Optimization of rhein-loaded polymeric nanoparticles using a factorial design and evaluation of the cytotoxic and anti-inflammatory effects.

The aim of the work was to develop rhein loaded polymeric nanoparticles (R-PNPs). Nanoparticles were prepared by three methods, solvent emulsion-evaporation, double emulsion, and nanoprecipitation, by means of experimental design. Additionally, the effects of the best formulation on in vitro cytotoxicity and inflammation were evaluated. The solvent emulsion-evaporation method presented the highest encapsulation efficiency of the three techniques (38.41%), as well as had a mean diameter of 189.33 nm and a polydispersity index of less than 0.1. Despite efforts to optimize the encapsulation of rhein, the drug release from nanoparticles was close to 50% during the first 5 min, followed by a continuous release within 60 min. It was observed that macrophages exposed to the highest concentration of R-PNPs showed cell viability about 80% and at the lowest nanoparticle concentrations was closed to 100%. IL-1ß in cell culture supernatants was decreased in the presence of R-PNPs and TNFα concentrations were lower than the sensitivity of the assay. ROS production was only inhibited with R-PNPs at concentrations of 2.5 and 5 µM. In conclusion, the solvent emulsion-evaporation was the best method evaluated to obtain nanoparticles with the desired specifications. It was possible to assess R-PNPs with low cytotoxicity and anti-inflammatory properties showed by the inhibition of IL-1ß production and a low decrease in ROS production.

Oxidative stress-induced CREB upregulation promotes DNA damage repair prior to neuronal cell death protection.

cAMP response element-binding (CREB) protein is a cellular transcription factor that mediates responses to different physiological and pathological signals. Using a model of human neuronal cells we demonstrate herein, that CREB is phosphorylated after oxidative stress induced by hydrogen peroxide. This phosphorylation is largely independent of PKA and of the canonical phosphoacceptor site at ser-133, and is accompanied by an upregulation of CREB expression at both mRNA and protein levels. In accordance with previous data, we show that CREB upregulation promotes cell survival and that its silencing results in an increment of apoptosis after oxidative stress. Interestingly, we also found that CREB promotes DNA repair after treatment with hydrogen peroxide. Using a cDNA microarray we found that CREB is responsible for the regulation of many genes involved in DNA repair and cell survival after oxidative injury. In summary, the neuroprotective effect mediated by CREB appears to follow three essential steps following oxidative injury. First, the upregulation of CREB expression that allows sufficient level of activated and phosphorylated protein is the primordial event that promotes the induction of genes of the DNA Damage Response. Then and when the DNA repair is effective, CREB induces detoxification and survival genes. This kinetics seems to be important to completely resolve oxidative-induced neuronal damages.

Phenotypic characterisation of RAB6A knockout mouse embryonic fibroblasts.

BACKGROUND INFORMATION: Rab6 is one of the most conserved Rab GTPaes throughout evolution and the most abundant Rab protein associated with the Golgi complex. The two ubiquitous Rab isoforms, Rab6A and Rab6A', that are generated by alternative splicing of the RAB6A gene, regulate several transport steps at the Golgi level, including retrograde transport between endosomes and Golgi, anterograde transport between Golgi and the plasma membrane, and intra-Golgi and Golgi to endoplasmic reticulum transport. RESULTS: We have generated mice with a conditional null allele of RAB6A. Mice homozygous for the RAB6A null allele died at an early stage of embryonic development. Mouse embryonic fibroblasts (MEFs) were isolated from RAB6A(loxP/loxP) Rosa26-CreERT2 and incubated with 4-hydroxy tamoxifen, resulting in the efficient depletion of Rab6A and Rab6A'. We show that Rab6 depletion affects cell growth, alters Golgi morphology and decreases the Golgi-associated levels of some known Rab6 effectors such as Bicaudal-D and myosin II. We also show that Rab6 depletion protects MEFs against ricin toxin and delays VSV-G secretion. CONCLUSIONS: Our study shows that RAB6 is an essential gene required for normal embryonic development. We confirm in MEF cells most of the functions previously attributed to the two ubiquitous Rab6 isoforms.

