a state of limited cell proliferation during a stress response or as part of a programmed process. When a senescent cell stops dividing, maintaining metabolic activity contributes to cellular homeostasis maintenance. In this process, the cell cycle is arrested at the G0/G1 phase. p16INK4A protein is a key regulator of this process via its cyclin­dependent kinase inhibitor (CDKI) function. CDKI 2A (CDKN2A)/p16 gene expression is regulated by DNA methylation and histone acetylation. Sirtuins (SIRTs) are nicotinamide dinucleotide (NAD+)­dependent deacetylases that have properties which prevent diseases and reverse certain aspects of aging (such as immune, metabolic and cardiovascular diseases). By performing quantitative PCR, Western blot, ChIP, and siRNAs assays, in this study it was demonstrated that CDKN2A/p16 gene transcriptional activation and repression were accompanied by selective deposition and elimination of histone acetylation during the senescence of MRC5 cells. Specifically, significant H3K9Ac and H3K18Ac enrichment in cells with a senescent phenotype concomitant with CDKN2A/p16 gene overexpression was demonstrated compared with the non­senescent phenotype. Furthermore, the presence of H3K18Ac in deacetyl­transferase SIRT7 knockdown MRC5 cells allowed CDKN2A/p16 promoter activation. These results suggested that SIRT7 served as a critical component of an epigenetic mechanism involved in senescence mediated by the CDKN2A/p16 gene.

Evolución de los accesos vasculares para hemodiálisis trombosados tras su reparación quirúrgica urgente / Outcomes after surgical thrombectomy in occluded vascular access used for hemodialysis

Resumen: Introducción: un acceso vascular (AV) que funcione bien es esencial para los pacientes en hemodiálisis. La trombosis es la principal causa de fallo de un AV. La trombectomía y la reparación del AV permiten mantenerlo funcional y prolongar su permeabilidad. Por lo tanto, este enfoque sigue siendo el objetivo principal para el rescate del acceso y la prolongación de la tasa de permeabilidad. Objetivo: describir la evolución natural del AV trombosado para HD tras reparación urgente. Métodos: estudio retrospectivo, observacional y unicéntrico. Se incluyeron todos los pacientes sometidos a cirugía urgente por trombosis AV para HD, nativa y protésica, entre 2013 y 2019. Análisis estadístico: en las variables categóricas se obtuvieron proporciones y razones de prevalencia, y en las variables cuantitativas, medias, medianas y desviación estándar. Se realizaron correlaciones de variables, análisis de supervivencia sin falla (SWF) del AV y log rank. Resultados: durante el periodo de estudio rescatamos 54 trombosados e incluimos 48 pacientes. Rescatamos 22 nativos (45,8 %) y 26 protésicos AV (54,2 %) mediante trombectomía urgente. Después de la reparación urgente, el SWF fue del 49 % a los 110 días (ES 0,07), del 25 % a los 478 días (ES 0,07) y del 15 % a los 659 días. Conclusiones: existe un porcentaje nada despreciable de AV que permanece en uso a medio plazo tras ser rescatados, lo que permite una HD eficaz.(AU)

Introduction: a well-functioning vascular access(VA) is essential for patients undergoing hemodialysis. Thrombosis is the leading cause of VA failure. Thrombectomy and the repair of the VA allow maintaining it functional and prolong its permeability. Thus, this approach remains the major goal toward access salvage and prolongation of patency rate. Objective: to describe the natural history of thrombosed VA for HD after urgent repair.Methods: retrospective, observational and single center study. All patients undergoing urgent surgery for VA thrombosis for HD, native and prosthetic, between 2013 and 2019, were included. Statistical analysis: in categorical variables, proportions and prevalence ratios were obtained; and in numerical variables, means, medians and standard deviation. Variable correlations, analysis of survival without failure (SWF) of the VA and log rank were performed. Results: during the study period we rescued 54 thrombosed, we included 48 patients. We rescued 22 native (45.8 %) and 26 prosthetic VA (54.2 %) by urgent thrombectomy. After urgent repair, the SWF was 49 % at 110 days (ES 0.07), 25 % at 478 days (ES 0.07) and 15 % at 659 days. Conclusions: there is a non-negligible percentage of VA that remain in use in the medium term after being rescued, allowing effective HD.(AU)

The serotype a-EmaA adhesin of Aggregatibacter actinomycetemcomitans does not require O-PS synthesis for collagen binding activity.