Beak deviations in the skull of Franciscana dolphins Pontoporia blainvillei from Argentina.

The Franciscana dolphin Pontoporia blainvillei is characterized by a long rostrum, a feature that is shared with the families formerly classified as river dolphins (Pontoporiidae, Platanistidae, Iniidae, Lipotidae). Although there are occasional reports on the existence of beak deformations, very little published information exists describing this process. The object of the present study was to describe and quantify the beak anomalies of Franciscana dolphins from the coastal waters of Argentina. Of 239 skulls analyzed 12% showed beak deviations (BD), affecting the premaxillary-maxillary and dentary bones to different extents. The occurrence of BD in the dentary bone represented 58%, whereas premaxillary-maxillary BDs were observed in 14% of the studied specimens, while the complete rostrum (dentary, premaxillary and maxillary) was affected in 28% of the skulls. Dorsoventral axis BD was more frequent than lateral BD (48 and 38%, respectively), and double BD was only observed in the dentary bone. Most of the BD observed in this study could be classified as mild/moderate, and we assume that it did not affect the feeding activities of individuals; however, 2 specimens (<1%) showed a severe and complex curvature that probably did affect them. The cause of these anomalies (natural or anthropogenic origins) is unknown but may be related to important parasite loads, heavy metal and organic contaminants and plastic ingestion that could affect the coastal dolphin in different ways. A more detailed and thorough study of these cranial anomalies is necessary.

p19INK4d is involved in the cellular senescence mechanism contributing to heterochromatin formation.

BACKGROUND: During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state. METHODS: Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated ß-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction. RESULTS: The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age. CONCLUSION: We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction. GENERAL SIGNIFICANCE: This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.

Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.

The Genomic and Cultural Diversity of the Inka Qhapaq Hucha Ceremony in Chile and Argentina.

The South American archaeological record has ample evidence of the socio-cultural dynamism of human populations in the past. This has also been supported through the analysis of ancient genomes, by showing evidence of gene flow across the region. While the extent of these signals is yet to be tested, the growing number of ancient genomes allows for more fine-scaled hypotheses to be evaluated. In this study, we assessed the genetic diversity of individuals associated with the Inka ritual, Qhapaq hucha. As part of this ceremony, one or more individuals were buried with Inka and local-style offerings on mountain summits along the Andes, leaving a very distinctive record. Using paleogenomic tools, we analyzed three individuals: two newly generated genomes from El Plomo Mountain (Chile) and El Toro Mountain (Argentina), and a previously published genome from Argentina (Aconcagua Mountain). Our results reveal a complex demographic scenario with each of the individuals showing different genetic affinities. Furthermore, while two individuals showed genetic similarities with present-day and ancient populations from the southern region of the Inka empire, the third individual may have undertaken long-distance movement. The genetic diversity we observed between individuals from similar cultural contexts supports the highly diverse strategies Inka implemented while incorporating new territories. More broadly, this research contributes to our growing understanding of the population dynamics in the Andes by discussing the implications and temporality of population movements in the region.

Novel competitive enzyme-linked immunosorbent assay for the detection of the high-risk Human Papillomavirus 18 E6 oncoprotein.

Cervical cancer represents a global concern with 604,000 new cases and 342,000 deaths reported annually, with the vast majority diagnosed in low income countries. Despite high-risk Human Papillomavirus (HR HPV)-induced cervical cancer has become highly preventable through prophylactic vaccines, screening programs are critical in the control of cervical carcinogenesis in populations with limited access to vaccination and in older generations of women who have already been exposed to HR HPV infection. The surge of HPV molecular tests has provided a more sensitive and accurate diagnostic alternative to cytology screening. Given that HPV DNA testing presents a low positive predicted value, leading to unnecessary treatment, the E6 oncoprotein from HR HPV types arises as a promising diagnostic marker for its overexpression in transformed HPV-positive cancer cells. For these reasons, this study aimed at obtaining monoclonal antibodies (mAbs) against the E6 oncoprotein of one of the most prevalent HR HPV types worldwide, HPV18, in order to develop a highly specific and sensitive indirect competitive ELISA (icELISA). The production of hybridomas secreting HPV18 E6 mAbs was carried out through a combined tolerization and immunization strategy, in order to avoid cross-reactivity with the E6 protein from low-risk HPV types 6 and 11. We selected the 7D2 hybridoma clone, which recognized HPV18 E6 and showed some cross-reactivity against the HR HPV45 E6 oncoprotein. The 7D2 mAb enabled the development of a sensitive, reliable and reproducible icELISA to detect and quantify small amounts of HPV18 E6 biomarker for cervical cancer progression. The present study establishes a valid 7D2-based icELISA that constitutes a promising bioanalytical method for the early detection and quantification of HPV18 E6 oncoprotein in cervical swab samples and cancer prevention.

Use of the first-acquired language modulates pupil size in the processing of island constraint violations.

Introduction: Traditional studies of the population called "heritage speakers" (HS) have treated this group as distinct from other bilingual populations, e.g., simultaneous or late bilinguals (LB), focusing on group differences in the competencies of the first-acquired language or "heritage language". While several explanations have been proposed for such differences (e.g., incomplete acquisition, attrition, differential processing mechanisms), few have taken into consideration the individual variation that must occur, due to the fluctuation of factors such as exposure and use that characterize all bilinguals. In addition, few studies have used implicit measures, e.g., psychophysiological methods (ERPs; Eye-tracking), that can circumvent confounding variables such as resorting to conscious metalinguistic knowledge. Methodology: This study uses pupillometry, a method that has only recently been used in psycholinguistic studies of bilingualism, to investigate pupillary responses to three syntactic island constructions in two groups of Spanish/English bilinguals: heritage speakers and late bilinguals. Data were analyzed using generalized additive mixed effects models (GAMMs) and two models were created and compared to one another: one with group (LB/HS) and the other with groups collapsed and current and historical use of Spanish as continuous variables. Results: Results show that group-based models generally yield conflicting results while models collapsing groups and having usage as a predictor yield consistent ones. In particular, current use predicts sensitivity to L1 ungrammaticality across both HS and LB populations. We conclude that individual variation, as measured by use, is a critical factor tha must be taken into account in the description of the language competencies and processing of heritage and late bilinguals alike.

[Report of two cases of anti-LGI1 autoimmune encephalitis in Mexico]. / Reporte de dos casos de encefalitis autoinmune anti-LGI1 en México.

Background: Anti-LGI1 encephalitis is characterized by a pattern of inflammation that predominantly affects the limbic system It is part of the autoimmune encephalitis that attack neuronal surface antigens. It is characterized by the triad of subacute dementia, faciobrachial dystonic crises, and hyponatremia, presenting an excellent response to immunotherapy. The aim of this article is to describe the clinical evolution and functional outcome at 6 months of two patients with anti-LGI1 encephalitis using clinical cases. Clinical cases: Case 1: 62-year-old man with 8-week symptoms manifested by changes in mood, disorientation, and focal motor seizures. Case 2 A 72-year-old woman with a 5-month evolution of rapidly progressive dementia, hyponatremia and bitemporal hyperintensities on MRI. In both, due to clinical suspicion, acute dual immunotherapy with steroid and immunoglobulin was given with substantial improvement. Subsequently, the existence of anti-LGI1 antibodies in cerebrospinal fluid was confirmed. Although both patients received a dose of rituximab during their hospitalization, only the patient in the first case continued biannual doses of rituximab. The second patient was not initially considered to continue long-term immunomodulatory treatment and experienced a relapse. Conclusions: These clinical vignettes present the reader with the classic characteristics of this disease. This can facilitate its recognition and timely initiation of treatment, improving the functional prognosis of patients.