Aggregatibacter actinomycetemcomitans, a causative agent of periodontitis and non-oral diseases, synthesizes a trimeric extracellular matrix protein adhesin A (EmaA) that mediates collagen binding and biofilm formation. EmaA is found as two molecular forms, which correlate with the serotype of the bacterium. The canonical protein (b-EmaA), associated with serotypes b and c, has a monomeric molecular mass of 202 kDa. The collagen binding activity of b-EmaA is dependent on the presence of O-polysaccharide (O-PS), whereas biofilm activity is independent of O-PS synthesis. The EmaA associated with serotype a strains (a-EmaA) has a monomeric molecular mass of 173 kDa and differs in the amino acid sequence of the functional domain of the protein. In this study, a-emaA was confirmed to encode a protein that forms antenna-like appendages on the surface of the bacterium, which were found to be important for both collagen binding and biofilm formation. In an O-PS-deficient talose biosynthetic (tld) mutant strain, the electrophoretic mobility of the a-EmaA monomers was altered and the amount of membrane-associated EmaA was decreased when compared to the parent strain. The mass of biofilm formed remained unchanged. Interestingly, the collagen binding activity of the mutant strain was similar to the activity associated with the parent strain, which differs from that observed with the canonical b-EmaA isoform. These data suggest that the properties of the a-EmaA isoform are like those of b-EmaA, with the exception that collagen binding activity is independent of the presence or absence of the O-PS.

Analysis of Global and Local DNA Methylation Patterns in Blood Samples of Patients With Autism Spectrum Disorder.

The goal of this investigation was to determine whether there are alterations in DNA methylation patterns in children with autism spectrum disorder (ASD). Material and Methods: Controlled prospective observational case-control study. Within the ASD group, children were sub-classified based on the presence (AMR subgroup) or absence (ANMR subgroup) of neurodevelopmental regression during the first 2 years of life. We analyzed the global levels of DNA methylation, reflected in LINE-1, and the local DNA methylation pattern in two candidate genes, Neural Cell Adhesion Molecule (NCAM1) and Nerve Growth Factor (NGF) that, according to our previous studies, might be associated to an increased risk for ASD. For this purpose, we utilized blood samples from pediatric patients with ASD (n = 53) and their corresponding controls (n = 45). Results: We observed a slight decrease in methylation levels of LINE-1 in the ASD group, compared to the control group. One of the CpG in LINE-1 (GenBank accession no.X58075, nucleotide position 329) was the main responsible for such reduction, highly significant in the ASD subgroup of children with AMR (p < 0.05). Furthermore, we detected higher NCAM1 methylation levels in ASD children, compared to healthy children (p < 0.001). The data, moreover, showed higher NGF methylation levels in the AMR subgroup, compared to the control group and the ANMR subgroup. These results are consistent with our prior study, in which lower plasma levels of NCAM1 and higher levels of NGF were found in the ANMR subgroup, compared to the subgroup that comprised neurotypically developing children. Conclusions: We have provided new clues about the epigenetic changes that occur in ASD, and suggest two potential epigenetic biomarkers that would facilitate the diagnosis of the disorder. We similarly present with evidence of a clear differentiation in DNA methylation between the ASD subgroups, with or without mental regression.

A propósito de un caso de endotensión asociada a apendicitis / Case report of endotension associated with appendicitis

El tratamiento endovascular del aneurisma de aorta abdominal (AAA) puede tener complicaciones durante su seguimiento; entre ellas, las endofugas. Presentamos el caso de un paciente de 55 años que fue tratado de manera programada con endoprótesis aórtica bifurcada por un AAA infrarrenal de 84 mm de diámetro. El paciente presentó una reducción progresiva del saco aneurismático hasta un diámetro de 42 mm tras treinta meses de seguimiento. Después de cuatro años de seguimiento y sin signos sugestivos de complicaciones en la ecografía de control, se encuentra un leve crecimiento del saco (46 mm), sin evidencia de endofugas. Ante el hallazgo, se solicita TAC en la que se evidencia crecimiento del saco de hasta 55 mm de diámetro, sin presencia de fugas. En un nuevo estudio a los 3 meses, el saco ha sufrido una rápida expansión, hasta 73 mm de diámetro, sin evidencia de fugas. Ante los hallazgos y el riesgo de rotura, se planifica reparación abierta por endofuga tipo V. Se encuentra una fístula apendicular comunicada con un saco aneurismático de aspecto inflamatorio en la zona de contacto aórtico-apendicular. El paciente evolucionó favorablemente con antibioticoterapia prolongada. A los siete meses no ha presentado complicaciones clínicas ni en las pruebas de imagen