Introducción: la encefalitis anti-LGI1 se caracteriza por un patrón de inflamación que afecta de forma predominante al sistema límbico. Forma parte de las encefalitis autoinmunes que atacan a antígenos de superficie neuronal. Se caracteriza por la tríada de demencia subaguda, crisis distónicas faciobraquiales e hiponatremia, presentando una respuesta excelente a la inmunoterapia. El objetivo de este trabajo es describir por casos clínicos la evolución clínica y resultado funcional a 6 meses de dos pacientes con encefalitis anti-LGI1. Casos clínicos: caso 1: hombre de 62 años con cuadro de 8 semanas, manifestado por cambios en el estado de ánimo, desorientación y crisis focales motoras. Caso 2: mujer de 72 años con una evolución de 5 meses de demencia rápidamente progresiva, hiponatremia e hiperintensidades bitemporales en RMN. En ambos, ante la sospecha clínica, se otorgó inmunoterapia dual aguda con esteroide e inmunoglobulina con mejoría sustancial, posteriormente se corroboró la existencia de anticuerpos anti-LGI1 en líquido cefalorraquídeo. Pese a que ambos pacientes recibieron una dosis de rituximab durante su hospitalización, solo el primer caso continuó dosis semestrales de rituximab. El segundo no fue considerado inicialmente para continuar con tratamiento inmunomodulador a largo plazo y presentó una recaída. Conclusiones: estos casos, presentan al lector las características clásicas de esta enfermedad. Esto puede facilitar su reconocimiento y la instauración oportuna del tratamiento, mejorando el pronóstico funcional de los pacientes.

[Neuromyelitis optica spectrum disorder in a patient with human immunodeficiency virus]. / Neuromielitis óptica en paciente con virus de inmunodeficiencia humana.

Background: Neuromyelitis optica spectrum (NMO) is an autoimmune condition with preferential target at the optic nerves and spinal cord. Although HIV infection can also cause neuritis and myelitis, the entity of HIV related to NMO has recently been elucidated, however, little is known about the context of this disease. Objective: To describe the clinical characteristics, imaging, treatment, and functional prognosis in an HIV-positive patient who developed an episode of longitudinally extensive transverse myelitis (LETM) with positive anti-AQP4 antibodies. Clinic case: 36-year-old man with a history of HIV diagnosed in 2017, on antiretroviral treatment. On March 2021 he was admitted for study due to complete spinal cord syndrome, corroborating in MRI a longitudinally extensive lesion from T8-L1, with CSF with and AQP4 seropositivity, a diagnosis of NMO was integrated by Wingerchuk criteria and rituximab is started with symptomatic improvement, objectifying it with the Expanded Disability Status Scale (EDSS) from 4 to 1. Conclusion: NMO entity related to HIV is rare, this phenomenon being classically found at the time of diagnosis or after the start of treatment when the immune system is still capable of developing an exaggerated immune response, however in the case we report the debut of NMO occurred 3 years after diagnosis, contrasting with previously reported cases, so we suggest that some other mechanisms could be involved, such as altered regulation of B cells and a direct viral effect.

Introducción: la neuromielitis óptica (NMO) es una condición autoinmune con blanco preferencial a nivel de nervios ópticos y médula espinal. Recientemente se ha elucidado la entidad de VIH relacionada a NMO; no obstante, aún se conoce poco sobre el contexto de esta enfermedad. El objetivo de este trabajo es describir las características clínicas, de imagen, tratamiento y el pronóstico funcional en un paciente VIH positivo, quien desarrolló un episodio de mielitis longitudinalmente extensa (LETM) con anticuerpos anti-AQP4 positivos. Caso clínico: hombre de 36 años con antecedente de VIH diagnosticado en 2017, en tratamiento antirretroviral y antecedente de sarcoma de Kaposi. Inicia su padecimiento en marzo de 2021 con dolor en región lumbar, acompañado de debilidad y anestesia en miembros pélvicos, agregándose incontinencia vesical y distensión abdominal. A la exploración se integra síndrome medular completo con nivel en T8-T9, corroborándose en RM lesión longitudinalmente extensa desde T8-L1, con LCR con proteinorraquia y seropositividad a AQP4. Se integra diagnóstico de NMO por criterios de Wingerchuk y se inicia tratamiento con rituximab con mejoría sintomática, objetivándola con una escala expandida del estado (EDSS) de 4 a 1. Conclusión: la entidad de NMO relacionada al VIH es poco frecuente, siendo clásicamente encontrado este fenómeno al momento del diagnóstico o posterior al inicio del tratamiento, cuando el sistema inmune aún resulta capaz de desarrollar una respuesta inmune exagerada. Sin embargo, en el caso que reportamos el debut de la NMO fue posterior a tres años del diagnóstico, contrastando con los casos previamente reportados, por lo que sugerimos que podrían intervenir algunos otros mecanismos, como la alteración en la regulación de las células B y un efecto viral directo.

Humoral immunity against SARS-CoV-2 evoked by heterologous vaccination groups using the CoronaVac (Sinovac) and BNT162b2 (Pfizer/BioNTech) vaccines in Chile.

Introduction: Severe acute respiratory syndrome virus 2 (SARS-CoV-2) has caused over million deaths worldwide, with more than 61,000 deaths in Chile. The Chilean government has implemented a vaccination program against SARS-CoV-2, with over 17.7 million people receiving a complete vaccination scheme. The final target is 18 million individuals. The most common vaccines used in Chile are CoronaVac (Sinovac) and BNT162b2 (Pfizer-Biotech). Given the global need for vaccine boosters to combat the impact of emerging virus variants, studying the immune response to SARS-CoV-2 is crucial. In this study, we characterize the humoral immune response in inoculated volunteers from Chile who received vaccination schemes consisting of two doses of CoronaVac [CoronaVac (2x)], two doses of CoronaVac plus one dose of BNT162b2 [CoronaVac (2x) + BNT162b2 (1x)], and three doses of BNT162b2 [BNT162b2 (3x)]. Methods: We recruited 469 participants from Clínica Dávila in Santiago and the Health Center Víctor Manuel Fernández in the city of Concepción, Chile. Additionally, we included participants who had recovered from COVID-19 but were not vaccinated (RCN). We analyzed antibodies, including anti-N, anti-S1-RBD, and neutralizing antibodies against SARS-CoV-2. Results: We found that antibodies against the SARS-CoV-2 nucleoprotein were significantly higher in the CoronaVac (2x) and RCN groups compared to the CoronaVac (2x) + BNT162b2 (1x) or BNT162b2 (3x) groups. However, the CoronaVac (2x) + BNT162b2 (1x) and BNT162b2 (3x) groups exhibited a higher concentration of S1-RBD antibodies than the CoronaVac (2x) group and RCN group. There were no significant differences in S1-RBD antibody titers between the CoronaVac (2x) + BNT162b2 (1x) and BNT162b2 (3x) groups. Finally, the group immunized with BNT162b2 (3x) had higher levels of neutralizing antibodies compared to the RCN group, as well as the CoronaVac (2x) and CoronaVac (2x) + BNT162b2 (1x) groups. Discussion: These findings suggest that vaccination induces the secretion of antibodies against SARS-CoV-2, and a booster dose of BNT162b2 is necessary to generate a protective immune response. In the current state of the pandemic, these data support the Ministry of Health of the Government of Chile's decision to promote heterologous vaccination as they indicate that a significant portion of the Chilean population has neutralizing antibodies against SARS-CoV-2.

Clinical and pulmonary function analysis in long-COVID revealed that long-term pulmonary dysfunction is associated with vascular inflammation pathways and metabolic syndrome.