Endovascular aneurysm repair (EVAR) is an important treatment option for abdominal aortic aneurysms. An endoleak is a common complication following EVAR. A 55-year-old man underwent elective EVAR with a bifurcated graft for a 84 mm infrarenal abdominal aortic aneurysm (AAA). Follow-up a showed appropriate reduction of the AAA to 42 mm. A duplex ultrasound at fourth year follow up showed an increase of AAA sac size to 46 mm with no observable endoleaks. A subsequent CTA showed an increase of AAA sac size to 55 mm with no observable endoleaks. A new CTA at 3 months follow up showed a rapid increase of AAA sac size to 73 mm with no observable endoleaks. We decided to perform open surgery. We found appendicular fistula in contact with aneurysmal sac with an inflammatory appearance in the aortic-appendicular contact area. The patient was successfully treated and no severe complications occurred during the 7 months follow-up

DNA Methylation Editing by CRISPR-guided Excision of 5-Methylcytosine.

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.

Results and complications of carotid endarterectomy in a hospital from Madrid, Spain.

OBJECTIVE: To study the incidence of cerebrovascular (transient ischemic attacks and stroke) and myocardial events (myocardial infarction) as well as early survival related to carotid endarterectomy. Our secondary aim is to establish possible risk factors associated with complications. METHOD: Retrospective observational case-control study within a cohort. All patients who underwent carotid endarterectomy by the angiology and vascular surgery service at the Hospital Universitario La Paz, in Madrid (Spain), in the period between January 2011 and December 2017 were included. Chi square was used to calculate differences. Kaplan-Meier and Cox regression was used for the survival analysis and patency. RESULTS: 111 procedures were performed on 108 patients, 95 (87,9%) male with an average age of 68.5 ± 8.75. The mean time of follow-up was 2.9 years. There was no 30-day post-surgical mortality, with a 30-day postoperative cerebral vascular event rate of 2.7%. Statistically significant correlation was found between the presence of 30-day postoperative cerebral vascular event and primary closure (p = 0.005) as well as between the smoking habit and 30-day postoperative myocardial infarction (p = 0.036) and restenosis (p = 0.008). In mid-term follow-up, the event rate for cerebral vascular events and myocardial infarction was 1.8%. CONCLUSION: carotid endarterectomy is the procedure of choice in carotid stenosis. The low rates of perioperative mortality, morbidity and complications have been demonstrated.

OBJETIVO: Conocer la incidencia de eventos cerebrovasculares y miocárdicos, y la supervivencia temprana, relacionados con la endarterectomía carotídea, y como objetivo secundario establecer los posibles factores de riesgo asociados a las complicaciones. MÉTODO: Estudio observacional de casos y controles anidado en una cohorte retrospectiva. Se incluyeron todos los pacientes que se sometieron a endarterectomía carotídea en el servicio de angiología y cirugía vascular del Hospital Universitario La Paz, de Madrid (España), en el periodo de enero de 2011 a diciembre de 2017. Para la estimación de diferencias se utilizó la prueba de ji al cuadrado. El análisis de supervivencia y permeabilidad se realizó mediante Kaplan-Meier y regresión de Cox. RESULTADOS: Se realizaron 111 procedimientos en 108 pacientes, 95 (87.9%) de ellos varones, con una edad media de 68.5 ± 8.75 años. La media de seguimiento fue de 2.9 años. No hubo mortalidad posquirúrgica a 30 días, y la tasa global de eventos vasculares cerebrales posoperatorios a 30 días fue del 2.7%. Se encontró asociación entre la presencia de eventos vasculares cerebrales posquirúrgicos a 30 días y el cierre arterial primario (p = 0.005), y del infarto agudo de miocardio posoperatorio a 30 días y la reestenosis carotídea con el hábito tabáquico (p = 0.036 y p = 0.008, respectivamente). En el seguimiento a mediano plazo se encontró una tasa de enfermedad vascular cerebral y de infarto agudo de miocardio del 1,8%. CONCLUSIÓN: La endarterectomía carotídea es el procedimiento de elección en la estenosis carotídea por enfermedad aterosclerótica. En nuestro estudio se demuestran sus bajas tasas de mortalidad, de morbilidad y de complicaciones perioperatorias.

Interactions between the Trimeric Autotransporter Adhesin EmaA and Collagen Revealed by Three-Dimensional Electron Tomography.

Bacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogen Aggregatibacter actinomycetemcomitans Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen.IMPORTANCE Bacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans.

On cross-correlations, averages and noise in electron microscopy.