Introduction: Long-term pulmonary dysfunction (L-TPD) is one of the most critical manifestations of long-COVID. This lung affection has been associated with disease severity during the acute phase and the presence of previous comorbidities, however, the clinical manifestations, the concomitant consequences and the molecular pathways supporting this clinical condition remain unknown. The aim of this study was to identify and characterize L-TPD in patients with long-COVID and elucidate the main pathways and long-term consequences attributed to this condition by analyzing clinical parameters and functional tests supported by machine learning and serum proteome profiling. Methods: Patients with L-TPD were classified according to the results of their computer-tomography (CT) scan and diffusing capacity of the lungs for carbon monoxide adjusted for hemoglobin (DLCOc) tests at 4 and 12-months post-infection. Results: Regarding the acute phase, our data showed that L-TPD was favored in elderly patients with hypertension or insulin resistance, supported by pathways associated with vascular inflammation and chemotaxis of phagocytes, according to computer proteomics. Then, at 4-months post-infection, clinical and functional tests revealed that L-TPD patients exhibited a restrictive lung condition, impaired aerobic capacity and reduced muscular strength. At this time point, high circulating levels of platelets and CXCL9, and an inhibited FCgamma-receptor-mediated-phagocytosis due to reduced FcγRIII (CD16) expression in CD14+ monocytes was observed in patients with L-TPD. Finally, 1-year post infection, patients with L-TPD worsened metabolic syndrome and augmented body mass index in comparison with other patient groups. Discussion: Overall, our data demonstrated that CT scan and DLCOc identified patients with L-TPD after COVID-19. This condition was associated with vascular inflammation and impair phagocytosis of virus-antibody immune complexes by reduced FcγRIII expression. In addition, we conclude that COVID-19 survivors required a personalized follow-up and adequate intervention to reduce long-term sequelae and the appearance of further metabolic diseases.

Atypical cristae morphology of human syncytiotrophoblast mitochondria: role for complex V.

Mitochondrial complexes I, III(2), and IV from human cytotrophoblast and syncytiotrophoblast associate to form supercomplexes or respirasomes, with the following stoichiometries: I(1):(III(2))(1) and I(1):(III(2))(1-2):IV(1-4). The content of respirasomes was similar in both cell types after isolating mitochondria. However, syncytiotrophoblast mitochondria possess low levels of dimeric complex V and do not have orthodox cristae morphology. In contrast, cytotrophoblast mitochondria show normal cristae morphology and a higher content of ATP synthase dimer. Consistent with the dimerizing role of the ATPase inhibitory protein (IF(1)) (García, J. J., Morales-Ríos, E., Cortés-Hernandez, P., and Rodríguez-Zavala, J. S. (2006) Biochemistry 45, 12695-12703), higher relative amounts of IF(1) were observed in cytotrophoblast when compared with syncytiotrophoblast mitochondria. Therefore, there is a correlation between dimerization of complex V, IF(1) expression, and the morphology of mitochondrial cristae in human placental mitochondria. The possible relationship between cristae architecture and the physiological function of the syncytiotrophoblast mitochondria is discussed.

Proinflammatory cytokine regulation of cyclic AMP-phosphodiesterase 4 signaling in microglia in vitro and following CNS injury.

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß on cyclic AMP-phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF-α or IL-1ß stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL-1ß but remained suppressed with TNF-α through 24 h. Cyclic AMP was also reduced in TNF-α-stimulated primary microglia, albeit to a lesser extent. Accompanying TNF-α-induced cyclic AMP reductions, but not IL-1ß, was increased cyclic AMP-PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF-α; IL-1ß increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF-α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF-α but not IL-1ß. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX-42(+) microglia; PDE4B was absent in OX-42(+) cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up-regulation from 24 h to 1 wk post-SCI, the peak of microglia activation. These studies show that TNF-α and IL-1ß differentially affect cyclic AMP-PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases.

E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

Impact of Obstructive Sleep Apnea (OSA) in COVID-19 Survivors, Symptoms Changes Between 4-Months and 1 Year After the COVID-19 Infection.