Biological samples are radiation-sensitive and require imaging under low-dose conditions to minimize damage. As a result, images contain a high level of noise and exhibit signal-to-noise ratios that are typically significantly smaller than 1. Averaging techniques, either implicit or explicit, are used to overcome the limitations imposed by the high level of noise. Averaging of 2D images showing the same molecule in the same orientation results in highly significant projections. A high-resolution structure can be obtained by combining the information from many single-particle images to determine a 3D structure. Similarly, averaging of multiple copies of macromolecular assembly subvolumes extracted from tomographic reconstructions can lead to a virtually noise-free high-resolution structure. Cross-correlation methods are often used in the alignment and classification steps of averaging processes for both 2D images and 3D volumes. However, the high noise level can bias alignment and certain classification results. While other approaches may be implicitly affected, sensitivity to noise is most apparent in multireference alignments, 3D reference-based projection alignments and projection-based volume alignments. Here, the influence of the image signal-to-noise ratio on the value of the cross-correlation coefficient is analyzed and a method for compensating for this effect is provided.

A Nonfimbrial Adhesin of Aggregatibacter actinomycetemcomitans Mediates Biofilm Biogenesis.

Periodontitis is an inflammatory disease caused by polymicrobial biofilms. The periodontal pathogen Aggregatibacter actinomycetemcomitans displays two proteinaceous surface structures, the fimbriae and the nonfimbrial extracellular matrix binding protein A (EmaA), as observed by electron microscopy. Fimbriae participate in biofilm biogenesis and the EmaA adhesins mediate collagen binding. However, in the absence of fimbriae, A. actinomycetemcomitans still retains the potential to form robust biofilms, suggesting that other surface macromolecules participate in biofilm development. Here, isogenic mutant strains lacking EmaA structures, but still expressing fimbriae, were observed to have reduced biofilm potential. In strains lacking both EmaA and fimbriae, biofilm mass was reduced by 80%. EmaA enhanced biofilm formation in different strains, independent of the fimbriation state or serotype. Confocal microscopy revealed differences in cell density within microcolonies between the EmaA positive and mutant strains. EmaA-mediated biofilm formation was found to be independent of the glycosylation state and the precise three-dimensional conformation of the protein, and thus this function is uncorrelated with collagen binding activity. The data suggest that EmaA is a multifunctional adhesin that utilizes different mechanisms to enhance bacterial binding to collagen and to enhance biofilm formation, both of which are important for A. actinomycetemcomitans colonization and subsequent infection.

Monitoring base excision repair in Chlamydomonas reinhardtii cell extracts.

Base excision repair (BER) is a major defense pathway against spontaneous DNA damage. This multistep process is initiated by DNA glycosylases that recognise and excise the damaged base, and proceeds by the concerted action of additional proteins that perform incision of the abasic site, gap filling and ligation. BER has been extensively studied in bacteria, yeasts and animals. Although knowledge of this pathway in land plants is increasing, there are no reports detecting BER in algae. We describe here an experimental in vitro system allowing the specific analysis of BER in the model alga Chlamydomonas reinhardtii. We show that C. reinhardtii cell-free extracts contain the enzymatic machinery required to perform BER of ubiquitous DNA lesions, such as uracil and abasic sites. Our results also reveal that repair can occur by both single-nucleotide insertion and long-patch DNA synthesis. The experimental system described here should prove useful in the biochemical and genetic dissection of BER in algae, and may contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways in photosynthetic eukaryotes.

DNA methylation reprogramming of human cancer cells by expression of a plant 5-methylcytosine DNA glycosylase.

Patterns of DNA methylation, an important epigenetic modification involved in gene silencing and development, are disrupted in cancer cells. Understanding the functional significance of aberrant methylation in tumors remains challenging, due in part to the lack of suitable tools to actively modify methylation patterns. DNA demethylation caused by mammalian DNA methyltransferase inhibitors is transient and replication-dependent, whereas that induced by TET enzymes involves oxidized 5mC derivatives that perform poorly understood regulatory functions. Unlike animals, plants possess enzymes that directly excise unoxidized 5mC from DNA, allowing restoration of unmethylated C through base excision repair. Here, we show that expression of Arabidopsis 5mC DNA glycosylase DEMETER (DME) in colon cancer cells demethylates and reactivates hypermethylated silenced loci. Interestingly, DME expression causes genome-wide changes that include both DNA methylation losses and gains, and partially restores the methylation pattern observed in normal tissue. Furthermore, such methylome reprogramming is accompanied by altered cell cycle responses and increased sensibility to anti-tumor drugs, decreased ability to form colonospheres, and tumor growth impairment in vivo. Our study shows that it is possible to reprogram a human cancer DNA methylome by expression of a plant DNA demethylase.