Objective: To determine the association between Obstructive Sleep Apnea (OSA) with long-term symptoms and inflammatory cytokines, exploring the changes between 4-months and 1-year after COVID-19 infection. Methods: We conducted an observational, prospective cohort study, including patients ≥18 years old with confirmed diagnosis of COVID-19 between April to July 2020. All participants underwent two clinical follow-up visits, the first at 4-months (Visit 1) and the second at 1 year, after SARS-CoV-2 infection (Visit 2). Plasma glucose, total cholesterol, HDL, and triglycerides. Regarding pulmonary function, spirometry and lung diffusion capacity tests were assessed. For mental and neurocognitive evaluation, a short-form (SF-12), Beck depression and Hospital-Anxiety depression questionnaires were conducted at both time-points, whereas the Montreal Cognitive assessment was conducted during the second follow-up. Regarding to sleep evaluation, Epworth Sleepiness Scale, Insomnia Severity index and STOP-BANG questionnaire were conducted. Additionally, a home sleep apnea test and 7-day wrist actigraphy were performed in all participants. Inflammatory cytokines were measured using an inflammatory cytokine bead array kit. p-values < 0.05 were considered statistically significant and statistical analyses were performed using R software. Results: A total of 60 patients were included in the first follow-up, from which 57 completed the second follow-up. The mean age was 46.4 years-old (SD ± 13.1) and 53.3% were male. 30% of cases reported mild COVID-19 infection, 28.3% with moderate illness, and 41.6% with severe illness. Moreover, 56.6% of them were admitted to the ICU. Regarding to metabolic values, the OSA group showed higher values of insulin resistance (IR) (27%), systolic blood pressure (SBP) 135.2 (±19.1), dyslipidemia (67.5%), total cholesterol 202.1 (±60.5), triglycerides 176.1 (±119.0) and HOMA-IR 9.0 (±18.8) in comparison with the non-OSA group. 1 year after COVID-19 infection, DLCO test remains abnormal in OSA patients (25% OSA vs. 3.6% non-OSA, p = 0.02). Finally, those participants with OSA who develop ARDS reported an adjusted OR 20.4 (95%-CI, 1.04-504) risk of neurocognitive impairment. Discussion: Among patients with previous COVID-19, OSA impact the development of incident glycemic, neurocognitive impairment, and abnormal functional pulmonary changes that persist up to 1 year since acute phase.

Undiagnosed sleep disorder breathing as a risk factor for critical COVID-19 and pulmonary consequences at the midterm follow-up.

INTRODUCTION: Patients with severe COVID-19 develops an acute respiratory distress syndrome (ARDS), requiring admission to the intensive care unit. COVID-19 also reports an increased prevalence of comorbidities, similar to patients with Sleep disorder breathing (SDB). OBJECTIVES: To evaluate the association between undiagnosed SDB and the risk of ARDS and pulmonary abnormalities in a cohort of patients' survivors of COVID-19 between 3 and 6 months after diagnosis. METHODS: Prospective cohort study of patients who developed ARDS during hospitalization due to COVID-19 compared with a control group of patients who had COVID-19 with mild to moderate symptoms. All patients were evaluated between the 12th and 24th week after SARS-CoV-2 infection. The evaluation includes persistent symptoms, lung diffusing capacity of carbon monoxide (DLCO), chest CT scan and home sleep apnea test. SDB was diagnosed by the respiratory disturbance index ≥5 ev/h. The association between SDB and ARDS, the hazards of lung impairment and the hazard ratios (HR) were analyzed. RESULTS: A total of 60 patients were included (ARDS: 34 patients, Control: 26 patients). The mean follow-up was 16 weeks (range 12-24). ARDS reported a high prevalence of SDB (79% vs. 38% in control group). A total of 35% reported DLCO impairment, and 67.6% abnormal chest CT. SDB was independently associated to ARDS, OR 6.72 (CI, 1.56-28.93), p < 0.01, and abnormal Chest CT, HR 17.2 (CI, 1.68-177.4, p = 0.01). Besides, ARDS, days in mechanical ventilation, male gender were also associated with an increased risk of abnormal chest CT. CONCLUSION: Undiagnosed SDB is prevalent and independently associated with ARDS. In addition, undiagnosed SDB increased the hazard of abnormal Chest CT in the midterm. STUDY REGISTER: ISRCTN16865246.

Metabolic syndrome after liver transplant in patients at the specialized Center San Vicente Fundación, Rionegro, Antioquia, Colombia, 2013-2017. / Síndrome metabólico postrasplante hepático en pacientes del Centro Especializado San Vicente Fundación, Rionegro, Antioquia, Colombia, 2013-2017.

OBJECTIVES.: Motivation for the study: there is a lack of studies in Latin America on the frequency of metabolic syndrome in patients who receive liver transplants. Main findings: two-thirds (66%) of patients who received liver transplantation between 2013 and 2017 at the Specialized Center San Vicente Fundación de Rionegro, Antioquia, Colombia, subsequently presented metabolic syndrome. Implications: this study confirms that liver transplant recipients very frequently develop metabolic syndrome; however, the frequency found by this study (66%) was almost double that reported in other regions of the world, suggesting that patients from the Specialized Center San Vicente Fundación de Rionegro, Antioquia, Colombia, may present some additional condition. The medical records of all liver transplant patients attended at the Centro Especializado San Vicente Fundación between January 2013 and June 2017 were reviewed in order to determine the frequency of post-transplant metabolic syndrome (MS). We collected sociodemographic data, pathological history, toxicological history, complications, and ATP III criteria in a validated instrument. The statistical analysis was carried out with OpenEpi 3.01; p<0.05 was considered as statistically significant. Of the 102 reviewed medical records, 73 met the inclusion criteria (no MS diagnosis prior to transplant and complete information for the instru-ment) and were analyzed. Most patients were male (59%), older adults (64%) and married (62%). The frequency of MS after liver transplant was 66%. The association between MS and history of hypertension and diabetes was significant. We confirmed that MS is a frequent complication in liver transplant recipients and that history of hypertension and diabetes are the most frequent associated factors.

OBJETIVOS.: Motivation for the study: there is a lack of studies in Latin America on the frequency of metabolic syndrome in patients who receive liver transplants. Main findings: two-thirds (66%) of patients who received liver transplantation between 2013 and 2017 at the Specialized Center San Vicente Fundación de Rionegro, Antioquia, Colombia, subsequently presented metabolic syndrome. Implications: this study confirms that liver transplant recipients very frequently develop metabolic syndrome; however, the frequency found by this study (66%) was almost double that reported in other regions of the world, suggesting that patients from the Specialized Center San Vicente Fundación de Rionegro, Antioquia, Colombia, may present some additional condition. Se revisaron las historias clínicas de todos los pacientes con trasplante hepático atendidos en el Centro Especializado San Vicente Fundación entre enero de 2013 y junio de 2017, para determinar la frecuencia de síndrome metabólico (SM) postrasplante. En instrumento validado se registraron datos sociodemográficos, antecedentes patológicos, antecedentes toxicológicos, complicaciones y criterios ATP III. Se utilizó OpenEpi 3,01 para análisis estadístico; se consideró significancia estadística con p<0,05. De 102 historias clínicas revisadas, se analizaron 73 que cumplieron criterios de inclusión (sin diagnóstico de SM pretrasplante y con información completa para el instrumento). La mayoría de los pacientes eran hombres (59%), adultos mayores (64%) y casados (62%). La frecuencia de SM postrasplante fue 66%; hubo asociación significativa entre SM y antecedentes de hipertensión arterial y diabetes. Se confirma que el SM es una complicación frecuente de los trasplantados hepáticos y los antecedentes de hipertensión y diabetes son los factores asociados más frecuentes.

Development of a novel NS1 competitive enzyme-linked immunosorbent assay for the early detection of Zika virus infection.

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.